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Liver Engineering

Arti Ahluwalia

Function

• The liver is the largest internal organ in the body, accounting for 2% of the weight of an adult (1.5-1.8 kg)

• Center for carbohydrate, lipid and amino acid regulation. • endocrine function secreting factors such as albumin and urea into

the blood, • exocrine function secreting bile to the intestine, • storage function in form of glycogen• V. important for its participation in systemic responses to trauma or

injury by producing acute phase proteins and assuming a hypermetabolic state. Produces coagulation factors, lymph..

• It is one of the body's first lines of defense, inactivating toxins and xenobiotics absorbed by the intestine and clearing particles from the blood

Problems

• The liver can regenerate, but the rate is too low

• 5° most common cause of death in the West

• Liver cancer is the no 3 cause of death due totumors

• Liver disorders are increasing rapidly

• Liver transplant is usually the only treatment.

• 90% patients with liver failure problems die forwant of an organ

Liver Anatomy

Normal anatomy of the liver. CBD = common bile duct, CD = cystic duct, CHD = common hepatic duct, HA = hepaticartery, IVC = inferior vena cava, LHA = left hepaticartery, LHD = left hepatic duct, LHV = left hepatic vein, LPV = left portal vein, MHV = middle hepatic vein, PV = portalvein, RHA= right hepatic artery, RHD = right hepaticduct, RHV = right hepatic vein, RPV= right portal vein.

75% PV

25 %, ha

DataHuman Data Values

Body mass, Mman 70 Kg

Basal metabolic rate (BMR) 88 J/s

Liver mass 1.8 Kg

# hepatocytes 2×1011

Rat Liver Data

Data from Hepatic Tissue Engineering for Adjunct and TemporaryLiver Support: Critical Technologies, Chan et al 2004. in Liver Transplatation

Complex but highly organizedstructure

Lobe, lobule

Liver Engineering

Therapuetic

• Hepatitis

• Cirrhosis

• Cancer

• Liver disease UCD

Toxicