CHAPTER 46

Hepatic TissueEngineering

Kelly R Stevens1 Robert E Schwartz1 Shengyong Ng1 Jing Shan1

and Sangeeta N Bhatia121 Harvard-MIT Division of Health Sciences and Technology and Electrical Engineeringand Computer Science Massachusetts Institute of Technology Cambridge Massachusetts2 Howard Hughes Medical Institute and Department of Medicine Brigham amp WomenrsquosHospital Boston Massachusetts

951

LIVER FAILURE AND CURRENT TREATMENTSLiver disease afflicts over 600 million people worldwide 30 million of whom are AmericansLiver disease leads to the death of over 40000 individuals in the United States every

year Liver failure can be generally separated into two major categories fulminant hepatic

failure also referred to as acute liver failure and chronic hepatic failure resulting fromchronic end-stage liver disorders The term fulminant hepatic failure is utilized for cases in

which hepatic encephalopathy and impaired synthetic function (ie coagulopathy) develops

within 26 weeks of the initial onset of jaundice Hepatic encephalopathy is a neuropsy-chiatric condition which can be divided into four stages ranging from minor effects such as

mild confusion and sleep disorder to deep coma Although fulminant hepatic failure is

relatively rare with approximately 2000 cases in the United States per year it exhibits a highmortality rate of approximately 28 [1] The major identified causes of fulminant hepatic

failure include acetaminophen overdose idiosyncratic drug reactions and viral hepatitis A

and B [1] Of note a recent multicenter etiology study showed that 17 of fulminant hepaticfailure cases remained of indeterminate origin [2] In addition to hepatic encephalopathy

other clinical manifestations of fulminant hepatic failure include bacterial and fungal

infection coagulopathy as well as metabolic cardiorespiratory and hemodynamic abnor-malities Though spontaneous recovery has been observed due to the regenerative capacity of

the liver this type of recovery is difficult to predict and rarely occurs in various etiologiessuch as idiosyncratic drug toxicity and hepatitis B [1] Liver transplantation is currently the

only therapy shown to directly alter mortality and therefore is the standard of care in most

clinical settings As a result all fulminant hepatic failure patients that meet the criteria fororthotopic liver transplant are immediately listed as United Network for Organ Sharing

Status 1 (highest priority) upon presentation Factors that preclude this designation are

irreversible brain damage unresponsive cerebral edema uncontrollable sepsis malignancyand multisystem organ failure Despite the effectiveness of liver transplant in improving

short-term survival of fulminant hepatic failure patients the utility of this approach remains

limited due to the scarcity of donor organs

Liver failure due to chronic liver diseases while exhibiting a longer time-course of disease

pathogenesis is much more common than fulminant failure with chronic liver disease

Principles of Tissue Engineering httpdxdoiorg101016B978-0-12-398358-900046-X

Copyright 2014 Elsevier Inc All rights reserved

952

PART 11Gastrointestinal System

and cirrhosis being the twelfth leading cause of death (13 of total deaths) in the UnitedStates in 2010 [3] The most common causes of chronic liver disease are hepatitis B virus

hepatitis C virus and alcohol-induced and non-alcoholic fatty liver disease (NAFLD) [4]

Other etiologies largely include metabolic and autoimmune etiliogies primary sclerosingcholangitis primary biliary cirrhosis a1-antitrypsin deficiency autoimmune hepatitis

hereditary hemochromatosis Wilsonrsquos disease and liver cancer The prevalence of hepatitis Cinfection in the United States population has been estimated at 18 or nearly 4 million

individuals [5] Notably cirrhosis initiated by hepatitis C infection is the most frequent cause

for liver transplantation accounting for 40e50 of both individuals who have undergonetransplant and those on the waiting list [6] In addition the long-term inflammation

precipitated by chronic hepatitis B and C infection can also promote the development of

hepatocellular carcinoma As a result substantial efforts are focused on understandinghepatitis B and C viral pathogenesis and the development of approaches for the improved

control of the viruses before and after transplantation Taken together so-called fatty liver

diseases also comprise a major proportion of chronic liver disease patients [4] In particularNAFLD is an increasingly prevalent condition in the United States present in approximately

20 of adults of which a subset (2e3 of adults) exhibit non-alcoholic steatohepatitis

(NASH) defined by the presence of characteristic injury and necroinflammatory changes inaddition to excessive fat accumulation NAFLD pathogenesis has been shown to be associated

with risk factors and conditions such as obesity type 2 diabetes mellitus hyperlipidemia

hyperinsulinemia and insulin resistance Collectively chronic liver disorders can progresstowards the eventual development of cirrhosis and sequelae of decompensated cirrhosis

ascites portal hypertension variceal bleeding and hepatic encephalopathy Unfortunately

these complications represent decompensation of liver function and evidence of liverdysfunction Medical management can minimize the impact on patient quality of life but

transplantation is the only currently effective therapy As a means to more accurately deter-

mine organ allocation to patients on the liver transplant waiting list the MELD (mathe-matical model for end-stage liver disease) system was implemented in 2002 which assigns a

priority score based on three prognostic indicators bilirubin level creatine level and INR

(a measure of blood clotting time) [6] Although overall improvements in liver allocationhave been achieved following the introduction of the MELD system regional variations

in MELD scores exist and the ability of this model to accurately predict outcomes across the

entire score distribution and for distinct pathologies is less clear

Unlike other major causes of mortality liver disease death rates are rising rather than declining

Hepatitis B and hepatitis C virus infect 370 million and 130 million people respectivelyCurrent hepatitis B vaccination campaigns may help to decrease hepatitis B virus (HBV)

associated morbidity although the rapid increase in HIV coinfected patients has represented a

challenge for clinical management The rising rates of obesity have also resulted in the rapidrise of NAFLD worldwide with recent epidemiologic studies suggesting a prevalence rate from

6 to 35 with a median of 20 Up to 20 of patients with NAFLD may progress to

cirrhosis raising the possibility that liver disease may become one of the most common causesof mortality worldwide Moreover patients with cirrhosis or hepatitis B are at greater risk of

developing hepatocellular carcinoma Consequently hepatocellular carcinoma is the third

and sixth most common cause of cancer death among men and women respectively

Given the steady rise in patients with liver disease the need for liver transplantation has

continued to increase However the number of available donor livers has not changed

significantly in five years As a result several different approaches have been undertaken toaddress this growing of donor organs Several surgical options have been pursued including

the use of non-heart-beating donors or split liver transplants from cadaveric or living

donors [7] Split liver transplants depend on the significant regeneration capacity of themammalian liver This regenerative process has been extensively examined via experiments

in rodent models which demonstrate that partial hepatectomy or chemical injury induces the

CHAPTER 46Hepatic Tissue Engineering

953

proliferation of the existing mature cell populations within the liver including hepatocytesbile duct epithelial cells and others resulting in the replacement of lost liver mass However

liver regeneration is difficult to predict clinically and although partial liver transplants have

demonstrated some effectiveness biliary and vascular complications are major concerns inthese procedures [7] In addition the surgical risk to the living donors (which has included

several donor deaths) has raised significant ethical questions Furthermore in spite of thesesurgical advances and improvements in organ allocation the increasing divergence

between the number of patients awaiting transplantation and the number of available organs

suggests that it is unlikely that liver transplantation procedures alone will expand the supply ofdonor livers to meet the increasing demand Alternative approaches are therefore needed

and are actively being pursued These include several non-biological extracorporeal support

systems that will be discussed in more detail later in this chapter such as plasma exchangeplasmapheresis hemodialysis molecular adsorbents recirculation system or hemoperfusion

over charcoal or various resins These systems have shown limited success likely due to the

narrow range of functions inherent to each of these devices relative to the complex array offunctions performed by the healthy liver which include detoxification synthetic and meta-

bolic processes Recapitulation of a substantial range of liver functions will be required to

provide sufficient liver support and it is unclear which known liver functions (eggluconeogenesis serum protein production coagulation factor production toxin-mediating

encephalopathy) should be prioritized to improve clinical outcomes Given this complexity

extracorporeal support systems that do not incorporate a hepatocyte component may notbe able to ameliorate and augment liver disease outcomes as illustrated by the failure of

several non-biological extracorporeal support systems In contrast limited hepatocyte trans-

plantation trials have been shown in some cases to ameliorate and improve liver functionThese cellular therapies encompass approaches at providing temporary support such as

extracorporeal bioartificial liver (BAL) devices as well as more permanent adjunct

interventions such as cell transplantation transgenic xenografts and implantable hepa-tocellular constructs (Fig 461) Collectively the development of these types of cell-based

therapies for liver disease is a major aim of liver tissue engineering and fundamental advances

and current status of these approaches are reviewed in this chapter

FIGURE 461Cell-based therapies for liver diseaseExtracorporeal devices perfuse patientrsquos

blood or plasma through bioreactors

containing hepatocytes Hepatocytes are

transplanted directly or implanted on

scaffolds Transgenic animals are being

raised in order to reduce complement-

mediated damage of the endothelium [114]

PART 11Gastrointestinal System

954

CELL SOURCES FOR LIVER CELL-BASED THERAPIESCell-based therapies hold great promise but their clinical use has been hindered by the

inability of hepatocytes isolated from the in vivo hepatic microenvironment to maintainhepatocyte-specific phenotype and function in vitro Due to the paucity of human liver tissue as

a cell source alternative cell sources have been explored (Table 461) with inherent strengths

and drawbacks However the criteria to characterize these alternative cell sources as a hepa-tocyte or hepatocyte-like cells have not been standardized and vary greatly among different

studies No single test has been demonstrated to be sufficient to determine whether a particular

cell type truly recapitulates hepatocyte function as a result several tests must be carried out toquery various domains of hepatocyte function including bile production detoxification

metabolic and synthetic functions (Table 462)

Cell lines

Immortalized hepatocyte cell lines such as HepG2 (human hepatoblastoma) [8] the HepG2

derived line C3A [9] HepLiu (SV40 immortalized) [10] or immortalized fetal humanhepatocytes [11] have been utilized as readily available surrogates for hepatic tissue However

these cell lines lack the full functional capacity of primary adult hepatocytes and for clinical

applications there is a risk that oncogenic factors or transformed cells could be transmittedto the patient Thus the generation of conditionally immortalized lines and the incorporation

of inducible suicide genes have been considered as potential precautionary measures

Primary cells

The use of primary hepatocyte-based systems could potentially eliminate the issues faced byimmortalized lines and provide the appropriate repertoire of liver functions Primary porcine

hepatocytes have been utilized in a range of BAL device configurations with some

encouraging results However the utility of xenogeneic porcine cells for human liver therapiesis restricted by immunogenicity and the potential for xenozoonotic transmission of infectious

agents such as porcine endogenous retrovirus (PERV) Recognizing these concerns recent

efforts have led to the development of PERV-free pigs as well as genetically modified pigs thatare transgenic for human proteins thereby decreasing their immunogenicity

Primary human hepatocytes are the ideal cell type for cell-based therapies and the develop-ment of primary hepatocyte-based approaches is the focus of substantial ongoing research

However progress has been hindered by the limited supply of primary human hepatocytes

and the difficulty of maintaining hepatocyte function in vitro Discussed in more detail laterhepatocytes exhibit a loss of liver-specific functions under many conditions in vitro and

despite their significant proliferative capacity during regenerative responses in vivo mature

hepatocyte proliferation in culture is limited [12] A variety of techniques have been developed

TABLE 461 Cell sources for liver therapies

Cell source Critical issues

Primary hepatocytesHuman adult and fetal xenogeneic

Sourcing expansion phenotypic instabilityimmunogenicity safety (xenozoonotic)

Immortalized hepatocyte linesTumor-derived SV40 telomerasespontaneously immortalized

Range of functions genomic instability safety(tumorigenicity)

Stem CellsEmbryonic liver progenitors (hepatoblastsoval cells) other lineages (HSC MAPC)Induced pluripotent stem cells directreprogramming to hepatocytes

Sourcing differentiation efficiencyphenotypic instability immunogenicity safety(tumorigenicity)

TABLE 462 Hepatic functions

Functional classification Examples

Synthetic Albumin SecretionAlpha-1-antitrypsin SecretionCoagulation Factor Production (II IX X)Lipoprotein and apoprotein synthesisCeruloplasmin productionFerritin productionComplement production

Metabolic Ureagenesis and metabolismBilirubin MetabolismSteroid MetabolismGluconeogenesisGlycogen ProductionLipid metabolism

Detoxification pluripotent stemcells direct reprogramming tohepatocytes

Metabolize detoxify and inactivate exogenous andendogenous compounds via cytochrome P450enzymes methyltransferases sulfotransferasesacetyltransferases UDP-glucuronosyltransferasesand Glutathione S-transferases

Bile Production

CHAPTER 46Hepatic Tissue Engineering

955

to enable the cryopreservation of human hepatocytes [13] This enables the large number of

hepatocytes that are prepared from a single liver to be stored and thawed with reproduciblecellular function This option has opened the door to a variety of in vitro pharmacologic

and infectious disease studies [14e16]

Due to the limitations in mature hepatocyte expansion in vitro alternative cell sources are

being pursued These include various stem cell and progenitor populations which can self-

renew in vitro and exhibit multipotency or pluripotency and thereby serve as a possible sourceof hepatocytes as well as other non-parenchymal liver cells

Fetal and adult progenitor cells

A variety of fetal and adult progenitor cell types have been explored Current investigations are

focused on determining the differentiation potential and lineage relationships of thesepopulations Fetal hepatoblasts are liver precursor cells present during development that

exhibit a bipotential differentiation capacity defined by the capability to generate both

hepatocytes and bile duct epithelial cells Sourcing problems associated with fetal cells haveled researchers to search for resident cells that have progenitor properties in the adult Towards

this end a few groups have argued that rare resident cells (which may represent embryonic

remnants) exhibit properties consistent with the hypothesized adult hepatic stem cells andshare phenotypic markers and functional properties with fetal hepatoblasts In adult animal

livers suffering certain types of severe and chronic injury such oval cells can mediate liver

repair through a program similar to hepatic development Whether or not these processesoccur in normal human liver injury processes remains controversial In either case there is

little evidence that cells other than adult hepatocytes participate in the daily turnover of

healthy undamaged rodent or human liver and therefore the existence and role of hepaticstem cells or progenitors in healthy rodent and human liver homeostasis remains

controversial

Along similar lines Weiss and colleagues have demonstrated the development of bipotential

mouse embryonic liver (BMEL) cell lines derived from mouse E14 embryos that exhibit

characteristics comparable to fetal hepatoblasts and oval cells [17] These BMEL cells areproliferative can be induced to be hepatocyte-like or bile duct epithelial-like in vitro and can

PART 11Gastrointestinal System

956

home to the liver to undergo bipotential differentiation in vivo within a regenerative envi-ronment More recently biopotential human embryonic liver cells have been isolated and

similar to mouse BMEL cells are proliferative and capable of bipotential differentiation [18]

Pluripotent stem cells

Human embryonic stem cells (hES) first isolated by James Thomson in 1998 offer the ability to

generate large numbers of pluripotent cells that have the potential to form all three germ layersand differentiated cells including hepatocytes Subsequently Yamanaka and colleagues

demonstrated that fully differentiated adult cells such as fibroblasts or skin cells could be

reprogrammed to a undifferentiated pluripotent state similar to embryonic stem cells throughthe forced expression of reprogramming factors Oct34 and Sox2 along with either Klf4 or

Nanog and Lin28 These reprogrammed cells are termed induced pluripotent stem (iPS) cells

and highly resemble hES cells sharing many characteristics such as self-renewal capabilitiesin vitro and pluripotent differentiation potential in vitro and in vivo (though epigenetic differ-

ences between hES and iPS have been identified) Since iPS cells are sourced from adult somatic

cells unlike embryonic stem cells ethical considerations associated with hES are eliminatedMoreover iPS cells permit the generation of patient-specific cell populations potentially

enabling therapies to be developed according to the characteristics of an individual patient or

to study a variety of metabolic disorders and genetic variations that only manifest in an adult

Inspired by the understanding of how a totipotent stem cell proceeds to become a hepatocyte

during normal development multiple protocols have been described that enable hES cell

differentiation into hepatocyte-like cells Duncan and colleagues as well as other researchersdemonstrated that through iPS reprogramming and a subsequent multistep differentiation

protocol skin fibroblasts can give rise to hepatocyte-like cells which not only exhibit a varietyof hepatocyte-specific functions in vitro but can also be induced to generate intact fetal livers in

mice in vivo via tetraploid complementation [19] Pluripotent stem cell-derived hepatocytes

exhibit both the phenotypic and functional characteristics associated with hepatocytes thoughthe resulting differentiated cells exhibit a more fetal state of maturation compared to adult

human hepatocytes [20] In addition as with adult primary hepatocytes it is not clear whether

stem cell-derived lsquohepatocyte-like cellsrsquo will maintain their phenotype in vitro To date only oneexample of in vivo engraftment of pluripotent stem cell-derived hepatocyte-like cells has been

reported this group utilized a unique and specific injury mouse model and the authors noted

little to no human-specific hepatic functions after engraftment [21] Future studies will need toverify that pluripotent stem cell-derived hepatocyte-like cells can engraft in vivo and will also

need to address safety concerns such as the potential for pluripotent cell-derived teratoma

formation and the oncogenic risks associated with integrating vectors used to generate someiPS lines Human embryonic stem (ES) cells have recently been approved by the FDA (Food

and Drug Administration) for safety trials to commence such studies

Over the past few years a variety of lineages have been generated via the direct reprogrammingof one adult cell type into another without an undifferentiated pluripotent intermediate

Similar to the use of master transcriptional regulators in the reprogramming to iPS cells the

expression of a key sets of genes have been used to directly generate skeletal muscle tissue cellsthat highly resemble b-cells and neurons [22] More recently mouse fibroblasts have been

directly reprogrammed to generate hepatocytes [2324] These findings raise future possibil-

ities for deriving human hepatocytes directly from another adult cell type although it isunclear how reprogrammed hepatocyte-like cells may behave compared to primary cells or

whether this approach will also be successful using human cells

Ultimately understanding the mechanisms governing the fates of stem and progenitor cell

populations can empower the development of cell-based therapies However many challenges

remain including the ability to program differentiation completely beyond a fetalhepatocyte stage Furthermore regardless of the cell source phenotypic stabilization of

CHAPTER 46Hepatic Tissue Engineering

957

hepatocytes ex vivo remains a primary issue Microenvironmental signals including solublemediators cell-extracellular matrix interactions and cell-cell interactions have been implicated

in the regulation of hepatocyte function Accordingly the development of robust in vitro

liver models is an essential stepping-stone towards a thorough understanding of hepatocytebiology and improved effectiveness of cell-based therapies for liver disease and failure

IN VITRO HEPATIC CULTURE MODELSAn extensive range of liver model systems have been developed some of which include

perfused whole organs and wedge biopsies precision cut liver slices isolated primary hepa-tocytes in suspension or cultured upon extracellular matrix immortalized liver cell lines

isolated organelles membranes or enzymes and recombinant systems expressing specific drugmetabolism enzymes [25] While perfused whole organs wedge biopsies and liver slices

maintainmany aspects of the normal in vivomicroenvironment and architecture they typically

suffer from short-term viability (lt 24 hours) and limited nutrientoxygen diffusion to innercell layers Purified liver fractions and single enzyme systems are routinely used in high-

throughput systems to identify enzymes involved in the metabolism of new pharmaceutical

compounds although they lack the complete spectrum of gene expression and cellularmachinery required for liver-specific functions In addition cell lines derived from hepato-

blastomas or from immortalization of primary hepatocytes are finding limited use as repro-

ducible inexpensive models of hepatic tissue However such lines are plagued by abnormallevels and repertoire of hepatic functions [26] perhaps most notably the divergence of

constitutive and inducible levels of cytochrome P450 enzymes [27] Though each of

these models has found utility for focused questions in drug metabolism research isolatedprimary hepatocytes are generally considered to be most physiologically relevant for

constructing in vitro platforms for a multitude of applications However a major limitation in

the use of primary hepatocytes is that they are notoriously difficult to maintain in culture dueto their precipitous decline in viability and liver-specific functions upon isolation from the

liver [25] Accordingly substantial research has been conducted over the last two decades

towards elucidating the specific molecular stimuli that can maintain phenotypic functions inhepatocytes In subsequent sections we present examples of strategies that have been

employed to improve the survival and liver-specific functions of primary hepatocytes in

culture

In vivo microenvironment of the liver

In order to engineer an optimal microenvironment for hepatocytes in vitro one can utilize as a

guide the precisely defined architecture of the liver in which hepatocytes interact with diverseextracellular matrix molecules non-parenchymal cells and soluble factors (ie hormones

oxygen) (Fig 462) Structurally the two lobes of the liver contain repeating functional units

called lobules which are centered on a draining central vein Portal triads at each corner of alobule contain portal venules arterioles and bile ductules The blood supply to the liver

comes from two major blood vessels on its right lobe the hepatic artery (one-third of the

blood) and the portal vein (two-thirds) The intrahepatic circulation consists of sinusoidswhich are small tortuous vessels lined by a fenestrated basement membrane lacking

endothelium that is separated from the hepatocyte compartment by a thin extracellular matrix

region termed the space of Disse The hepatocytes constituting w70 of the liver mass arearranged in unicellular plates along the sinusoid where they experience homotypic cell

interactions Several types of junctions (ie gap junctions cadherins and tight junctions) and

bile canaliculi at the interface of hepatocytes facilitate the coordinated excretion of bile tothe bile duct and subsequently to the gall bladder Non-parenchymal cells including stellate

cells cholangiocytes (biliary ductal cells) sinusoidal endothelial cells Kupffer cells (liver

macrophages) natural killer cells and pit cells (large granular lymphocytes) interact withhepatocytes to modulate their diverse functions In the space of Disse hepatocytes are

FIGURE 462The precisely defined architecture of therepeating unit of the liver the lobuleHepatocytes are arranged in cords along the

length of the sinusoid where they interact

with extracellular matrix molecules

non-parenchymal cells and gradients of

soluble factors Nutrient and oxygen rich

blood from the intestine flows into the

sinusoid via the portal vein After being

processed by the hepatocytes the blood

enters the central vein and into the systemic

circulation (Reproduced with permission

from J Daugherty)

PART 11Gastrointestinal System

958

sandwiched between layers of extracellular matrix (collagen types I II III IV laminin

fibronectin heparan sulfate proteoglycans) the composition of which varies from the portaltriad to the central vein [28]

Within the liver lobule hepatocytes are partitioned into three zones based on morphologicaland functional variations along the length of the sinusoid (zonation) Zonal differences have

been observed in virtually all hepatocyte functions For instance compartmentalization of

gene expression is thought to underlie the capacity of the liver to operate as a lsquoglucostatrsquoFurthermore zonal differences in expression of cytochrome-P450 enzymes have also been

implicated in the zonal hepatotoxicity observed with some xenobiotics [29] Possible

modulators of zonation include blood-borne hormones oxygen tension pH levelsextracellular matrix composition and innervation [30] Therefore a precisely defined

microarchitecture coupled with specific cell-cell cell-soluble factor and cell-matrix

interactions allows the liver to carry out its many diverse functions which can be broadlycategorized into protein synthesis (ie albumin clotting factors) cholesterol metabolism bile

production glucose and fatty acid metabolism and detoxification of endogenous (ie bili-

rubin ammonia) and exogenous (drugs and environmental compounds) substances

Two-dimensional cultures

Numerous studies have improved primary hepatocyte survival and liver-specific functions

in vitro through modifications in microenvironmental signals including soluble factors(medium composition) cell-matrix interactions as well as heterotypic cell-cell interactions

with non-parenchymal cells (Fig 463) The supplementation of culture media with

FIGURE 463Cell sourcing of human hepatocytes Approaches have focused on modulating in vitro culture conditions such as the additionof soluble factors such as growth factors and small molecules extracellular matrix proteins heterotypic cell-cell interactions

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

953

proliferation of the existing mature cell populations within the liver including hepatocytesbile duct epithelial cells and others resulting in the replacement of lost liver mass However

liver regeneration is difficult to predict clinically and although partial liver transplants have

demonstrated some effectiveness biliary and vascular complications are major concerns inthese procedures [7] In addition the surgical risk to the living donors (which has included

several donor deaths) has raised significant ethical questions Furthermore in spite of thesesurgical advances and improvements in organ allocation the increasing divergence

between the number of patients awaiting transplantation and the number of available organs

suggests that it is unlikely that liver transplantation procedures alone will expand the supply ofdonor livers to meet the increasing demand Alternative approaches are therefore needed

and are actively being pursued These include several non-biological extracorporeal support

systems that will be discussed in more detail later in this chapter such as plasma exchangeplasmapheresis hemodialysis molecular adsorbents recirculation system or hemoperfusion

over charcoal or various resins These systems have shown limited success likely due to the

narrow range of functions inherent to each of these devices relative to the complex array offunctions performed by the healthy liver which include detoxification synthetic and meta-

bolic processes Recapitulation of a substantial range of liver functions will be required to

provide sufficient liver support and it is unclear which known liver functions (eggluconeogenesis serum protein production coagulation factor production toxin-mediating

encephalopathy) should be prioritized to improve clinical outcomes Given this complexity

extracorporeal support systems that do not incorporate a hepatocyte component may notbe able to ameliorate and augment liver disease outcomes as illustrated by the failure of

several non-biological extracorporeal support systems In contrast limited hepatocyte trans-

plantation trials have been shown in some cases to ameliorate and improve liver functionThese cellular therapies encompass approaches at providing temporary support such as

extracorporeal bioartificial liver (BAL) devices as well as more permanent adjunct

interventions such as cell transplantation transgenic xenografts and implantable hepa-tocellular constructs (Fig 461) Collectively the development of these types of cell-based

therapies for liver disease is a major aim of liver tissue engineering and fundamental advances

and current status of these approaches are reviewed in this chapter

FIGURE 461Cell-based therapies for liver diseaseExtracorporeal devices perfuse patientrsquos

blood or plasma through bioreactors

containing hepatocytes Hepatocytes are

transplanted directly or implanted on

scaffolds Transgenic animals are being

raised in order to reduce complement-

mediated damage of the endothelium [114]

PART 11Gastrointestinal System

954

CELL SOURCES FOR LIVER CELL-BASED THERAPIESCell-based therapies hold great promise but their clinical use has been hindered by the

inability of hepatocytes isolated from the in vivo hepatic microenvironment to maintainhepatocyte-specific phenotype and function in vitro Due to the paucity of human liver tissue as

a cell source alternative cell sources have been explored (Table 461) with inherent strengths

and drawbacks However the criteria to characterize these alternative cell sources as a hepa-tocyte or hepatocyte-like cells have not been standardized and vary greatly among different

studies No single test has been demonstrated to be sufficient to determine whether a particular

cell type truly recapitulates hepatocyte function as a result several tests must be carried out toquery various domains of hepatocyte function including bile production detoxification

metabolic and synthetic functions (Table 462)

Cell lines

Immortalized hepatocyte cell lines such as HepG2 (human hepatoblastoma) [8] the HepG2

derived line C3A [9] HepLiu (SV40 immortalized) [10] or immortalized fetal humanhepatocytes [11] have been utilized as readily available surrogates for hepatic tissue However

these cell lines lack the full functional capacity of primary adult hepatocytes and for clinical

applications there is a risk that oncogenic factors or transformed cells could be transmittedto the patient Thus the generation of conditionally immortalized lines and the incorporation

of inducible suicide genes have been considered as potential precautionary measures

Primary cells

The use of primary hepatocyte-based systems could potentially eliminate the issues faced byimmortalized lines and provide the appropriate repertoire of liver functions Primary porcine

hepatocytes have been utilized in a range of BAL device configurations with some

encouraging results However the utility of xenogeneic porcine cells for human liver therapiesis restricted by immunogenicity and the potential for xenozoonotic transmission of infectious

agents such as porcine endogenous retrovirus (PERV) Recognizing these concerns recent

efforts have led to the development of PERV-free pigs as well as genetically modified pigs thatare transgenic for human proteins thereby decreasing their immunogenicity

Primary human hepatocytes are the ideal cell type for cell-based therapies and the develop-ment of primary hepatocyte-based approaches is the focus of substantial ongoing research

However progress has been hindered by the limited supply of primary human hepatocytes

and the difficulty of maintaining hepatocyte function in vitro Discussed in more detail laterhepatocytes exhibit a loss of liver-specific functions under many conditions in vitro and

despite their significant proliferative capacity during regenerative responses in vivo mature

hepatocyte proliferation in culture is limited [12] A variety of techniques have been developed

TABLE 461 Cell sources for liver therapies

Cell source Critical issues

Primary hepatocytesHuman adult and fetal xenogeneic

Sourcing expansion phenotypic instabilityimmunogenicity safety (xenozoonotic)

Immortalized hepatocyte linesTumor-derived SV40 telomerasespontaneously immortalized

Range of functions genomic instability safety(tumorigenicity)

Stem CellsEmbryonic liver progenitors (hepatoblastsoval cells) other lineages (HSC MAPC)Induced pluripotent stem cells directreprogramming to hepatocytes

Sourcing differentiation efficiencyphenotypic instability immunogenicity safety(tumorigenicity)

TABLE 462 Hepatic functions

Functional classification Examples

Synthetic Albumin SecretionAlpha-1-antitrypsin SecretionCoagulation Factor Production (II IX X)Lipoprotein and apoprotein synthesisCeruloplasmin productionFerritin productionComplement production

Metabolic Ureagenesis and metabolismBilirubin MetabolismSteroid MetabolismGluconeogenesisGlycogen ProductionLipid metabolism

Detoxification pluripotent stemcells direct reprogramming tohepatocytes

Metabolize detoxify and inactivate exogenous andendogenous compounds via cytochrome P450enzymes methyltransferases sulfotransferasesacetyltransferases UDP-glucuronosyltransferasesand Glutathione S-transferases

Bile Production

CHAPTER 46Hepatic Tissue Engineering

955

to enable the cryopreservation of human hepatocytes [13] This enables the large number of

hepatocytes that are prepared from a single liver to be stored and thawed with reproduciblecellular function This option has opened the door to a variety of in vitro pharmacologic

and infectious disease studies [14e16]

Due to the limitations in mature hepatocyte expansion in vitro alternative cell sources are

being pursued These include various stem cell and progenitor populations which can self-

renew in vitro and exhibit multipotency or pluripotency and thereby serve as a possible sourceof hepatocytes as well as other non-parenchymal liver cells

Fetal and adult progenitor cells

A variety of fetal and adult progenitor cell types have been explored Current investigations are

focused on determining the differentiation potential and lineage relationships of thesepopulations Fetal hepatoblasts are liver precursor cells present during development that

exhibit a bipotential differentiation capacity defined by the capability to generate both

hepatocytes and bile duct epithelial cells Sourcing problems associated with fetal cells haveled researchers to search for resident cells that have progenitor properties in the adult Towards

this end a few groups have argued that rare resident cells (which may represent embryonic

remnants) exhibit properties consistent with the hypothesized adult hepatic stem cells andshare phenotypic markers and functional properties with fetal hepatoblasts In adult animal

livers suffering certain types of severe and chronic injury such oval cells can mediate liver

repair through a program similar to hepatic development Whether or not these processesoccur in normal human liver injury processes remains controversial In either case there is

little evidence that cells other than adult hepatocytes participate in the daily turnover of

healthy undamaged rodent or human liver and therefore the existence and role of hepaticstem cells or progenitors in healthy rodent and human liver homeostasis remains

controversial

Along similar lines Weiss and colleagues have demonstrated the development of bipotential

mouse embryonic liver (BMEL) cell lines derived from mouse E14 embryos that exhibit

characteristics comparable to fetal hepatoblasts and oval cells [17] These BMEL cells areproliferative can be induced to be hepatocyte-like or bile duct epithelial-like in vitro and can

PART 11Gastrointestinal System

956

home to the liver to undergo bipotential differentiation in vivo within a regenerative envi-ronment More recently biopotential human embryonic liver cells have been isolated and

similar to mouse BMEL cells are proliferative and capable of bipotential differentiation [18]

Pluripotent stem cells

Human embryonic stem cells (hES) first isolated by James Thomson in 1998 offer the ability to

generate large numbers of pluripotent cells that have the potential to form all three germ layersand differentiated cells including hepatocytes Subsequently Yamanaka and colleagues

demonstrated that fully differentiated adult cells such as fibroblasts or skin cells could be

reprogrammed to a undifferentiated pluripotent state similar to embryonic stem cells throughthe forced expression of reprogramming factors Oct34 and Sox2 along with either Klf4 or

Nanog and Lin28 These reprogrammed cells are termed induced pluripotent stem (iPS) cells

and highly resemble hES cells sharing many characteristics such as self-renewal capabilitiesin vitro and pluripotent differentiation potential in vitro and in vivo (though epigenetic differ-

ences between hES and iPS have been identified) Since iPS cells are sourced from adult somatic

cells unlike embryonic stem cells ethical considerations associated with hES are eliminatedMoreover iPS cells permit the generation of patient-specific cell populations potentially

enabling therapies to be developed according to the characteristics of an individual patient or

to study a variety of metabolic disorders and genetic variations that only manifest in an adult

Inspired by the understanding of how a totipotent stem cell proceeds to become a hepatocyte

during normal development multiple protocols have been described that enable hES cell

differentiation into hepatocyte-like cells Duncan and colleagues as well as other researchersdemonstrated that through iPS reprogramming and a subsequent multistep differentiation

protocol skin fibroblasts can give rise to hepatocyte-like cells which not only exhibit a varietyof hepatocyte-specific functions in vitro but can also be induced to generate intact fetal livers in

mice in vivo via tetraploid complementation [19] Pluripotent stem cell-derived hepatocytes

exhibit both the phenotypic and functional characteristics associated with hepatocytes thoughthe resulting differentiated cells exhibit a more fetal state of maturation compared to adult

human hepatocytes [20] In addition as with adult primary hepatocytes it is not clear whether

stem cell-derived lsquohepatocyte-like cellsrsquo will maintain their phenotype in vitro To date only oneexample of in vivo engraftment of pluripotent stem cell-derived hepatocyte-like cells has been

reported this group utilized a unique and specific injury mouse model and the authors noted

little to no human-specific hepatic functions after engraftment [21] Future studies will need toverify that pluripotent stem cell-derived hepatocyte-like cells can engraft in vivo and will also

need to address safety concerns such as the potential for pluripotent cell-derived teratoma

formation and the oncogenic risks associated with integrating vectors used to generate someiPS lines Human embryonic stem (ES) cells have recently been approved by the FDA (Food

and Drug Administration) for safety trials to commence such studies

Over the past few years a variety of lineages have been generated via the direct reprogrammingof one adult cell type into another without an undifferentiated pluripotent intermediate

Similar to the use of master transcriptional regulators in the reprogramming to iPS cells the

expression of a key sets of genes have been used to directly generate skeletal muscle tissue cellsthat highly resemble b-cells and neurons [22] More recently mouse fibroblasts have been

directly reprogrammed to generate hepatocytes [2324] These findings raise future possibil-

ities for deriving human hepatocytes directly from another adult cell type although it isunclear how reprogrammed hepatocyte-like cells may behave compared to primary cells or

whether this approach will also be successful using human cells

Ultimately understanding the mechanisms governing the fates of stem and progenitor cell

populations can empower the development of cell-based therapies However many challenges

remain including the ability to program differentiation completely beyond a fetalhepatocyte stage Furthermore regardless of the cell source phenotypic stabilization of

CHAPTER 46Hepatic Tissue Engineering

957

hepatocytes ex vivo remains a primary issue Microenvironmental signals including solublemediators cell-extracellular matrix interactions and cell-cell interactions have been implicated

in the regulation of hepatocyte function Accordingly the development of robust in vitro

liver models is an essential stepping-stone towards a thorough understanding of hepatocytebiology and improved effectiveness of cell-based therapies for liver disease and failure

IN VITRO HEPATIC CULTURE MODELSAn extensive range of liver model systems have been developed some of which include

perfused whole organs and wedge biopsies precision cut liver slices isolated primary hepa-tocytes in suspension or cultured upon extracellular matrix immortalized liver cell lines

isolated organelles membranes or enzymes and recombinant systems expressing specific drugmetabolism enzymes [25] While perfused whole organs wedge biopsies and liver slices

maintainmany aspects of the normal in vivomicroenvironment and architecture they typically

suffer from short-term viability (lt 24 hours) and limited nutrientoxygen diffusion to innercell layers Purified liver fractions and single enzyme systems are routinely used in high-

throughput systems to identify enzymes involved in the metabolism of new pharmaceutical

compounds although they lack the complete spectrum of gene expression and cellularmachinery required for liver-specific functions In addition cell lines derived from hepato-

blastomas or from immortalization of primary hepatocytes are finding limited use as repro-

ducible inexpensive models of hepatic tissue However such lines are plagued by abnormallevels and repertoire of hepatic functions [26] perhaps most notably the divergence of

constitutive and inducible levels of cytochrome P450 enzymes [27] Though each of

these models has found utility for focused questions in drug metabolism research isolatedprimary hepatocytes are generally considered to be most physiologically relevant for

constructing in vitro platforms for a multitude of applications However a major limitation in

the use of primary hepatocytes is that they are notoriously difficult to maintain in culture dueto their precipitous decline in viability and liver-specific functions upon isolation from the

liver [25] Accordingly substantial research has been conducted over the last two decades

towards elucidating the specific molecular stimuli that can maintain phenotypic functions inhepatocytes In subsequent sections we present examples of strategies that have been

employed to improve the survival and liver-specific functions of primary hepatocytes in

culture

In vivo microenvironment of the liver

In order to engineer an optimal microenvironment for hepatocytes in vitro one can utilize as a

guide the precisely defined architecture of the liver in which hepatocytes interact with diverseextracellular matrix molecules non-parenchymal cells and soluble factors (ie hormones

oxygen) (Fig 462) Structurally the two lobes of the liver contain repeating functional units

called lobules which are centered on a draining central vein Portal triads at each corner of alobule contain portal venules arterioles and bile ductules The blood supply to the liver

comes from two major blood vessels on its right lobe the hepatic artery (one-third of the

blood) and the portal vein (two-thirds) The intrahepatic circulation consists of sinusoidswhich are small tortuous vessels lined by a fenestrated basement membrane lacking

endothelium that is separated from the hepatocyte compartment by a thin extracellular matrix

region termed the space of Disse The hepatocytes constituting w70 of the liver mass arearranged in unicellular plates along the sinusoid where they experience homotypic cell

interactions Several types of junctions (ie gap junctions cadherins and tight junctions) and

bile canaliculi at the interface of hepatocytes facilitate the coordinated excretion of bile tothe bile duct and subsequently to the gall bladder Non-parenchymal cells including stellate

cells cholangiocytes (biliary ductal cells) sinusoidal endothelial cells Kupffer cells (liver

macrophages) natural killer cells and pit cells (large granular lymphocytes) interact withhepatocytes to modulate their diverse functions In the space of Disse hepatocytes are

FIGURE 462The precisely defined architecture of therepeating unit of the liver the lobuleHepatocytes are arranged in cords along the

length of the sinusoid where they interact

with extracellular matrix molecules

non-parenchymal cells and gradients of

soluble factors Nutrient and oxygen rich

blood from the intestine flows into the

sinusoid via the portal vein After being

processed by the hepatocytes the blood

enters the central vein and into the systemic

circulation (Reproduced with permission

from J Daugherty)

PART 11Gastrointestinal System

958

sandwiched between layers of extracellular matrix (collagen types I II III IV laminin

fibronectin heparan sulfate proteoglycans) the composition of which varies from the portaltriad to the central vein [28]

Within the liver lobule hepatocytes are partitioned into three zones based on morphologicaland functional variations along the length of the sinusoid (zonation) Zonal differences have

been observed in virtually all hepatocyte functions For instance compartmentalization of

gene expression is thought to underlie the capacity of the liver to operate as a lsquoglucostatrsquoFurthermore zonal differences in expression of cytochrome-P450 enzymes have also been

implicated in the zonal hepatotoxicity observed with some xenobiotics [29] Possible

modulators of zonation include blood-borne hormones oxygen tension pH levelsextracellular matrix composition and innervation [30] Therefore a precisely defined

microarchitecture coupled with specific cell-cell cell-soluble factor and cell-matrix

interactions allows the liver to carry out its many diverse functions which can be broadlycategorized into protein synthesis (ie albumin clotting factors) cholesterol metabolism bile

production glucose and fatty acid metabolism and detoxification of endogenous (ie bili-

rubin ammonia) and exogenous (drugs and environmental compounds) substances

Two-dimensional cultures

Numerous studies have improved primary hepatocyte survival and liver-specific functions

in vitro through modifications in microenvironmental signals including soluble factors(medium composition) cell-matrix interactions as well as heterotypic cell-cell interactions

with non-parenchymal cells (Fig 463) The supplementation of culture media with

FIGURE 463Cell sourcing of human hepatocytes Approaches have focused on modulating in vitro culture conditions such as the additionof soluble factors such as growth factors and small molecules extracellular matrix proteins heterotypic cell-cell interactions

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

954

CELL SOURCES FOR LIVER CELL-BASED THERAPIESCell-based therapies hold great promise but their clinical use has been hindered by the

inability of hepatocytes isolated from the in vivo hepatic microenvironment to maintainhepatocyte-specific phenotype and function in vitro Due to the paucity of human liver tissue as

a cell source alternative cell sources have been explored (Table 461) with inherent strengths

and drawbacks However the criteria to characterize these alternative cell sources as a hepa-tocyte or hepatocyte-like cells have not been standardized and vary greatly among different

studies No single test has been demonstrated to be sufficient to determine whether a particular

cell type truly recapitulates hepatocyte function as a result several tests must be carried out toquery various domains of hepatocyte function including bile production detoxification

metabolic and synthetic functions (Table 462)

Cell lines

Immortalized hepatocyte cell lines such as HepG2 (human hepatoblastoma) [8] the HepG2

derived line C3A [9] HepLiu (SV40 immortalized) [10] or immortalized fetal humanhepatocytes [11] have been utilized as readily available surrogates for hepatic tissue However

these cell lines lack the full functional capacity of primary adult hepatocytes and for clinical

applications there is a risk that oncogenic factors or transformed cells could be transmittedto the patient Thus the generation of conditionally immortalized lines and the incorporation

of inducible suicide genes have been considered as potential precautionary measures

Primary cells

The use of primary hepatocyte-based systems could potentially eliminate the issues faced byimmortalized lines and provide the appropriate repertoire of liver functions Primary porcine

hepatocytes have been utilized in a range of BAL device configurations with some

encouraging results However the utility of xenogeneic porcine cells for human liver therapiesis restricted by immunogenicity and the potential for xenozoonotic transmission of infectious

agents such as porcine endogenous retrovirus (PERV) Recognizing these concerns recent

efforts have led to the development of PERV-free pigs as well as genetically modified pigs thatare transgenic for human proteins thereby decreasing their immunogenicity

Primary human hepatocytes are the ideal cell type for cell-based therapies and the develop-ment of primary hepatocyte-based approaches is the focus of substantial ongoing research

However progress has been hindered by the limited supply of primary human hepatocytes

and the difficulty of maintaining hepatocyte function in vitro Discussed in more detail laterhepatocytes exhibit a loss of liver-specific functions under many conditions in vitro and

despite their significant proliferative capacity during regenerative responses in vivo mature

hepatocyte proliferation in culture is limited [12] A variety of techniques have been developed

TABLE 461 Cell sources for liver therapies

Cell source Critical issues

Primary hepatocytesHuman adult and fetal xenogeneic

Sourcing expansion phenotypic instabilityimmunogenicity safety (xenozoonotic)

Immortalized hepatocyte linesTumor-derived SV40 telomerasespontaneously immortalized

Range of functions genomic instability safety(tumorigenicity)

Stem CellsEmbryonic liver progenitors (hepatoblastsoval cells) other lineages (HSC MAPC)Induced pluripotent stem cells directreprogramming to hepatocytes

Sourcing differentiation efficiencyphenotypic instability immunogenicity safety(tumorigenicity)

TABLE 462 Hepatic functions

Functional classification Examples

Synthetic Albumin SecretionAlpha-1-antitrypsin SecretionCoagulation Factor Production (II IX X)Lipoprotein and apoprotein synthesisCeruloplasmin productionFerritin productionComplement production

Metabolic Ureagenesis and metabolismBilirubin MetabolismSteroid MetabolismGluconeogenesisGlycogen ProductionLipid metabolism

Detoxification pluripotent stemcells direct reprogramming tohepatocytes

Metabolize detoxify and inactivate exogenous andendogenous compounds via cytochrome P450enzymes methyltransferases sulfotransferasesacetyltransferases UDP-glucuronosyltransferasesand Glutathione S-transferases

Bile Production

CHAPTER 46Hepatic Tissue Engineering

955

to enable the cryopreservation of human hepatocytes [13] This enables the large number of

hepatocytes that are prepared from a single liver to be stored and thawed with reproduciblecellular function This option has opened the door to a variety of in vitro pharmacologic

and infectious disease studies [14e16]

Due to the limitations in mature hepatocyte expansion in vitro alternative cell sources are

being pursued These include various stem cell and progenitor populations which can self-

renew in vitro and exhibit multipotency or pluripotency and thereby serve as a possible sourceof hepatocytes as well as other non-parenchymal liver cells

Fetal and adult progenitor cells

A variety of fetal and adult progenitor cell types have been explored Current investigations are

focused on determining the differentiation potential and lineage relationships of thesepopulations Fetal hepatoblasts are liver precursor cells present during development that

exhibit a bipotential differentiation capacity defined by the capability to generate both

hepatocytes and bile duct epithelial cells Sourcing problems associated with fetal cells haveled researchers to search for resident cells that have progenitor properties in the adult Towards

this end a few groups have argued that rare resident cells (which may represent embryonic

remnants) exhibit properties consistent with the hypothesized adult hepatic stem cells andshare phenotypic markers and functional properties with fetal hepatoblasts In adult animal

livers suffering certain types of severe and chronic injury such oval cells can mediate liver

repair through a program similar to hepatic development Whether or not these processesoccur in normal human liver injury processes remains controversial In either case there is

little evidence that cells other than adult hepatocytes participate in the daily turnover of

healthy undamaged rodent or human liver and therefore the existence and role of hepaticstem cells or progenitors in healthy rodent and human liver homeostasis remains

controversial

Along similar lines Weiss and colleagues have demonstrated the development of bipotential

mouse embryonic liver (BMEL) cell lines derived from mouse E14 embryos that exhibit

characteristics comparable to fetal hepatoblasts and oval cells [17] These BMEL cells areproliferative can be induced to be hepatocyte-like or bile duct epithelial-like in vitro and can

PART 11Gastrointestinal System

956

home to the liver to undergo bipotential differentiation in vivo within a regenerative envi-ronment More recently biopotential human embryonic liver cells have been isolated and

similar to mouse BMEL cells are proliferative and capable of bipotential differentiation [18]

Pluripotent stem cells

Human embryonic stem cells (hES) first isolated by James Thomson in 1998 offer the ability to

generate large numbers of pluripotent cells that have the potential to form all three germ layersand differentiated cells including hepatocytes Subsequently Yamanaka and colleagues

demonstrated that fully differentiated adult cells such as fibroblasts or skin cells could be

reprogrammed to a undifferentiated pluripotent state similar to embryonic stem cells throughthe forced expression of reprogramming factors Oct34 and Sox2 along with either Klf4 or

Nanog and Lin28 These reprogrammed cells are termed induced pluripotent stem (iPS) cells

and highly resemble hES cells sharing many characteristics such as self-renewal capabilitiesin vitro and pluripotent differentiation potential in vitro and in vivo (though epigenetic differ-

ences between hES and iPS have been identified) Since iPS cells are sourced from adult somatic

cells unlike embryonic stem cells ethical considerations associated with hES are eliminatedMoreover iPS cells permit the generation of patient-specific cell populations potentially

enabling therapies to be developed according to the characteristics of an individual patient or

to study a variety of metabolic disorders and genetic variations that only manifest in an adult

Inspired by the understanding of how a totipotent stem cell proceeds to become a hepatocyte

during normal development multiple protocols have been described that enable hES cell

differentiation into hepatocyte-like cells Duncan and colleagues as well as other researchersdemonstrated that through iPS reprogramming and a subsequent multistep differentiation

protocol skin fibroblasts can give rise to hepatocyte-like cells which not only exhibit a varietyof hepatocyte-specific functions in vitro but can also be induced to generate intact fetal livers in

mice in vivo via tetraploid complementation [19] Pluripotent stem cell-derived hepatocytes

exhibit both the phenotypic and functional characteristics associated with hepatocytes thoughthe resulting differentiated cells exhibit a more fetal state of maturation compared to adult

human hepatocytes [20] In addition as with adult primary hepatocytes it is not clear whether

stem cell-derived lsquohepatocyte-like cellsrsquo will maintain their phenotype in vitro To date only oneexample of in vivo engraftment of pluripotent stem cell-derived hepatocyte-like cells has been

reported this group utilized a unique and specific injury mouse model and the authors noted

little to no human-specific hepatic functions after engraftment [21] Future studies will need toverify that pluripotent stem cell-derived hepatocyte-like cells can engraft in vivo and will also

need to address safety concerns such as the potential for pluripotent cell-derived teratoma

formation and the oncogenic risks associated with integrating vectors used to generate someiPS lines Human embryonic stem (ES) cells have recently been approved by the FDA (Food

and Drug Administration) for safety trials to commence such studies

Over the past few years a variety of lineages have been generated via the direct reprogrammingof one adult cell type into another without an undifferentiated pluripotent intermediate

Similar to the use of master transcriptional regulators in the reprogramming to iPS cells the

expression of a key sets of genes have been used to directly generate skeletal muscle tissue cellsthat highly resemble b-cells and neurons [22] More recently mouse fibroblasts have been

directly reprogrammed to generate hepatocytes [2324] These findings raise future possibil-

ities for deriving human hepatocytes directly from another adult cell type although it isunclear how reprogrammed hepatocyte-like cells may behave compared to primary cells or

whether this approach will also be successful using human cells

Ultimately understanding the mechanisms governing the fates of stem and progenitor cell

populations can empower the development of cell-based therapies However many challenges

remain including the ability to program differentiation completely beyond a fetalhepatocyte stage Furthermore regardless of the cell source phenotypic stabilization of

CHAPTER 46Hepatic Tissue Engineering

957

hepatocytes ex vivo remains a primary issue Microenvironmental signals including solublemediators cell-extracellular matrix interactions and cell-cell interactions have been implicated

in the regulation of hepatocyte function Accordingly the development of robust in vitro

liver models is an essential stepping-stone towards a thorough understanding of hepatocytebiology and improved effectiveness of cell-based therapies for liver disease and failure

IN VITRO HEPATIC CULTURE MODELSAn extensive range of liver model systems have been developed some of which include

perfused whole organs and wedge biopsies precision cut liver slices isolated primary hepa-tocytes in suspension or cultured upon extracellular matrix immortalized liver cell lines

isolated organelles membranes or enzymes and recombinant systems expressing specific drugmetabolism enzymes [25] While perfused whole organs wedge biopsies and liver slices

maintainmany aspects of the normal in vivomicroenvironment and architecture they typically

suffer from short-term viability (lt 24 hours) and limited nutrientoxygen diffusion to innercell layers Purified liver fractions and single enzyme systems are routinely used in high-

throughput systems to identify enzymes involved in the metabolism of new pharmaceutical

compounds although they lack the complete spectrum of gene expression and cellularmachinery required for liver-specific functions In addition cell lines derived from hepato-

blastomas or from immortalization of primary hepatocytes are finding limited use as repro-

ducible inexpensive models of hepatic tissue However such lines are plagued by abnormallevels and repertoire of hepatic functions [26] perhaps most notably the divergence of

constitutive and inducible levels of cytochrome P450 enzymes [27] Though each of

these models has found utility for focused questions in drug metabolism research isolatedprimary hepatocytes are generally considered to be most physiologically relevant for

constructing in vitro platforms for a multitude of applications However a major limitation in

the use of primary hepatocytes is that they are notoriously difficult to maintain in culture dueto their precipitous decline in viability and liver-specific functions upon isolation from the

liver [25] Accordingly substantial research has been conducted over the last two decades

towards elucidating the specific molecular stimuli that can maintain phenotypic functions inhepatocytes In subsequent sections we present examples of strategies that have been

employed to improve the survival and liver-specific functions of primary hepatocytes in

culture

In vivo microenvironment of the liver

In order to engineer an optimal microenvironment for hepatocytes in vitro one can utilize as a

guide the precisely defined architecture of the liver in which hepatocytes interact with diverseextracellular matrix molecules non-parenchymal cells and soluble factors (ie hormones

oxygen) (Fig 462) Structurally the two lobes of the liver contain repeating functional units

called lobules which are centered on a draining central vein Portal triads at each corner of alobule contain portal venules arterioles and bile ductules The blood supply to the liver

comes from two major blood vessels on its right lobe the hepatic artery (one-third of the

blood) and the portal vein (two-thirds) The intrahepatic circulation consists of sinusoidswhich are small tortuous vessels lined by a fenestrated basement membrane lacking

endothelium that is separated from the hepatocyte compartment by a thin extracellular matrix

region termed the space of Disse The hepatocytes constituting w70 of the liver mass arearranged in unicellular plates along the sinusoid where they experience homotypic cell

interactions Several types of junctions (ie gap junctions cadherins and tight junctions) and

bile canaliculi at the interface of hepatocytes facilitate the coordinated excretion of bile tothe bile duct and subsequently to the gall bladder Non-parenchymal cells including stellate

cells cholangiocytes (biliary ductal cells) sinusoidal endothelial cells Kupffer cells (liver

macrophages) natural killer cells and pit cells (large granular lymphocytes) interact withhepatocytes to modulate their diverse functions In the space of Disse hepatocytes are

FIGURE 462The precisely defined architecture of therepeating unit of the liver the lobuleHepatocytes are arranged in cords along the

length of the sinusoid where they interact

with extracellular matrix molecules

non-parenchymal cells and gradients of

soluble factors Nutrient and oxygen rich

blood from the intestine flows into the

sinusoid via the portal vein After being

processed by the hepatocytes the blood

enters the central vein and into the systemic

circulation (Reproduced with permission

from J Daugherty)

PART 11Gastrointestinal System

958

sandwiched between layers of extracellular matrix (collagen types I II III IV laminin

fibronectin heparan sulfate proteoglycans) the composition of which varies from the portaltriad to the central vein [28]

Within the liver lobule hepatocytes are partitioned into three zones based on morphologicaland functional variations along the length of the sinusoid (zonation) Zonal differences have

been observed in virtually all hepatocyte functions For instance compartmentalization of

gene expression is thought to underlie the capacity of the liver to operate as a lsquoglucostatrsquoFurthermore zonal differences in expression of cytochrome-P450 enzymes have also been

implicated in the zonal hepatotoxicity observed with some xenobiotics [29] Possible

modulators of zonation include blood-borne hormones oxygen tension pH levelsextracellular matrix composition and innervation [30] Therefore a precisely defined

microarchitecture coupled with specific cell-cell cell-soluble factor and cell-matrix

interactions allows the liver to carry out its many diverse functions which can be broadlycategorized into protein synthesis (ie albumin clotting factors) cholesterol metabolism bile

production glucose and fatty acid metabolism and detoxification of endogenous (ie bili-

rubin ammonia) and exogenous (drugs and environmental compounds) substances

Two-dimensional cultures

Numerous studies have improved primary hepatocyte survival and liver-specific functions

in vitro through modifications in microenvironmental signals including soluble factors(medium composition) cell-matrix interactions as well as heterotypic cell-cell interactions

with non-parenchymal cells (Fig 463) The supplementation of culture media with

FIGURE 463Cell sourcing of human hepatocytes Approaches have focused on modulating in vitro culture conditions such as the additionof soluble factors such as growth factors and small molecules extracellular matrix proteins heterotypic cell-cell interactions

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

TABLE 462 Hepatic functions

Functional classification Examples

Synthetic Albumin SecretionAlpha-1-antitrypsin SecretionCoagulation Factor Production (II IX X)Lipoprotein and apoprotein synthesisCeruloplasmin productionFerritin productionComplement production

Metabolic Ureagenesis and metabolismBilirubin MetabolismSteroid MetabolismGluconeogenesisGlycogen ProductionLipid metabolism

Detoxification pluripotent stemcells direct reprogramming tohepatocytes

Metabolize detoxify and inactivate exogenous andendogenous compounds via cytochrome P450enzymes methyltransferases sulfotransferasesacetyltransferases UDP-glucuronosyltransferasesand Glutathione S-transferases

Bile Production

CHAPTER 46Hepatic Tissue Engineering

955

to enable the cryopreservation of human hepatocytes [13] This enables the large number of

hepatocytes that are prepared from a single liver to be stored and thawed with reproduciblecellular function This option has opened the door to a variety of in vitro pharmacologic

and infectious disease studies [14e16]

Due to the limitations in mature hepatocyte expansion in vitro alternative cell sources are

being pursued These include various stem cell and progenitor populations which can self-

renew in vitro and exhibit multipotency or pluripotency and thereby serve as a possible sourceof hepatocytes as well as other non-parenchymal liver cells

Fetal and adult progenitor cells

A variety of fetal and adult progenitor cell types have been explored Current investigations are

focused on determining the differentiation potential and lineage relationships of thesepopulations Fetal hepatoblasts are liver precursor cells present during development that

exhibit a bipotential differentiation capacity defined by the capability to generate both

hepatocytes and bile duct epithelial cells Sourcing problems associated with fetal cells haveled researchers to search for resident cells that have progenitor properties in the adult Towards

this end a few groups have argued that rare resident cells (which may represent embryonic

remnants) exhibit properties consistent with the hypothesized adult hepatic stem cells andshare phenotypic markers and functional properties with fetal hepatoblasts In adult animal

livers suffering certain types of severe and chronic injury such oval cells can mediate liver

repair through a program similar to hepatic development Whether or not these processesoccur in normal human liver injury processes remains controversial In either case there is

little evidence that cells other than adult hepatocytes participate in the daily turnover of

healthy undamaged rodent or human liver and therefore the existence and role of hepaticstem cells or progenitors in healthy rodent and human liver homeostasis remains

controversial

Along similar lines Weiss and colleagues have demonstrated the development of bipotential

mouse embryonic liver (BMEL) cell lines derived from mouse E14 embryos that exhibit

characteristics comparable to fetal hepatoblasts and oval cells [17] These BMEL cells areproliferative can be induced to be hepatocyte-like or bile duct epithelial-like in vitro and can

PART 11Gastrointestinal System

956

home to the liver to undergo bipotential differentiation in vivo within a regenerative envi-ronment More recently biopotential human embryonic liver cells have been isolated and

similar to mouse BMEL cells are proliferative and capable of bipotential differentiation [18]

Pluripotent stem cells

Human embryonic stem cells (hES) first isolated by James Thomson in 1998 offer the ability to

generate large numbers of pluripotent cells that have the potential to form all three germ layersand differentiated cells including hepatocytes Subsequently Yamanaka and colleagues

demonstrated that fully differentiated adult cells such as fibroblasts or skin cells could be

reprogrammed to a undifferentiated pluripotent state similar to embryonic stem cells throughthe forced expression of reprogramming factors Oct34 and Sox2 along with either Klf4 or

Nanog and Lin28 These reprogrammed cells are termed induced pluripotent stem (iPS) cells

and highly resemble hES cells sharing many characteristics such as self-renewal capabilitiesin vitro and pluripotent differentiation potential in vitro and in vivo (though epigenetic differ-

ences between hES and iPS have been identified) Since iPS cells are sourced from adult somatic

cells unlike embryonic stem cells ethical considerations associated with hES are eliminatedMoreover iPS cells permit the generation of patient-specific cell populations potentially

enabling therapies to be developed according to the characteristics of an individual patient or

to study a variety of metabolic disorders and genetic variations that only manifest in an adult

Inspired by the understanding of how a totipotent stem cell proceeds to become a hepatocyte

during normal development multiple protocols have been described that enable hES cell

differentiation into hepatocyte-like cells Duncan and colleagues as well as other researchersdemonstrated that through iPS reprogramming and a subsequent multistep differentiation

protocol skin fibroblasts can give rise to hepatocyte-like cells which not only exhibit a varietyof hepatocyte-specific functions in vitro but can also be induced to generate intact fetal livers in

mice in vivo via tetraploid complementation [19] Pluripotent stem cell-derived hepatocytes

exhibit both the phenotypic and functional characteristics associated with hepatocytes thoughthe resulting differentiated cells exhibit a more fetal state of maturation compared to adult

human hepatocytes [20] In addition as with adult primary hepatocytes it is not clear whether

stem cell-derived lsquohepatocyte-like cellsrsquo will maintain their phenotype in vitro To date only oneexample of in vivo engraftment of pluripotent stem cell-derived hepatocyte-like cells has been

reported this group utilized a unique and specific injury mouse model and the authors noted

little to no human-specific hepatic functions after engraftment [21] Future studies will need toverify that pluripotent stem cell-derived hepatocyte-like cells can engraft in vivo and will also

need to address safety concerns such as the potential for pluripotent cell-derived teratoma

formation and the oncogenic risks associated with integrating vectors used to generate someiPS lines Human embryonic stem (ES) cells have recently been approved by the FDA (Food

and Drug Administration) for safety trials to commence such studies

Over the past few years a variety of lineages have been generated via the direct reprogrammingof one adult cell type into another without an undifferentiated pluripotent intermediate

Similar to the use of master transcriptional regulators in the reprogramming to iPS cells the

expression of a key sets of genes have been used to directly generate skeletal muscle tissue cellsthat highly resemble b-cells and neurons [22] More recently mouse fibroblasts have been

directly reprogrammed to generate hepatocytes [2324] These findings raise future possibil-

ities for deriving human hepatocytes directly from another adult cell type although it isunclear how reprogrammed hepatocyte-like cells may behave compared to primary cells or

whether this approach will also be successful using human cells

Ultimately understanding the mechanisms governing the fates of stem and progenitor cell

populations can empower the development of cell-based therapies However many challenges

remain including the ability to program differentiation completely beyond a fetalhepatocyte stage Furthermore regardless of the cell source phenotypic stabilization of

CHAPTER 46Hepatic Tissue Engineering

957

hepatocytes ex vivo remains a primary issue Microenvironmental signals including solublemediators cell-extracellular matrix interactions and cell-cell interactions have been implicated

in the regulation of hepatocyte function Accordingly the development of robust in vitro

liver models is an essential stepping-stone towards a thorough understanding of hepatocytebiology and improved effectiveness of cell-based therapies for liver disease and failure

IN VITRO HEPATIC CULTURE MODELSAn extensive range of liver model systems have been developed some of which include

perfused whole organs and wedge biopsies precision cut liver slices isolated primary hepa-tocytes in suspension or cultured upon extracellular matrix immortalized liver cell lines

isolated organelles membranes or enzymes and recombinant systems expressing specific drugmetabolism enzymes [25] While perfused whole organs wedge biopsies and liver slices

maintainmany aspects of the normal in vivomicroenvironment and architecture they typically

suffer from short-term viability (lt 24 hours) and limited nutrientoxygen diffusion to innercell layers Purified liver fractions and single enzyme systems are routinely used in high-

throughput systems to identify enzymes involved in the metabolism of new pharmaceutical

compounds although they lack the complete spectrum of gene expression and cellularmachinery required for liver-specific functions In addition cell lines derived from hepato-

blastomas or from immortalization of primary hepatocytes are finding limited use as repro-

ducible inexpensive models of hepatic tissue However such lines are plagued by abnormallevels and repertoire of hepatic functions [26] perhaps most notably the divergence of

constitutive and inducible levels of cytochrome P450 enzymes [27] Though each of

these models has found utility for focused questions in drug metabolism research isolatedprimary hepatocytes are generally considered to be most physiologically relevant for

constructing in vitro platforms for a multitude of applications However a major limitation in

the use of primary hepatocytes is that they are notoriously difficult to maintain in culture dueto their precipitous decline in viability and liver-specific functions upon isolation from the

liver [25] Accordingly substantial research has been conducted over the last two decades

towards elucidating the specific molecular stimuli that can maintain phenotypic functions inhepatocytes In subsequent sections we present examples of strategies that have been

employed to improve the survival and liver-specific functions of primary hepatocytes in

culture

In vivo microenvironment of the liver

In order to engineer an optimal microenvironment for hepatocytes in vitro one can utilize as a

guide the precisely defined architecture of the liver in which hepatocytes interact with diverseextracellular matrix molecules non-parenchymal cells and soluble factors (ie hormones

oxygen) (Fig 462) Structurally the two lobes of the liver contain repeating functional units

called lobules which are centered on a draining central vein Portal triads at each corner of alobule contain portal venules arterioles and bile ductules The blood supply to the liver

comes from two major blood vessels on its right lobe the hepatic artery (one-third of the

blood) and the portal vein (two-thirds) The intrahepatic circulation consists of sinusoidswhich are small tortuous vessels lined by a fenestrated basement membrane lacking

endothelium that is separated from the hepatocyte compartment by a thin extracellular matrix

region termed the space of Disse The hepatocytes constituting w70 of the liver mass arearranged in unicellular plates along the sinusoid where they experience homotypic cell

interactions Several types of junctions (ie gap junctions cadherins and tight junctions) and

bile canaliculi at the interface of hepatocytes facilitate the coordinated excretion of bile tothe bile duct and subsequently to the gall bladder Non-parenchymal cells including stellate

cells cholangiocytes (biliary ductal cells) sinusoidal endothelial cells Kupffer cells (liver

macrophages) natural killer cells and pit cells (large granular lymphocytes) interact withhepatocytes to modulate their diverse functions In the space of Disse hepatocytes are

FIGURE 462The precisely defined architecture of therepeating unit of the liver the lobuleHepatocytes are arranged in cords along the

length of the sinusoid where they interact

with extracellular matrix molecules

non-parenchymal cells and gradients of

soluble factors Nutrient and oxygen rich

blood from the intestine flows into the

sinusoid via the portal vein After being

processed by the hepatocytes the blood

enters the central vein and into the systemic

circulation (Reproduced with permission

from J Daugherty)

PART 11Gastrointestinal System

958

sandwiched between layers of extracellular matrix (collagen types I II III IV laminin

fibronectin heparan sulfate proteoglycans) the composition of which varies from the portaltriad to the central vein [28]

Within the liver lobule hepatocytes are partitioned into three zones based on morphologicaland functional variations along the length of the sinusoid (zonation) Zonal differences have

been observed in virtually all hepatocyte functions For instance compartmentalization of

gene expression is thought to underlie the capacity of the liver to operate as a lsquoglucostatrsquoFurthermore zonal differences in expression of cytochrome-P450 enzymes have also been

implicated in the zonal hepatotoxicity observed with some xenobiotics [29] Possible

modulators of zonation include blood-borne hormones oxygen tension pH levelsextracellular matrix composition and innervation [30] Therefore a precisely defined

microarchitecture coupled with specific cell-cell cell-soluble factor and cell-matrix

interactions allows the liver to carry out its many diverse functions which can be broadlycategorized into protein synthesis (ie albumin clotting factors) cholesterol metabolism bile

production glucose and fatty acid metabolism and detoxification of endogenous (ie bili-

rubin ammonia) and exogenous (drugs and environmental compounds) substances

Two-dimensional cultures

Numerous studies have improved primary hepatocyte survival and liver-specific functions

in vitro through modifications in microenvironmental signals including soluble factors(medium composition) cell-matrix interactions as well as heterotypic cell-cell interactions

with non-parenchymal cells (Fig 463) The supplementation of culture media with

FIGURE 463Cell sourcing of human hepatocytes Approaches have focused on modulating in vitro culture conditions such as the additionof soluble factors such as growth factors and small molecules extracellular matrix proteins heterotypic cell-cell interactions

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

956

home to the liver to undergo bipotential differentiation in vivo within a regenerative envi-ronment More recently biopotential human embryonic liver cells have been isolated and

similar to mouse BMEL cells are proliferative and capable of bipotential differentiation [18]

Pluripotent stem cells

Human embryonic stem cells (hES) first isolated by James Thomson in 1998 offer the ability to

generate large numbers of pluripotent cells that have the potential to form all three germ layersand differentiated cells including hepatocytes Subsequently Yamanaka and colleagues

demonstrated that fully differentiated adult cells such as fibroblasts or skin cells could be

reprogrammed to a undifferentiated pluripotent state similar to embryonic stem cells throughthe forced expression of reprogramming factors Oct34 and Sox2 along with either Klf4 or

Nanog and Lin28 These reprogrammed cells are termed induced pluripotent stem (iPS) cells

and highly resemble hES cells sharing many characteristics such as self-renewal capabilitiesin vitro and pluripotent differentiation potential in vitro and in vivo (though epigenetic differ-

ences between hES and iPS have been identified) Since iPS cells are sourced from adult somatic

cells unlike embryonic stem cells ethical considerations associated with hES are eliminatedMoreover iPS cells permit the generation of patient-specific cell populations potentially

enabling therapies to be developed according to the characteristics of an individual patient or

to study a variety of metabolic disorders and genetic variations that only manifest in an adult

Inspired by the understanding of how a totipotent stem cell proceeds to become a hepatocyte

during normal development multiple protocols have been described that enable hES cell

differentiation into hepatocyte-like cells Duncan and colleagues as well as other researchersdemonstrated that through iPS reprogramming and a subsequent multistep differentiation

protocol skin fibroblasts can give rise to hepatocyte-like cells which not only exhibit a varietyof hepatocyte-specific functions in vitro but can also be induced to generate intact fetal livers in

mice in vivo via tetraploid complementation [19] Pluripotent stem cell-derived hepatocytes

exhibit both the phenotypic and functional characteristics associated with hepatocytes thoughthe resulting differentiated cells exhibit a more fetal state of maturation compared to adult

human hepatocytes [20] In addition as with adult primary hepatocytes it is not clear whether

stem cell-derived lsquohepatocyte-like cellsrsquo will maintain their phenotype in vitro To date only oneexample of in vivo engraftment of pluripotent stem cell-derived hepatocyte-like cells has been

reported this group utilized a unique and specific injury mouse model and the authors noted

little to no human-specific hepatic functions after engraftment [21] Future studies will need toverify that pluripotent stem cell-derived hepatocyte-like cells can engraft in vivo and will also

need to address safety concerns such as the potential for pluripotent cell-derived teratoma

formation and the oncogenic risks associated with integrating vectors used to generate someiPS lines Human embryonic stem (ES) cells have recently been approved by the FDA (Food

and Drug Administration) for safety trials to commence such studies

Over the past few years a variety of lineages have been generated via the direct reprogrammingof one adult cell type into another without an undifferentiated pluripotent intermediate

Similar to the use of master transcriptional regulators in the reprogramming to iPS cells the

expression of a key sets of genes have been used to directly generate skeletal muscle tissue cellsthat highly resemble b-cells and neurons [22] More recently mouse fibroblasts have been

directly reprogrammed to generate hepatocytes [2324] These findings raise future possibil-

ities for deriving human hepatocytes directly from another adult cell type although it isunclear how reprogrammed hepatocyte-like cells may behave compared to primary cells or

whether this approach will also be successful using human cells

Ultimately understanding the mechanisms governing the fates of stem and progenitor cell

populations can empower the development of cell-based therapies However many challenges

remain including the ability to program differentiation completely beyond a fetalhepatocyte stage Furthermore regardless of the cell source phenotypic stabilization of

CHAPTER 46Hepatic Tissue Engineering

957

hepatocytes ex vivo remains a primary issue Microenvironmental signals including solublemediators cell-extracellular matrix interactions and cell-cell interactions have been implicated

in the regulation of hepatocyte function Accordingly the development of robust in vitro

liver models is an essential stepping-stone towards a thorough understanding of hepatocytebiology and improved effectiveness of cell-based therapies for liver disease and failure

IN VITRO HEPATIC CULTURE MODELSAn extensive range of liver model systems have been developed some of which include

perfused whole organs and wedge biopsies precision cut liver slices isolated primary hepa-tocytes in suspension or cultured upon extracellular matrix immortalized liver cell lines

isolated organelles membranes or enzymes and recombinant systems expressing specific drugmetabolism enzymes [25] While perfused whole organs wedge biopsies and liver slices

maintainmany aspects of the normal in vivomicroenvironment and architecture they typically

suffer from short-term viability (lt 24 hours) and limited nutrientoxygen diffusion to innercell layers Purified liver fractions and single enzyme systems are routinely used in high-

throughput systems to identify enzymes involved in the metabolism of new pharmaceutical

compounds although they lack the complete spectrum of gene expression and cellularmachinery required for liver-specific functions In addition cell lines derived from hepato-

blastomas or from immortalization of primary hepatocytes are finding limited use as repro-

ducible inexpensive models of hepatic tissue However such lines are plagued by abnormallevels and repertoire of hepatic functions [26] perhaps most notably the divergence of

constitutive and inducible levels of cytochrome P450 enzymes [27] Though each of

these models has found utility for focused questions in drug metabolism research isolatedprimary hepatocytes are generally considered to be most physiologically relevant for

constructing in vitro platforms for a multitude of applications However a major limitation in

the use of primary hepatocytes is that they are notoriously difficult to maintain in culture dueto their precipitous decline in viability and liver-specific functions upon isolation from the

liver [25] Accordingly substantial research has been conducted over the last two decades

towards elucidating the specific molecular stimuli that can maintain phenotypic functions inhepatocytes In subsequent sections we present examples of strategies that have been

employed to improve the survival and liver-specific functions of primary hepatocytes in

culture

In vivo microenvironment of the liver

In order to engineer an optimal microenvironment for hepatocytes in vitro one can utilize as a

guide the precisely defined architecture of the liver in which hepatocytes interact with diverseextracellular matrix molecules non-parenchymal cells and soluble factors (ie hormones

oxygen) (Fig 462) Structurally the two lobes of the liver contain repeating functional units

called lobules which are centered on a draining central vein Portal triads at each corner of alobule contain portal venules arterioles and bile ductules The blood supply to the liver

comes from two major blood vessels on its right lobe the hepatic artery (one-third of the

blood) and the portal vein (two-thirds) The intrahepatic circulation consists of sinusoidswhich are small tortuous vessels lined by a fenestrated basement membrane lacking

endothelium that is separated from the hepatocyte compartment by a thin extracellular matrix

region termed the space of Disse The hepatocytes constituting w70 of the liver mass arearranged in unicellular plates along the sinusoid where they experience homotypic cell

interactions Several types of junctions (ie gap junctions cadherins and tight junctions) and

bile canaliculi at the interface of hepatocytes facilitate the coordinated excretion of bile tothe bile duct and subsequently to the gall bladder Non-parenchymal cells including stellate

cells cholangiocytes (biliary ductal cells) sinusoidal endothelial cells Kupffer cells (liver

macrophages) natural killer cells and pit cells (large granular lymphocytes) interact withhepatocytes to modulate their diverse functions In the space of Disse hepatocytes are

FIGURE 462The precisely defined architecture of therepeating unit of the liver the lobuleHepatocytes are arranged in cords along the

length of the sinusoid where they interact

with extracellular matrix molecules

non-parenchymal cells and gradients of

soluble factors Nutrient and oxygen rich

blood from the intestine flows into the

sinusoid via the portal vein After being

processed by the hepatocytes the blood

enters the central vein and into the systemic

circulation (Reproduced with permission

from J Daugherty)

PART 11Gastrointestinal System

958

sandwiched between layers of extracellular matrix (collagen types I II III IV laminin

fibronectin heparan sulfate proteoglycans) the composition of which varies from the portaltriad to the central vein [28]

Within the liver lobule hepatocytes are partitioned into three zones based on morphologicaland functional variations along the length of the sinusoid (zonation) Zonal differences have

been observed in virtually all hepatocyte functions For instance compartmentalization of

gene expression is thought to underlie the capacity of the liver to operate as a lsquoglucostatrsquoFurthermore zonal differences in expression of cytochrome-P450 enzymes have also been

implicated in the zonal hepatotoxicity observed with some xenobiotics [29] Possible

modulators of zonation include blood-borne hormones oxygen tension pH levelsextracellular matrix composition and innervation [30] Therefore a precisely defined

microarchitecture coupled with specific cell-cell cell-soluble factor and cell-matrix

interactions allows the liver to carry out its many diverse functions which can be broadlycategorized into protein synthesis (ie albumin clotting factors) cholesterol metabolism bile

production glucose and fatty acid metabolism and detoxification of endogenous (ie bili-

rubin ammonia) and exogenous (drugs and environmental compounds) substances

Two-dimensional cultures

Numerous studies have improved primary hepatocyte survival and liver-specific functions

in vitro through modifications in microenvironmental signals including soluble factors(medium composition) cell-matrix interactions as well as heterotypic cell-cell interactions

with non-parenchymal cells (Fig 463) The supplementation of culture media with

FIGURE 463Cell sourcing of human hepatocytes Approaches have focused on modulating in vitro culture conditions such as the additionof soluble factors such as growth factors and small molecules extracellular matrix proteins heterotypic cell-cell interactions

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

957

hepatocytes ex vivo remains a primary issue Microenvironmental signals including solublemediators cell-extracellular matrix interactions and cell-cell interactions have been implicated

in the regulation of hepatocyte function Accordingly the development of robust in vitro

liver models is an essential stepping-stone towards a thorough understanding of hepatocytebiology and improved effectiveness of cell-based therapies for liver disease and failure

IN VITRO HEPATIC CULTURE MODELSAn extensive range of liver model systems have been developed some of which include

perfused whole organs and wedge biopsies precision cut liver slices isolated primary hepa-tocytes in suspension or cultured upon extracellular matrix immortalized liver cell lines

isolated organelles membranes or enzymes and recombinant systems expressing specific drugmetabolism enzymes [25] While perfused whole organs wedge biopsies and liver slices

maintainmany aspects of the normal in vivomicroenvironment and architecture they typically

suffer from short-term viability (lt 24 hours) and limited nutrientoxygen diffusion to innercell layers Purified liver fractions and single enzyme systems are routinely used in high-

throughput systems to identify enzymes involved in the metabolism of new pharmaceutical

compounds although they lack the complete spectrum of gene expression and cellularmachinery required for liver-specific functions In addition cell lines derived from hepato-

blastomas or from immortalization of primary hepatocytes are finding limited use as repro-

ducible inexpensive models of hepatic tissue However such lines are plagued by abnormallevels and repertoire of hepatic functions [26] perhaps most notably the divergence of

constitutive and inducible levels of cytochrome P450 enzymes [27] Though each of

these models has found utility for focused questions in drug metabolism research isolatedprimary hepatocytes are generally considered to be most physiologically relevant for

constructing in vitro platforms for a multitude of applications However a major limitation in

the use of primary hepatocytes is that they are notoriously difficult to maintain in culture dueto their precipitous decline in viability and liver-specific functions upon isolation from the

liver [25] Accordingly substantial research has been conducted over the last two decades

towards elucidating the specific molecular stimuli that can maintain phenotypic functions inhepatocytes In subsequent sections we present examples of strategies that have been

employed to improve the survival and liver-specific functions of primary hepatocytes in

culture

In vivo microenvironment of the liver

In order to engineer an optimal microenvironment for hepatocytes in vitro one can utilize as a

guide the precisely defined architecture of the liver in which hepatocytes interact with diverseextracellular matrix molecules non-parenchymal cells and soluble factors (ie hormones

oxygen) (Fig 462) Structurally the two lobes of the liver contain repeating functional units

called lobules which are centered on a draining central vein Portal triads at each corner of alobule contain portal venules arterioles and bile ductules The blood supply to the liver

comes from two major blood vessels on its right lobe the hepatic artery (one-third of the

blood) and the portal vein (two-thirds) The intrahepatic circulation consists of sinusoidswhich are small tortuous vessels lined by a fenestrated basement membrane lacking

endothelium that is separated from the hepatocyte compartment by a thin extracellular matrix

region termed the space of Disse The hepatocytes constituting w70 of the liver mass arearranged in unicellular plates along the sinusoid where they experience homotypic cell

interactions Several types of junctions (ie gap junctions cadherins and tight junctions) and

bile canaliculi at the interface of hepatocytes facilitate the coordinated excretion of bile tothe bile duct and subsequently to the gall bladder Non-parenchymal cells including stellate

cells cholangiocytes (biliary ductal cells) sinusoidal endothelial cells Kupffer cells (liver

macrophages) natural killer cells and pit cells (large granular lymphocytes) interact withhepatocytes to modulate their diverse functions In the space of Disse hepatocytes are

FIGURE 462The precisely defined architecture of therepeating unit of the liver the lobuleHepatocytes are arranged in cords along the

length of the sinusoid where they interact

with extracellular matrix molecules

non-parenchymal cells and gradients of

soluble factors Nutrient and oxygen rich

blood from the intestine flows into the

sinusoid via the portal vein After being

processed by the hepatocytes the blood

enters the central vein and into the systemic

circulation (Reproduced with permission

from J Daugherty)

PART 11Gastrointestinal System

958

sandwiched between layers of extracellular matrix (collagen types I II III IV laminin

fibronectin heparan sulfate proteoglycans) the composition of which varies from the portaltriad to the central vein [28]

Within the liver lobule hepatocytes are partitioned into three zones based on morphologicaland functional variations along the length of the sinusoid (zonation) Zonal differences have

been observed in virtually all hepatocyte functions For instance compartmentalization of

gene expression is thought to underlie the capacity of the liver to operate as a lsquoglucostatrsquoFurthermore zonal differences in expression of cytochrome-P450 enzymes have also been

implicated in the zonal hepatotoxicity observed with some xenobiotics [29] Possible

modulators of zonation include blood-borne hormones oxygen tension pH levelsextracellular matrix composition and innervation [30] Therefore a precisely defined

microarchitecture coupled with specific cell-cell cell-soluble factor and cell-matrix

interactions allows the liver to carry out its many diverse functions which can be broadlycategorized into protein synthesis (ie albumin clotting factors) cholesterol metabolism bile

production glucose and fatty acid metabolism and detoxification of endogenous (ie bili-

rubin ammonia) and exogenous (drugs and environmental compounds) substances

Two-dimensional cultures

Numerous studies have improved primary hepatocyte survival and liver-specific functions

in vitro through modifications in microenvironmental signals including soluble factors(medium composition) cell-matrix interactions as well as heterotypic cell-cell interactions

with non-parenchymal cells (Fig 463) The supplementation of culture media with

FIGURE 463Cell sourcing of human hepatocytes Approaches have focused on modulating in vitro culture conditions such as the additionof soluble factors such as growth factors and small molecules extracellular matrix proteins heterotypic cell-cell interactions

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

FIGURE 462The precisely defined architecture of therepeating unit of the liver the lobuleHepatocytes are arranged in cords along the

length of the sinusoid where they interact

with extracellular matrix molecules

non-parenchymal cells and gradients of

soluble factors Nutrient and oxygen rich

blood from the intestine flows into the

sinusoid via the portal vein After being

processed by the hepatocytes the blood

enters the central vein and into the systemic

circulation (Reproduced with permission

from J Daugherty)

PART 11Gastrointestinal System

958

sandwiched between layers of extracellular matrix (collagen types I II III IV laminin

fibronectin heparan sulfate proteoglycans) the composition of which varies from the portaltriad to the central vein [28]

Within the liver lobule hepatocytes are partitioned into three zones based on morphologicaland functional variations along the length of the sinusoid (zonation) Zonal differences have

been observed in virtually all hepatocyte functions For instance compartmentalization of

gene expression is thought to underlie the capacity of the liver to operate as a lsquoglucostatrsquoFurthermore zonal differences in expression of cytochrome-P450 enzymes have also been

implicated in the zonal hepatotoxicity observed with some xenobiotics [29] Possible

modulators of zonation include blood-borne hormones oxygen tension pH levelsextracellular matrix composition and innervation [30] Therefore a precisely defined

microarchitecture coupled with specific cell-cell cell-soluble factor and cell-matrix

interactions allows the liver to carry out its many diverse functions which can be broadlycategorized into protein synthesis (ie albumin clotting factors) cholesterol metabolism bile

production glucose and fatty acid metabolism and detoxification of endogenous (ie bili-

rubin ammonia) and exogenous (drugs and environmental compounds) substances

Two-dimensional cultures

Numerous studies have improved primary hepatocyte survival and liver-specific functions

in vitro through modifications in microenvironmental signals including soluble factors(medium composition) cell-matrix interactions as well as heterotypic cell-cell interactions

with non-parenchymal cells (Fig 463) The supplementation of culture media with

FIGURE 463Cell sourcing of human hepatocytes Approaches have focused on modulating in vitro culture conditions such as the additionof soluble factors such as growth factors and small molecules extracellular matrix proteins heterotypic cell-cell interactions

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

959

physiological factors such as hormones corticosteroids growth factors vitamins amino acidsor trace elements [2531] or non-physiological small molecules such as phenobarbital and

dimethylsulfoxide [3233] have been shown to modulate hepatocyte function More recently

oxygen tension has also been shown to modulate hepatocyte function [34] Certain mitogenicfactors like epidermal growth factor and hepatocyte growth factor can induce limited prolif-

eration in rat hepatocytes in vitro but these mitogens result in negligible proliferation ofhuman hepatocytes in vitro [3536] The impact of extracellular matrix composition and to-

pology on hepatocyte phenotype is widely recognized A variety of extracellular matrix (ECM)

coatings (most commonly collagen type I) have been shown to enhance hepatocyte attach-ment to the substrate but this usually occurs concomitantly with hepatocyte spreading and a

subsequent loss of hepatocyte function [37] Studies investigating the benefits of complex

mixtures of ECM components have utilized strategies including Matrigel liver-derived bio-matrix [37e40] or bottom-up approaches such as a combinatorial high-throughput ECM

microarray where the latter has revealed specific combinations of ECM molecules that

improve hepatocyte function compared tomonolayer cultures on collagen I [41] In contrast toculturing hepatocytes on a monolayer of ECM molecules hepatocytes have also been sand-

wiched between two layers of type I collagen gel In this classic lsquosandwichrsquo culture format

hepatocytes exhibit desirable morphology with polarized bile canaliculi as well as stablefunctions for several weeks [4243] However phase III detoxification processes have been

shown to become imbalanced over time in this format [44] Also the presence of an overlaid

layer of extracellular matrix may present diffusion barriers for molecular stimuli (ie drugcandidates) and the fragility of the gelled matrix may hinder scale-up within BAL devices or

multiwell in vitro models

Heterotypic interactions between hepatocytes and their non-parenchymal neighbors areknown to be important at multiple stages in vivo Liver specification from the endodermal

foregut and mesenchymal vasculature during development is believed to be mediated by

heterotypic interactions [4546] Similarly non-parenchymal cells of several types modulatecell fate processes of hepatocytes under both physiologic and pathophysiologic conditions

within the adult liver [4748] Substantial work pioneered by Guguen-Guillouzo and col-

leagues has demonstrated that a wide variety of non-parenchymal cells from both within andoutside the liver are capable of supporting hepatocyte function for several weeks in co-culture

contexts in vitro even across species barriers suggesting that the mechanisms responsiblefor non-parenchymal cell-mediated stabilization of hepatocyte phenotype may be conserved

[4950] Microfabrication approaches have been employed to control tissue microarchitecture

in hepatic co-cultures so as to achieve an optimal balance of homotypic and heterotypiccellular interactions to promote hepatocyte function which will be described in a later section

Hepatic co-cultures have been utilized to investigate various physiologic and pathophysiologic

processes including the acute phase response mutagenesis xenobiotic toxicity oxidativestress lipid and drug metabolism [50] For example co-cultures of hepatocytes and Kupffer

cells have been used to examine mechanisms of hepatocellular damage [5152] while

co-cultures with liver sinusoidal endothelial cells (which are also phenotypically unstable inmonoculture upon isolation from the liver) have highlighted the importance of hepatocyte-

endothelial cell interactions in the bidirectional stabilization of these cell types [5356]

Studies focused on the underlying mechanisms of hepatocyte stabilization in co-culture haveidentified several cell surface and secreted factors that play a role including T-cadherin

E-cadherin decorin and TGF-b1 which demonstrate the potential for highly functional

hepatocyte-only culture platforms [5758]

Three-dimensional cultures

Culture of hepatocytes on substrates that promote aggregation into three-dimensional

spheroids has also been extensively explored Numerous technologies including non-adhesivesurfaces (such as poly(2-hydroxyethyl methacrylate) and positively charged Primaria dishes)

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

960

rotational or rocking cultures and encapsulation in macroporous scaffolds have been devel-oped for hepatocyte spheroid formation [59e64] Under spheroidal culture conditions he-

patocyte survival and functions are improved over standard monolayers on collagen [65]

Potential mechanisms underlying these effects include an increased extent of homotypiccell-cell contacts between hepatocytes the retention of a three-dimensional cytoarchitecture

and the three-dimensional presentation of ECM molecules around the spheroids [59] Somelimitations of conventional spheroidal culture include a tendency for secondary aggrega-

tion of spheroids and the resultant development of a necrotic core in the larger aggregates due

to diffusion-limited transport of nutrients and waste and the lack of control over the cellnumbers within each spheroid [25] A variety of methods are being developed to prevent

secondary aggregation of initially-formed spheroids and control cell-cell interactions

including microfabricated scaffolds bioreactor systems encapsulation techniques and syn-thetic linkers [266667e73] which are discussed in further detail in the following sections

Bioreactor cultures

A wide range of small-scale bioreactor platforms have been developed for in vitro liver appli-

cations For example perfusion systems containing hepatocellular aggregates exhibit desirablecell morphology and liver-specific functions for several weeks in culture [266874e75]

and the incorporation of multiple reactors in parallel has been explored as an approach for

high-throughput drug screening studies [76e79] In order to promote oxygen delivery whileprotecting hepatocytes from deleterious shear effects gas-permeable membranes with

endothelial-like physical parameters grooved substrates and microfluidic microchannel

networks have been integrated into several bioreactor designs [7680e82] In vivo hepatocytefunctions are heterogeneously segregated along the hepatic sinusoid and this zonation is

thought to be modulated by gradients in oxygen hormones nutrients and extracellular matrix

molecules Using a parallel-plate bioreactor it was demonstrated that steady state oxygengradients characteristic of those found in liver sinuosoids could contribute to a heterogeneous

expression of drug metabolism enzymes CYP2B and CYP3A in hepatocyte-non-parenchymal

co-cultures which mimics the expression gradients present in vivo (Fig 464) [83] Further-more the localization of acetaminophen-induced hepatic toxicity in the region experiencing

low oxygen in this model system recapitulates the perivenous location of toxicity in vivo

A zonal microbioreactor array based on microfluidic serpentine mixing regions feeding intoseveral microbioreactors in parallel has also been designed for potential in vitro liver zonation

applications (Fig 464) Overall the ability to decouple oxygen tension from gradients of

other soluble stimuli and cell-cell interaction effects within this platform represents animportant tool for the systematic investigation of the role of extracellular stimuli in zonation

Bioreactors may also be used to studymore dynamic physiological processes than is possible inconventional culture platforms For example a recent bioreactor device describes the ability

to monitor invasion of metastatic cells into hepatic parenchyma by recreating relevant features

of the liver tissue such as fluid flow and length scales [84] Collectively by providing dynamiccontrol over hepatocyte culture parameters and hence hepatocyte function bioreactors will

continue to be useful in studying liver biology and facilitating drug development applications

Microtechnology tools to optimize and miniaturize liver cultures

The ability to control tissue architecture along with cell-cell and cell-matrix interactions on theorder of single cell dimensions represents another important tool in the investigation of

mechanisms underlying tissue development and ultimately the realization of tissue-

engineered systems Semiconductor-driven microtechnology tools enable micrometer-scalecontrol over cell adhesion shape and multicellular interactions [8586] Thus over the last

decade microtechnology tools have emerged both to probe biomedical phenomena at rele-

vant length scales and to miniaturize and parallelize biomedical assays (eg DNAmicroarraysmicrofluidics)

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

(a)

(e)(d)

(c)

(b)

FIGURE 464Bioreactors for in vitro liver applications (a) Zonation and toxicity in a hepatocyte bioreactor Co-cultures of hepatocytes and non-parenchymal cells are

created on collagen-coated glass slides and placed in a bioreactor circuit where the oxygen concentration at the inlet is held at a constant value Depletion of

oxygen by cells creates a gradient of oxygen tensions along the length of the chamber similar to that observed in vivo (b) Two-dimensional contour plot of the

medial cross section of the reactor depicting the cell surface oxygen gradient formed with inlet pO2 of 76 mmHg and flow rate of 03 mLmin

(c) Bright-field images of perfused cultures treated with 15mM acetaminophen (APAP a hepatotoxin) and stained with MTT (3-(45-dimethylthiazol-2-yl)-25-

diphenyltetrazolium bromide) as a measure of cell viability from five regions along the length of the bioreactor The intensity of MTT staining is reported as

relative optical density (ROD) values The zonal pattern of APAP toxicity seen here is consistent with that observed in vivo [83] (d) A zonal microbioreactor array

that incorporates serpentine mixing regions and two sources is able to create a gradient of fluorescein (shown in blue on right) in an array of microbioreactors

containing random co-cultures of hepatocytes and non-parenchymal cells (bottom) (e) A bilayer microfluidics device designed with a microchannel network that

mimics liver vasculature so as to support the large metabolic needs of hepatocytes contained within an adjacent chamber (Figure panels reproduced with

permission from [82])

CHAPTER 46Hepatic Tissue Engineering

961

In the context of the liver a photolithographic cell patterning technique (lsquomicropatterned co-

culturersquo) has enabled the optimization of liver-specific functions in co-cultures via engineeringof the balance between homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-non-

parenchymal) cell-cell interactions [50] Specifically micropatterned co-cultures were created

in which hepatocyte islands of controlled diameters were surrounded by supporting non-parenchymal cells (Fig 465) This pattern was miniaturized into a multiwall format using soft

lithography techniques and has resulted in micropatterned co-cultures optimized for human

hepatocyte function [14] The maintenance of hepatic functions has been shown to be highlydependent upon precise optimization of island size in systems utilizing both rat and human

hepatocytes This platform has been utilized extensively for both drug development andpathogen model systems as discussed in detail in the next section

As described above several culture techniques have been explored for the formation of hepatic

spheroids These methods exhibit certain limitations including an inability to immobilize thespheroids at defined locations and the heterogeneity of the structures which can result in

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

FIGURE 465Micropatterned co-cultures (a) Morphology of micropatterned co-cultures containing primary human hepatocytes (representative images at day 1 and

week 1) Scale bars 250 mm (b) Albumin secretion and urea synthesis in micropatterned co-cultures and pure hepatocyte cultures [14]

PART 11Gastrointestinal System

962

cell necrosis at the core of large and coalesced spheroids due to depletion of oxygen and

nutrients Recently microcontact printing robot spotting techniques and micromolded

hydrogels have been used to fabricate immobilized microarrays of hepatic cells or spheroids[417387e90] Hepatocytes in these platforms typically retain a liver-specific phenotype as

assessed by the expression of liver-enriched transcription factors secretion of albumin and the

presence of urea cycle enzymes Microtechnological tools have also been applied to theanalysis of dynamic processes related to hepatocyte biology For example microfluidic devices

that recreate physiologically relevant fluid flows and lengths scales have been used to study

drug clearance toxicity and inflammation-mediated gene expression changes [7891e93] andmechanically actuated microfabricated substrates have been used to deconstruct the role of

contact and short-range paracrine signals in interactions between hepatocytes and stromal cells

or liver endothelial cells [5394] Overall the fine spatial and temporal control of molecularsignals provided by microtechnological approaches continue to reveal important mechanisms

in liver biology and accelerate the development of therapeutic strategies

Drug and disease model systems

Advances in the development of liver cell-based culture systems have begun to provideimportant insights into human-specific liver processes that were not previously accessible with

standard in vitro models Specifically engineered tissue systems which promote long-term

functional stabilization of human hepatocytes in vitro have enabled novel studies into drug-drug interactions and hepatocellular toxicity For example micropatterned co-culture-based

platforms have been demonstrated to support human hepatocyte phenotypic function for

several weeks including maintenance of gene expression profiles canalicular transport phaseIII metabolism and the secretion of liver-specific products [1495] Furthermore drug-

mediated modulation of CYP450 expression and activity and resultant changes in toxicity

profiles were observed illustrating the utility of the platform for human ADMETox(adsorption distribution metabolism excretion and toxicity) applications (Table 463)

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

TABLE 463 Generation of major human drug metabolites by different in vitro systems [95254]

In Vivo Microsomesa S-9b Hepatocytesuspensionb

Micropatternedco-culture

48 h 7 Days

Excretory metabolites gt 10 of doseAll excretory metabolites 39 19 (49) 22 (56) 25 (64) 27 (69) 32 (82)Metabolites arising by phase 1reactions only

29 17 (59) 19 (66) 19 (66) 20 (69) 24 (83)

Metabolites arising by a phase 2reaction

10 2 (30) 3 (30) 6 (60) 7 (70) 8 (80)

Metabolites that are one reactionfrom parent (primary)

16 12 (69) 11 (69) 12 (75) 13 (81) 15 (94)

Metabolites that are two or morereactions from parent (secondary)

23 7 (48) 11 (48) 13 (57) 14 (61) 17 (74)

Circulatory metabolites gt 10 oftotal drug-related materialAll circulating metabolites 40 17 (43) 19 (48) 21 (53) 28 (70) 30 (75)Metabolites arising by a phase 1reactions only

31 14 (52) 16 (52) 14 (45) 22 (71) 23 (74)

Metabolites arising by a phase 2reaction

9 3 (33) 3 (33) 7 (78) 6 (67) 7 (78)

Metabolites that are one reactionfrom parent (primary)

16 11 (69) 11 (69) 12 (75) 12 (75) 14 (88)

Metabolites that are two or morereactions from parent (secondary)

24 6 (25) 8 (33) 9 (38) 16 (57) 16 (67)

CHAPTER 46Hepatic Tissue Engineering

963

Several other engineered model systems incorporating 3D hepatocellular aggregates or

perfusion methods have also been designed for drug screening purposes and tested with eitherrodent or human hepatocytes [26689697]

Engineered in vitro liver models can also facilitate studies into the behavior of pathogens that

exclusively target human hepatocytes including two such pathogens with profound globalhealth implications hepatitis C virus (HCV) and malaria Early experiments examining HCV

replication in vitro employed carcinoma cells stably transfected with a subgenomic viral

replicon [98] While these studies provided important information on viral replication andpotential small molecule inhibitors of viral replicative enzymes the entire viral life cycle could

not be completed due to the absence of structural proteins Furthermore prior to 2005 there

was no known viral genotype that could complete the viral life cycle in vitro Following theidentification of a genotype-2a strain of HCV responsible for fulminant hepatitis in a Japanese

patient termed JFH-1 [99] it was demonstrated that JFH-1 and a chimeric variant could

complete a full viral life cycle in the Huh7 carcinoma cell line [100e102] Subsequently recentapproaches have made it possible to examine HCV infection in primary human hepatocytes

[16103104] In particular the stabilization of human hepatocytes in a micropatterned co-

culture platform was demonstrated to allow the recapitulation of the full viral life cycle in vitro[16] (Fig 466) In this platform human hepatocytes expressed all known entry factors for

HCVand supported viral replication for several weeks illustrating the potential of such in vitro

systems for screening drug candidates that suppress HCV replication and the ultimate iden-tification of non-hepatotoxic anti-HCV compounds Furthermore a recent study demon-

strating HCV infection of human iPS-derived hepatocyte-like cells establishes a foundation forthe development of personalized disease models that allow the study of the impact of host

genetics on viral pathogenesis [105]

In vitro culture platforms have also been explored for the study of hepatocyte infection byPlasmodium sporozoites in order to model the liver stage of malaria To date the primary cell

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

(a) (b)

(c) (d)

(e) (f)

(g)

(h) (i)

FIGURE 466Modeling hepatotropic pathogenic diseases with human liver platforms Primary human hepatocyte in micropatterned co-cultures express host entry

factors for HCV (a) CD81 (b) SCARB1 (c) CDLN1 and (d) OCLDN and (g) can be persistently infectible with HCV (green line) and is decreased by

various inhibitors (orange red blue lines) Human iPS cell-derived hepatocyte-like cells (iHLCs) express host entry factors for HCV like (e) CD81 SRB1

(f) CLDN1 and OCLN iHLCs are infectible with HCV (red) as determined by (h) a luciferase reporter assay and (i) RT-PCR and infection can be inhibited with

a polymerase inhibitor 2rsquoCMA (blue) and a protease inhibitor VX-950 (green) [16105]

PART 11Gastrointestinal System

964

culture models that have been utilized include primary human hepatocyte monolayers and

human hepatoma cells [106e108] and have provided important information regardinghepatocyte invasion and the role of SR-BI and CD81 [109] and potential targets for attenu-

ating parasite growth [110] Recently micropatterned co-cultures which support long-term

functional maintenance of primary human hepatocytes have been shown to recapitulate theliver stage of two human malaria species Plasmodium falciparum and Plasmodium vivax in vitro

[111] By enhancing the efficiency and duration of Plasmodium infection in vitro such

engineered hepatocyte culture platforms could form the basis for drug screening and vaccinevalidation assays against Plasmodium in an era of renewed interest and focus on global malaria

eradication

The field of hepatic tissue engineering continues to evolve towards creating an optimal

microenvironment for liver cells in vitro Overall it is evident that many different culture

conditions can preserve at least some phenotypic features of fully functional hepatocytes Sincedetailed differences can exist in these model systems current strategies have focused on

selecting a platform that is appropriate for a particular application Although much progress

has been made further work is required to obtain a more complete picture of the molecularsignals that provide phenotypic stability of liver cultures Indeed it is likely that many path-

ways contribute to the differentiated state of the hepatocyte via a systems-level network

Stimulation of this network via different inputs (eg using different culture systems) mayresult in distinct signal transduction trajectories each of which could lead to a lsquophenotypically

stablersquo hepatocyte It is also possible that differences between such stabilized hepatic popu-

lations will emerge as newmethods to probe phenotype and function in greater detail (eg viaepigenetics proteomics metabolomics) are utilized Future challenges therefore will be to

identify such differences and understand them in the context of network-level (in vivo) hepatic

phenotype The development of highly functional and systems-level [112113] in vitro liverplatforms will ultimately facilitate both the clinical effectiveness of cell-based therapies as well

as enable high-throughput screening of candidate drugs for liver-specific metabolism and

toxicity earlier in the drug discovery pipeline and for human hepatotropic infectious diseasessuch as HCV and malaria

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

965

EXTRACORPOREAL BIOARTIFICIAL LIVER DEVICESOne promising approach for cell-based therapies for liver diseases is the development of

extracorporeal support devices which would process the blood or plasma of liver failurepatients These devices are principally aimed at providing temporary support for patients

suffering from acute or acute-on-chronic liver failure to enable sufficient regeneration of the

host liver tissue or serve as a bridge to transplantation Early extracorporeal device designsutilized primarily non-biological mechanisms such as hemoperfusion hemodialysis plas-

mapheresis and plasma exchange [114] Hemoperfusion removes toxins but also captures

other useful metabolites by passage of blood or plasma through a charcoal column Amodification of this approach referred to as hemodiadsorbtion reduces direct contact with

charcoal components through the utilization of a flat membrane dialyzer containing

charcoal and exchange resin particles [115] In general charcoal perfusion systems have beenthe most extensively studied non-biological configuration including clinical evaluation in

patients with acute liver failure although no clear survival improvement has been observed

[116] More recent configurations of artificial support systems have focused on the eliminationof albumin-bound toxins utilizing a method termed albumin dialysis These devices such

as the Molecular Adsorbent Recirculating System (MARS) and the Prometheus platform clear

albumin-bound toxins through interaction with an albumin impregnated dialysis membrane[117] They have been shown to offer some clinical benefit by reducing plasma bile acids

bilirubin and other albumin-bound toxins [118] but additional randomized controlled trials

are required to fully evaluate their effectiveness

In order to provide amore complete array of synthetic metabolic and detoxification functions

which are lacking in strictly artificial support systems biological approaches including crosshemodialysis whole liver perfusion and liver slice perfusion have been explored [116] While

these methods have shown minor improvements for patients with acute hepatic failure they

have not demonstrated any clinically meaningful survival benefits Consequently substantialefforts have been placed towards the development of extracorporeal BAL devices containing

hepatic cells BAL devices can be categorized into four main types summarized in Fig 467

hollow fiber devices flat plate andmonolayer systems perfusion bed or porous matrix devicesand suspension reactors Broadly several criteria have emerged as central to the design of an

effective BAL device these include issues of cell sourcing maintenance of cell viability and

hepatic functions sufficient bidirectional mass transfer and scalability to therapeutic levelsIn this section we discuss these issues and review recent advances in BAL device design

Cell sourcing

As discussed earlier the sourcing of hepatic cells is a fundamental challenge for all liver

cell-based therapies Most BAL devices are forced to employ xenogeneic sources (primarilyporcine) or immortalized human hepatocyte cell lines [119e122] Porcine hepatocytes are well

studied easily sourced and offer a functional profile quite similar to human hepatocytes

but are limited by safety concerns such as the possibility of transmitting pathogens likeporcine endogenous retrovirus (PERV) across species However patients treated with porcine-

derived therapies have all been negative for PERV infection [123] Human hepatocyte cell

FIGURE 467Schematics of cell-based bioreactordesigns The majority of liver cell-basedbioreactor designs fall into these four

general categories each with inherent

advantages and disadvantages [253]

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

966

lines while expandable exhibit an abnormal repertoire of liver functions which limits theirclinical impact Primary human hepatocytes present the best functional output but are

scarce and rapidly lose viability and phenotype after procurement and isolation although

recent advances in cryopreservation fetal cell procurement and stem cell differentiation havehelped primary human cells become increasingly available [124e127] Magnifying the cell

sourcing challenge are scalability requirements for translation which will be discussed indetail in the scale-up section of this chapter

Cell viability and function

The development of effective BAL systems is dependent on the incorporation of appropriate

environmental and organizational cues which enable maximal survival and function of thehepatocellular component Hollow fiber devices are themost common BAL design and contain

hepatic cells within cartridge units similar to those utilized in hemodialysis systems [128] The

hollow fiber membranes serve as a scaffold for cell attachment and compartmentalizationalthough adequate nutrient transport and proper environmental stimuli are potentially limited

in these configurations Multiple modifications aimed at optimizing cellular performance

have been explored In particular due to the enhanced function of hepatocyte spheroids relativeto dispersed cells many device configurations contain either attached or encapsulated hepa-

tocyte aggregates [128] For example the original HepatAssist system previously developed by

Circe Biomedicalwas ahollowfiber device containingmicrocarrier-attachedporcinehepatocyteaggregates within the extracapillary space [129] Collagen gel entrapment of hepatocyte ag-

gregates has also been added to some hollow fiber designs to improve function and many

perfusion bed systems integrate hepatocyte aggregates within a polymeric network of pores orcapillaries [120] Furthermore encapsulation of pre-formed hepatocyte spheroids within

calcium-alginate beads has been explored as a means of promoting hepatocyte stability while

simultaneously providing an immunoisolation barrier [128] In the modular extracorporealliver support (MELS) system (Charite Germany) hepatocytes are aggregated in co-culture with

non-parenchymal cells resulting in the formation of tissue-like organoid structures [130]

Bile canaliculi and bile duct structures as well as matrix deposition have been observed in thissystem which utilized both porcine cells and primary human hepatocytes isolated from livers

unsuitable for transplant [121] Platforms based on collagen gel lsquosandwich culturersquo a stable

hepatocyte-only culture configuration have also been developed recently [131] Moreoverexposure of hepatocytes to the plasma or blood of a sick patient may necessitate alterations in

hepatocyte culture conditions Specifically pre-conditioning with physiologic levels of insulin

lower than that present in normal culturemedium has been shown to prevent fat accumulationin hepatocytes upon exposure to plasma [132] Additionally the supplementation of plasma

with amino acids has been demonstrated to increase albumin and urea synthesis [133]

Mass transfer

Bidirectional mass transfer is another primary consideration in the design of BAL systems and

is required to provide vital nutrients to the incorporated cells and simultaneously allow export

of therapeutic cellular products Device configuration determines both the convective anddiffusive properties of the system thereby dictating the exchange of soluble components In

particular diffusion resistance is often a major constraint in BAL devices Factors commonly

limiting diffusive transport are membrane structures collagen gels and non-viable cells Forexample semipermeable membranes are often utilized in BAL devices in order to enable

selectivity in the size of exchanged factors Inherent to most hollow fiber devices but also

utilized in some flat plate and perfusion bed systems such membranes with a designatedmolecular weight cutoff act to prevent the transport of immunologic components and larger

xenogeneic substances while maintaining transport of carrier proteins such as albumin

Although clearly not ideal in order to maximize mass transfer immunologic barriers havebeen eliminated from some device designs [134] with the assumption that the short duration

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

967

of contact with xenogeneic cells will result in minimal immunological complicationsPerfusion bed systems allow for enhanced mass transfer due to the direct contact with the

perfusing media although fluid flow distribution is highly contingent on the type of packing

material Encapsulation of dispersed or aggregated cells represents another strategy forimmunoisolation but can increase diffusion resistance [135136] The development of novel

methods for the microencapsulation of hepatocytes is an active research area and is discussedin more detail in the implantable applications section of this chapter

Oxygen tension is an important mediator of hepatocyte function [137e139] thus the

improved regulation of oxygen delivery is a major goal of many recently developed BALplatforms Strategies have included the incorporation of additional fiber compartments that

carry oxygen directly into the device [140141] and more recently the incorporation of bovine

red blood cells to the circulating culture medium [142] Studies have confirmed that hepa-tocyte viability and function are more effectively maintained in devices with enhanced oxygen

transport [143144] In contrast with other designs flat plate geometries can be perfused in a

relatively uniform manner although this configuration may result in the exposure of cellsto shear stress causing deleterious effects on cellular function [131145e147] Several ap-

proaches for minimizing shear stress exposure in the flat plate geometry have been explored

including the fabrication of grooved substrates for shear protection as well as the integration ofadjacent channels separated by a gas-permeable membrane as a means to decouple oxygen

exchange and volumetric flow rate [80147148] Notably as discussed earlier oxygen has been

implicated in the heterogeneous distribution of hepatocyte functions along the liver sinusoidand this zonation can be recapitulated in vitro within a bioreactor system [149] The eventual

incorporation of oxygen gradients as well as gradients of other diverse stimuli such as

hormones and growth factors into BAL designs could provide a means to more closelysimulate the range of hepatocyte functions exhibited in vivo and further enhance the effec-

tiveness of BAL devices

Scale-up

The successful clinical implementation of any BAL device is dependent on the ability to scale

the device to a level that provides effective therapy It is estimated that such BAL devices would

require approximately 1 1010 hepatocytes representing roughly 10 of total liver weight[150] although distinct categories of liver disease such as acute liver failure end stage

cirrhosis inherited metabolic disorders will have varied scale requirements Hepatocyte

transplantation experiments in humans as well as rat models have demonstrated someimprovement in various blood parameters following the transplantation of cell numbers

representing 1e10 of total liver mass [151e154] BAL devices that have been tested clinically

have used approximately 05 109 to 1 1011 porcine hepatocytes or 4 1010 C3A hepato-blastoma cells [120] Ultimately to achieve clinical efficacy approaches will require a)

scaled-up systems with efficient nutrient transport and b) expandable cell sources as discussed

in earlier sections of this chapter Accordingly increasing cartridge size and use of multiplecartridges have been utilized as a means to scale-up hollow fiber-based systems Perfusion

systems [155] or devices utilizing encapsulated cells can be scaled fairly easily to the desired

size however such expanded configurations normally present a large priming or lsquodeadrsquovolume Stacked plate designs have been suggested as a means of scaling-up flat plate systems

although these modifications may introduce channeling effects and heterogeneous flow dis-

tribution [114] Overall the development of BAL devices exhibiting therapeutic levels offunction is a major challenge and modifications aimed at further improving the capacity and

efficiency of these systems is a central goal in the field

Regulation and safety

Similar to other tissue-engineering-based therapies the regulation of BAL devices is complexdue to the hybrid nature of these systems Currently BAL devices are being regulated as drugs

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

968

through the Center for Biologics Evaluation and Research of the Food and Drug Adminis-tration The primary safety concerns for BAL systems are similar to those for other cell-based

therapies and include the escape of tumorigenic cells immune reactions to foreign antigens

and xenozoonosis Other potential complications generally associated with extracorporealblood treatment include hemodynamic metabolic and hypothermia-related abnormalities

as well as problems linked with catheterization and anticoagulation [118] In order toexplicitly prevent escape of tumorigenic cells such as the C3A cell line used in the ELAD

system downstream filters have been added to BAL designs [119] an approach which is

generally acknowledged as an adequate precautionary measure

With regard to the utilization of porcine hepatocytes there is some evidence for the presence of

antibodies directed against porcine antigens in the serum of patients treated with BAL

devices [156] However the clinical significance of these findings remains unclear since hightiters are not observed until one to three weeks for IgM and IgG isotypes respectively

These results suggest that immune rejection may not be a significant problem in the context of

BAL therapy except in cases of repetitive treatments Still appropriate modifications throughcell sourcing or device design aimed at limiting immunologic complications would likely

be important in expansion of BAL treatment options to chronic liver disease patients and

patients with repetitive episodes of acute decompensation As mentioned earlier exposure toporcine cells can also represent a risk of xenozoonotic transmission of PERV ubiquitous in

the genome of bred pigs PERV has been shown to infect human cell lines in vitro [157]

although studies examining transmission to BAL-treated patients have not demonstrated anyevidence of infection [158] While specific transmission of PERV may not occur during the

course of BAL therapy the use of xenogeneic cells in BAL devices will always incur a note of

caution

Ongoing clinical trials

A number of BAL devices have been tested clinically and the characteristics of these systems areprovided in Table 464 Important practical issues include the use ofwhole blood versus plasma

and the type of anticoagulation regimen The use of whole blood exhibits the advantage of

including oxygen-containing erythrocytes however undesirable leukocyte activation and celldamage may arise In contrast perfusion of plasma prevents hematopoietic cell injury but the

solubility of oxygen in plasma devoid of oxygen carriers is quite low Furthermore heparin

coagulation is normally used in BAL systems although deleterious effects of heparin exposureon hepatocyte morphology and function have been suggested [159]

The design of clinical trials for BAL devices poses a significant challenge In particular liverfailure progression is highly variable and etiology dependent Additionally hepatic encepha-

lopathy one of the major manifestations of liver failure is difficult to quantify clinically As a

result patients in clinical trials must be randomized while still controlling for the stage atwhich support was initiated as well as individual etiology Similarly the determination of the

relevant control therapy can be difficult Ideally in order to minimize non-specific effects of

extracorporeal treatment a non-biological control such as veno-venous dialysis would beutilized Another challenge is the choice of clinical end point Most clinical trials utilize end

points of 30-day survival and 30-day transplant-free survival however trials can often be

confounded by the fact that acute liver failure patients receive transplants variably dependingon organ availability and the eligibility criteria in place at a given center Furthermore

interpretation of the specific role of incorporated live functional hepatocytes can be

complicated by the presence of non-biological adjuncts such as charcoal perfusion in somedesigns A direct comparison of the effect of non-biological systems alone dead or non-

hepatocyte cells and live hepatocytes would provide substantial insight concerning the

effectiveness of the cellular components particularly given that dead hepatocytes and non-hepatocyte cells have been shown to offer some survival benefit in various animal models of

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

TABLE 464 Characteristics of eight bioartificial liver systems

BAL system Configuration Cell source amount Trial phase Comments

HepatAssist(Arbios WalthamMA)

Hollow fiberpolysulphonemicrocarrier attached

Cryopreservedporcine (7 109)

IIIIIPrimaryendpointnot reached

Plasma citrate anticoagulation015e020mm pore size7 hsession daily

ELAD (Vitaltherapies SanDiego CA)

Hollow fiberpolysulfone celluloseacetate largeaggregates

C3A human cell line(200e400g)

IIIII Plasma heparinanticoagulation 020 um porescontinuous up to 107 h

MELS (ChariteVirchow BerlinGermany)

Hollow fiberinterwovenmulticompartmenttissue organoids

Freshly isolatedporcine or human(up to 650g)

III Plasma heparinanticoagulation 400 kDacutoff continuous up to 6d

BLSS (ExcorpMedical OakdaleMN)

Hollow fiber celluloseacetate collagen gelentrapped

Freshly isolatedporcine (70e120 g)

IIIII Whole blood heparinanticoagulation 100-kDacutoff 12 hsession forup to 2 session

RFB-BAL (Univof Ferrara Italy)

Radial flow bioreactoraggregates

Freshly isolatedporcine (200e230 g)

III Plasma heparincitrateanticoagulation 1 um polyesterscreen 6e24 h treatments

AMC-BAL (Univof AmsterdamNetherlands)

Non-woven polyestermatrix spirally woundaggregates

Freshly isolatedporcine(10e14 109)

I Plasma heparinanticoagulation directcell-plasma contact up to18 hsession for up to 2sessions

LiverX-2000(Algenix IncMinneapolis MN)

Hollow fiber collagenentrapped

Freshly isolatedporcine (40e80g)

I Whole blood heparinanticoagulation

HBAL (NanjingUniv NanjingChina)

Hollow fiberpolysulfoneadsorption column

Freshly isolatedporcine (10 109)

I Plasma 100k Da cutoff one totwo 60 h treatments

CHAPTER 46Hepatic Tissue Engineering

969

acute liver failure [160] Notably the ability to more accurately assess viability and function of

cells during BAL treatment would be a major advance Such information would be crucial indetermining treatment time and the potential requirement for device replacement both

important considerations due to the instability of hepatocellular function in many contexts

and the demonstrated detrimental effect of plasma from liver failure patients on culturedhepatocytes [161] Finally even if clinical trials of current BAL devices do not prove efficacy

information obtained from these studies coupled with improvements in cell sourcing and

functional stabilization will represent the foundation for the next generation of devices

IMPLANTABLE TECHNOLOGIES FOR LIVER THERAPIESAND MODELINGIn addition to temporary extracorporeal support the development of cell-based therapies for

liver treatment aimed at the eventual replacement of damaged or diseased tissue is an active

area of investigation In many cases hepatocytes have been injected into animal hostsand exhibit substantial proliferative capacity as well as the ability to replace diseased tissue

and correct metabolic liver deficiencies in those models [162e165] However the clinical

efficacy of these procedures is currently limited due in part to technical hurdles in celldelivery and animal models These limitations might be addressed in part by engineering

three-dimensional liver tissue ex vivo prior to implantation Here we detail the state-of-art in

cell transplantation in the context of applicable animal models as well as in the constructionand application of engineered liver tissue

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

970

Cell transplantation and animal models

Hepatocytes have been transplanted into rodents via injection into the spleen or splenic artery

intraperitoneal space peripheral veins or portal vein Cell transplantation can improvehost survival in animals with acute liver failure induced both chemically and surgically as well

as end stage liver failure due to cirrhosis [165e169] Transplantation also can correct meta-

bolic deficiencies in several models of liver-based metabolic diseases [165166] Despiteencouraging results in cell transplantation in animal models translation of cell trans-

plantation to the clinic has failed to result in sustained improvements in patient outcome

These results demonstrate the need for further improvements in cell delivery (eg throughmodalities such as tissue engineering) andor better animal model systems

Models of acute liver failure include administration of clinically relevant toxic doses of carbon

tetrachloride (CCl4) or acetaminophen which can induce localized centrilobular necrosis orsurgical resection of two thirds of the liver in a partial hepatectomy model [170] In these

models animals develop severe liver injury but if animals can be kept alive for as little as

72 hours following injury host regeneration can rapidly correct the damage This observationis unlike the human situation in which the repair of damage due to acute liver failure in

patients has been estimated to take weeks or months [171172] This discrepancy could in part

explain contradictory results in which animals can be lsquocuredrsquo following acute liver failureafter hepatocyte transplantation whereas humans are not it is possible that injected hepato-

cytes are able to provide short-term support to animals but not the long-term replacement

of hepatic function necessary for clinical therapy Of note parabiotic systems have demon-strated that after partial hepatectomy factors regulating liver cell proliferation are present in

the circulation [173174] Consequently this model may serve as a well-controlled system

to examine the importance of regenerative cues in the engraftment and proliferation of hepaticconstructs implanted in extrahepatic sites

Chronic liver failure represents a more substantial clinical burden as outlined above The mostcommon animal models of experimental fibrosis are toxic damage due to CCl4 administration

and bile duct ligation [175] In a rat cirrhosis model induced by CCl4 treatment

hepatocytes transplanted into the spleen result in improved survival over controls for a periodof months [176e178] To date hepatocyte transplantation has resulted in improvements in

liver function and encephalopathy in some patients with acute and chronic liver failure but no

change in outcome and survival of these patients has been observed [179] Engineeringstrategies to improve engraftment and survival in extrahepatic sites may improve the efficacy of

these therapies

Finally hepatocytes have been transplanted into several animal models of metabolic liver

diseases In most cases transplantation only partially corrected the genetic abnormalities

However in the fumaryl acetoacetate hydrolase (FAH)-deficient mouse model of familialtyrosinemia [180] the albumin-uPA transgenic mouse [181] and in the transgenic mouse

model of human alpha-1-antitrypsin deficiency [182] the inherited metabolic defect results in

reduced survival of host hepatocytes and transplanted hepatocytes are able to engraft andreplace the hepatocytes in the host liver over the course of weeks or months These findings led

to the generation of lsquochimericrsquo mice in which a substantial portion of the liver is composed of

human hepatocytes (Fig 468) For this reason FAH-deficient and albumin-uPA transgenicmice are the most frequently used animal models for studying hepatocyte engraftment in host

liver Additionally such mice promise to be useful as model systems for investigating human-

specific drug metabolism and toxicity as well as infectious diseases that exhibit humantropism The most convincing data for the therapeutic use of hepatocyte transplantation to

date has been demonstrated in patients primarily pediatric with liver-based metabolic dis-

orders Patients with certain disorders such as urea cycle disorders experience greaterimprovement than others but even in these cases the function of hepatocytes deteriorates

with time such that liver transplantation is typically necessary by six months [183]

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

(a) (b)

(c)

(d) (e) (f)

10

1

01

001

00010 2 4 6 8 10 12 14 16

Weeks after transplantation

Hum

an a

lbum

in (m

gm

l)

FIGURE 468Mice with humanized livers (a) FAH-positive human hepatocytes injected into the spleen of Fah mice can engraft and integrate in the mouse livers

resulting in mice with lsquohumanizedrsquo livers (b) Human albumin can be detected in the blood of these mice (c) Engineered human liver tissue (lsquoectopic liver

tissuersquo) could also be constructed ex vivo and then implanted into mice (d) Ectopic liver tissue can survive and function in mice that are both immunocompromised

(left) and immunocompetent (right) (e) Human albumin can be detected in the blood of these mice (f) Mice with ectopic human liver tissue can be used to identify

disproportionate human drug metabolites that would not have been identified by wild-type mice (Reproduced with permission from [15180])

CHAPTER 46Hepatic Tissue Engineering

971

In all of these animal models a regenerative stimulation is provided by transgenic injury

partial hepatectomy portocaval shunting or the administration of hepatotoxic agents prior to

cell transplantation However as evidenced by recent clinical trials that have seen limitedsuccess these approaches are difficult to adapt to a clinical setting Major limitations of

isolated cell transplantation include the inefficient engraftment and limited survival of

transplanted hepatocytes which has been collectively reported at only 10e30 of injectedcells [184] Several studies have demonstrated methods to improve engraftment and

enhance the selective proliferation of transplanted hepatocytes [185e187] although analo-

gous to regeneration models the clinical utility of these approaches remains to be determinedThe time for engraftment and proliferation of transplanted hepatocytes also can create a

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

972

substantial lag time (48 hours in one study [152]) before clinical benefit is observed whichcould restrict the utility of cell transplantation for certain clinical conditions such as fulminant

hepatic failure

In summary clinical trials of hepatocyte transplantation have recently demonstrated long-term

safety but donor hepatocyte engraftment and restoration of failing host livers has not been

adequate to obviate the need for organ transplantation [188e190] Additionally only partialcorrection of metabolic disorders has been accomplished to date in the clinic [188e190]

Future studies that improve cell delivery survival and engraftment as well as reduce the time

required for integration of the grafted cells with the host could greatly improve the effective-ness of cell-based therapies and animal model systems

Implantable engineered tissue for humanized mouse models

Implantation of human engineered liver tissue into animal hosts may also provide alternative in

vivo model systems for human disease Despite their promise as model systems for investigating

human-specific drug responses and infectious diseases with human tropism current humanizedmousemodels (eg FAH() and albumin-uPAmodels detailed above) are limited in that animals

must be both immunodeficient and exhibit significant host liver injury Additionally the process of

human hepatocyte injection homing to the liver and expansion can takeweeks tomonths creatinghumanized mice using lsquoclassicrsquo cell transplantation is therefore tedious and time-consuming [15]

As one example of a candidate alternative a recent study generated humanized mice by implanting

hepatocytes and supporting non-parenchymal cells within a three-dimensional hydrogel scaffoldinto the intraperitoneal space of uninjuredmice [15] (Fig 468) The engineered human liver tissue

synthesized human liver proteins as well as human-specific drug metabolism drug-drug interac-

tion and drug-induced hepatocellular toxicity The polyethylene glycol (PEG)-based engineeredtissue was shown to survive and functionwithin immunocompetent hosts for a period of time after

implantation suggesting that encapsulation of cells in this material system may have the potential

to delay immune rejection and enable studies that require both human liver systems and intactimmune processes

Implantable therapeutic engineered liver tissue

The development of implantable engineered hepatic tissue is a promising strategy for thetreatment of liver disease due to its potential to mitigate the limitations in current cell

transplantation strategies including inefficient seeding and engraftment poor long-term

hepatocyte survival a required donor cell repopulation advantage and the inherent lag phasebefore clinical benefit is attained [165191] Implantable engineered hepatic tissues are

typically fabricated by immobilizing or encapsulating hepatic cells in biomaterial scaffolds

in conjunction with strategies to optimize hepatocyte survival and function leading to thegeneration of liver-like tissue in vitro prior to in vivo implantation

Design criteria for implantable systems

To achieve therapeutic levels of liver function to treat liver failure the development of engi-

neered hepatic tissues that contain high densities of stable and functional hepatocytes with

efficient transport of nutrients and secreted therapeutic factors is necessary Furthermoreintegration of the engineered tissue with the host upon implantation is critical in ensuring

its long-term survival The potential tunability of engineered implantable systems offers

attractive prospects for the optimization of hepatocyte survival and function as well as sub-sequent host integration Scaffold parameters that are customizable include porosity me-

chanical and chemical properties and three-dimensional architecture Additionally relevant

microenvironmental cues like paracrine and juxtacrine cell-cell interactions cell-matrix in-teractions and soluble factors can be incorporated into implantable engineered hepatic tissue

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

973

by translating either biological or biomimetic strategies from in vitro culture models so as torecapitulate important aspects of the in vivo hepatocyte microenvironment

Natural scaffold chemistry and modifications

A wide variety of naturally-derived material scaffolds have been explored for liver

tissue-engineering applications including materials like collagen peptides fibrin alginatechitosan hyaluronic acid cellulose decellularized liver matrix and composites of these

[70192e197] (Table 465) The choice of material determines the physicochemical and bio-

logical properties of the scaffold For example early efforts in developing implantable hepaticconstructs utilized collagen-coated dextran microcarriers that enabled hepatocyte attachment

since hepatocytes are known to be anchorage-dependent cells The intraperitoneal trans-plantation of these hepatocyte-attached microcarriers resulted in successful replacement of liver

functions in two different rodentmodels of genetic liver disorders [198] Subsequently collagen-

coated or peptide-modified cellulose [196199] gelatin [200] and gelatin-chitosan composite[201] microcarrier chemistries have also been explored for their capacity to promote hepatocyte

attachment On the other hand materials that are poorly cell adhesive like alginate [70] have

been exploited for their utility in promoting hepatocyte-hepatocyte aggregation (eg spheroidformation) and hence hepatocyte stabilization within these scaffolds Collectively the size

of engineered tissues created by these approaches is limited by oxygen and nutrient diffusion to

only a few hundred microns in thickness To address this constraint recent work has sought touse decellularized whole organ tissue as a matrix for liver tissue engineering (Fig 469) The

decellularization process removes cells from donor tissues but preserves the structural and

functional characteristics of much of the tissue microarchitecture and vasculature of theunderlying extracellular matrix Decellularized livermatrices can be seededwith hepatocytes and

vascular cells exhibit liver-specific functions and survive after transplantation into rodents

[197202] Future work in this area will likely focus on improving cell seeding protocols whichto date have achieved only 20e40 of endogenous liver mass after recellularization as well as

co-seeding with a both hepatocytes and the various non-parenchymal cell types found in liver

(eg stellate cells Kupffer cells) In general the advantages of biologically-derived materialsinclude their biocompatibility naturally occurring cell adhesive moieties and in the case of

decellularization native architectural presentation of extracellular matrix molecules

Synthetic scaffold chemistry

In contrast to biologically-derived material systems synthetic materials enable preciselycustomized architecture (porosity and topography) mechanical and chemical properties and

degradation modality and kinetics Synthetic materials that have been explored for liver tissue

engineering include poly(L-lactic acid) (PLLA) poly(DL-lactide-co-glycolide) (PLGA) poly(ε-caprolactone) (PCL) and poly(ethylene glycol) (PEG) [1571203e207] Polyesters like

PLLA and PLGA are the most common synthetic polymers utilized in the generation of porous

tissue-engineering constructs These materials are biocompatible biodegradable and havebeen used as scaffolds for hepatocyte transplantation [203208] A key advantage of PLGA is

the potential to finely tune its degradation time due to differences in susceptibility to

TABLE 465 Scaffolds utilized for hepatocellular constructs

Category Composition

Natural Collagen chitosan collagenchitosancomposites alginate alginate compositesliver-derived biomatrix peptides hyaluronic acidfibrin gelatin agarose

Synthetic PLLA PLGA PLLAPLGA composite PGAPEG PCL PET PVA

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

(a) (b) (c) (d) (e)

(h) (i)(g)(f)

FIGURE 469Decellularized liver scaffolds Representative photographs of ischemic rat livers during decellularization at (a) 0 h (b) 18 h (c) 48 h (d) 52 h and (e) 72 h

(f) Corrosion cast model of a decellularized liver with portal (red) and venous (blue) vasculature demonstrates that vasculature is intact (g) SEM images

of extracellular matrix within the parenchyma with hepatocyte-size free spaces (h) Decellularized livers can be re-cellularized to a total cell mass of 20e40

of native liver mass (i) Recellularization with human fetal liver cells can result in self-organization of hepatocytes (green) and biliary cells (red) (Reproduced with

permission from [197202])

PART 11Gastrointestinal System

974

hydrolysis of the ester groups of its monomeric components (lactic acid and glycolic acid)However the accumulation of hydrolytic degradation products has been shown to produce an

acidic environment within the scaffold which initiates peptide degradation and stimulates

inflammation which may affect hepatocyte function [209] Consequently as alternatives tomacroporous scaffold systems approaches aimed at the efficient and homogeneous

encapsulation of hepatocytes within a fully 3D structure have been explored In particular

hydrogels that exhibit high water content and thus similar mechanical properties to tissues arewidely utilized for various tissue-engineering applications including hepatocellular platforms

Synthetic PEG-based hydrogels have been increasingly utilized in liver tissue-engineering

applications due to their high water content hydrophilicity resistance to protein adsorptionbiocompatibility ease of chemical modification and the ability to be polymerized in the

presence of cells thereby enabling the fabrication of 3D networks with uniform cellular

distribution [210] PEG-based hydrogels have been used for the encapsulation of diverse celltypes including immortalized and primary hepatocytes and hepatoblastoma cell lines

[15206207] The encapsulation of primary hepatocytes requires distinct material modifica-

tions (eg 10 wv polyethylene glycol (PEG) hydrogel inclusion of RGD adhesive motifs) asdetailed below as well as analogous to 2D co-culture systems the inclusion of non-

parenchymal supporting cell types such as fibroblasts and endothelial cells [207]

Modifications in scaffold chemistry

The relatively inert nature of synthetic scaffolds allows for the controlled incorporation of

chemicalpolymer moieties or biologically active factors to regulate different aspects of cellular

function Chemical modifications like oxygen plasma treatment or alkali hydrolysis of PLGA[211212] or the incorporation of polymers like poly(vinyl alcohol) (PVA) or poly(N-p-

vinylbenzyl-4-O-b-D-galactopyranosyl-D-glucoamide) (PVLA) into poly(lactic-co-glycolic

acid) (PLGA) or poly-l-lactide acid (PLLA) scaffolds [203213214] have improved hepatocyteadhesion bymodulating the hydrophilicity of the scaffold surface [215] Biological factors may

include whole biomolecules or short bioactive peptides Whole biomolecules are typically

incorporated by non-specific adsorption of extracellular matrix molecules like collagenlaminin or fibronectin [211216] and covalent conjugation of sugar molecules like heparin

[217218] galactose [71219] lactose [217] or fructose [220] or growth factors like EGF [221]

Alternatively short bioadhesive peptides that interact with cell surface integrin receptors havebeen extensively utilized to promote hepatocyte attachment in synthetic scaffolds

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

975

For example conjugation of the RGD peptide to PLLA has been shown to enhance hepatocyteattachment [222] whereas RGDmodification significantly improved the stability of long-term

hepatocyte function in PEG hydrogels [15207] The additional incorporation of adhesive

peptides that bind other integrins may serve as a way to further modulate and enhancehepatocyte function within synthetic polymer substrates Moreover although not yet applied

to hepatocellular systems the integration of matrix metalloproteinase-sensitive peptidesequences into hydrogel networks as degradable linkages has been shown to enable cell-

mediated remodeling of the gel [223e225] The capacity to modify biomaterial scaffold

chemistry through the introduction of biologically active factors will likely enable the finelytuned regulation of cell function and interactions with host tissues important for implantable

systems

Porosity

A common feature of many implantable tissue-engineering approaches is the use of porousscaffolds that provide mechanical support often in conjunction with cues for growth and

morphogenesis Collagen sponges various alginate and chitosan composites and PLGA are

the most commonly used porous scaffolds for hepatocyte culture and are generally synthe-sized using freeze-dry or gas-foaming techniques Pore size has been found to regulate cell

spreading and cell-cell interactions both of which can influence hepatocyte functions [192]

and may also influence angiogenesis and tissue ingrowth [226] Porous acellular scaffolds arenormally seeded using gravity or centrifugal forces capillary action convective flow or

through cellular recruitment with chemokines but hepatocyte seeding is generally heteroge-

neous in these scaffolds [227228]

Controlling 3D architecture and cellular organization

Another approach to improving the functionality of tissue-engineered constructs is to more

closely mimic in vivo microarchitecture by generating scaffolds with a highly defined materialand cellular architecture which would provide better control over the 3D environment at the

microscale

A range of rapid prototyping and patterning strategies have been developed for polymersusing multiple modes of assembly including fabrication using heat light adhesives or

molding and these techniques have been extensively reviewed elsewhere [229] For example

3D printing with adhesives combined with particulate leaching has been utilized to generateporous PLGA scaffolds for hepatocyte attachment [230] and microstructured ceramic [231]

and silicon scaffolds [232233] have been proposed as platforms for hepatocyte culture

Furthermore molding and microsyringe deposition have been demonstrated to be robustmethods for fabricating specified 3D PLGA structures towards the integration into

implantable systems [234]

Microfabrication techniques have similarly been employed for the generation of patterned

cellular hydrogel constructs For instance microfluidic molding has been used to form

biological gels containing cells into various patterns [235] In addition syringe deposition inconjunction with micropositioning was recently illustrated as a means to generate patterned

gelatin hydrogels containing hepatocytes [236] Patterning of synthetic hydrogel systems has

also recently been explored Specifically the photopolymerization property of PEG hydrogelsenables the adaptation of photolithographic techniques to generate patterned hydrogel

networks In this process patterned masks printed on transparencies act to localize the UV

(ultraviolet) exposure of the pre-polymer solution and thus dictate the structure of theresultant hydrogel The major advantages of photolithography-based techniques for

patterning of hydrogel structures are its simplicity and flexibility Photopatterning has been

employed to surface pattern biological factors [237] produce hydrogel structures with arange of sizes and shapes [238239] as well as build multilayer cellular networks [240241]

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

976

Consequently hydrogel photopatterning technology is ideally suited for the regulation ofscaffold architecture at the multiple length scales required for implantable hepatocellular

constructs As a demonstration of these capabilities photopatterning of PEG hydrogels was

utilized to generate hepatocytefibroblast co-culture hydrogels with a defined 3D branchednetwork resulting in improved hepatocyte viability and functions under perfusion [206]

More recently a rsquobottom-uprsquo approach for fabricating multicellular tissue constructs utilizingDNA-templated assembly of 3D cell-laden hydrogel microtissues demonstrates robust

patterning of cellular hydrogel constructs containing numerous cell types [242] Also the

additional combination of photopatterning with dielectrophoresis-mediated cell patterningenabled the construction of hepatocellular hydrogel structures organized at at the cellular

scale (Fig 468) Overall the ability to dictate scaffold architecture coupled with other ad-

vances in scaffold material properties chemistries and the incorporation of bioactive ele-ments will serve as the foundation for the future development of improved tissue-engineered

liver constructs that can be customized spatially physically and chemically

Host interactions

Further challenges in the design of therapeutic implantable liver devices are more specifically

associated with the interactions with the host environment These include issues related to

vascularization remodeling the biliary system and immunologic considerations Notably asignificant challenge for the design of implantable liver constructs is the need to overcome

transport limitations within the grafted construct due to the lack of functional vasculature

Within the normal liver environment hepatocytes are supplied by an extensive sinusoidalvasculature with minimal extracellular matrix and a lining of fenestrated (sinusoidal) endo-

thelial cells Together these features allow for the efficient transport of nutrients to the highly

metabolic hepatocytes Strategies to incorporate vasculature into engineered constructsinclude the microfabrication of vascular units with accompanying surgical anastomosis during

implantation [232243] For example polymer molding using microetched silicon has been

shown to generate extensive channel networks with capillary dimensions [244] The incor-poration of angiogenic factors within the implanted scaffolds has also been explored Spe-

cifically integration of cytokines important in angiogenesis such as VEGF [208] basic

fibroblast growth factor (bFGF) [245] and Vascular endothelial growth factor (VEGF) incombination with Platelet-derived growth factor (PDGF) [246] has been shown to promote

the recruitment of host vasculature to implanted constructs Furthermore preimplantation of

VEGF releasing alginate scaffolds prior to hepatocyte seeding was demonstrated to enhancecapillary density and improve engraftment [247]

In addition to interactions with the vasculature integration with other aspects of host tissuewill constitute important future design parameters For instance incorporation of hydrolytic

or protease-sensitive domains into hepatocellular hydrogel constructs could enable the

degradation of these systems following implantation Of note liver regeneration proceedsin conjunction with a distinctive array of remodeling processes such as protease expression and

extracellular matrix deposition Interfacing with these features could provide a mechanism for

the efficient integration of implantable constructs Similarly to whole liver or cell trans-plantation the host immune response following the transplant of tissue-engineered constructs

is also a major consideration Immunosuppresive treatments will likely play an important role

in initial therapies although stem cell-based approaches hold the promise of implantablesystems with autologous cells Furthermore harnessing the liverrsquos unique ability to induce

antigen-specific tolerance [248] could potentially represent another means for improving the

acceptance of engineered grafts Finally incorporation of excretory functions associated withthe biliary system will ultimately be required in future designs Towards this end current

studies are focused on the development of in vitromodels which exhibit biliary morphogenesis

and recapitulate appropriate polarization and bile canaliculi organization [249e251] as wellas platforms for the engineering of artificial bile duct structures [252]

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

CHAPTER 46Hepatic Tissue Engineering

CONCLUSIONAlthough many challenges remain for the improvement of tissue-engineered liver therapies

substantial progress has been made towards achieving a thorough understanding of thenecessary components The parallel development of highly functional in vitro systems as well

as extracorporeal and implantable therapeutic devices is based on contributions from

diverse disciplines including regenerative medicine developmental biology transplantmedicine and bioengineering In particular novel technologies such as hydrogel chemistries

high-throughput platforms and microfabrication techniques represent enabling tools for

investigating the critical role of the microenvironment in liver function and subsequently thedevelopment of structurally complex and clinically effective engineered liver systems

977

References[1] Lee WM Acute liver failure in the United States Semin Liver Dis 200323217e26

[2] Ostapowicz G et al Results of a prospective study of acute liver failure at 17 tertiary care centers in the United

States Ann Intern Med 2002137947e54

[3] Murphy SL Xu J Kochanek KD Deaths Preliminary Data for 2010 Natl Vital Stat Rep 201260

[4] Kim WR Brown Jr RS Terrault NA El-Serag H Burden of liver disease in the United States summary of aworkshop Hepatology 200236227e42

[5] Alter MJ et al The prevalence of hepatitis C virus infection in the United States 1988 through 1994 N Engl J

Med 1999341556e62

[6] Brown RS Hepatitis C and liver transplantation Nature 2005436973e8

[7] Brown KA Liver transplantation Curr Opin Gastroenterol 200521331e6

[8] Kelly JH Darlington GJ Modulation of the liver specific phenotype in the human hepatoblastoma line

Hep G2 In vitro Cell Dev Biol 198925217e22

[9] Ellis AJ et al Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure Hepatology

1996241446e51

[10] Liu J et al Characterization and evaluation of detoxification functions of a nontumorigenic immortalized

porcine hepatocyte cell line (HepLiu) Cell Transplant 19998219e32

[11] Yoon JH Lee HV Lee JS Park JB Kim CY Development of a non-transformed human liver cell line with

differentiated-hepatocyte and urea-synthetic functions applicable for bioartificial liver Int J Artif Organs

199922769e77

[12] Mitaka T The current status of primary hepatocyte culture Int J Exp Pathol 199879393e409

[13] Terry C Dhawan A Mitry RR Hughes RD Cryopreservation of isolated human hepatocytes for trans-

plantation State of the art Cryobiology 200653149e59 doiS0011e2240(06)00087e3 [pii] 101016jcryobiol200605004

[14] Khetani SR Bhatia SN Microscale culture of human liver cells for drug development Nat Biotechnol200826120e6 doinbt1361 [pii] 101038nbt1361

[15] Chen AA et al Humanized mice with ectopic artificial liver tissues Proc Natl Acad Sci U S A

201110811842e7 doi1101791108 [pii] 101073pnas1101791108

[16] Ploss A et al Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures Proc

Natl Acad Sci U S A 20101073141e5 doi0915130107 [pii] 101073pnas0915130107

[17] Strick-Marchand H Weiss MC Inducible differentiation and morphogenesis of bipotential liver cell lines

from wild-type mouse embryos Hepatology 200236794e804 doiS027091390200085X [pii] 101053

jhep200236123

[18] Strick-Marchand H Personal Communication

[19] Espejel S et al Induced pluripotent stem cell-derived hepatocytes have the functional and proliferativecapabilities needed for liver regeneration in mice J Clin Invest 20101203120e6 doi43267 [pii] 101172

JCI43267

[20] Si-Tayeb K et al Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem

cells Hepatology 201051297e305 doi101002hep23354

[21] Liu H Kim Y Sharkis S Marchionni L Jang YY In vivo liver regeneration potential of human inducedpluripotent stem cells from diverse origins Sci Transl Med 2011382ra39 doi38282ra39 [pii] 101126

scitranslmed3002376

[22] Chambers SM Studer L Cell fate plug and play direct reprogramming and induced pluripotency Cell2011145827e30 doiS0092e8674(11)00597e6 [pii] 101016jcell201105036

PART 11Gastrointestinal System

978

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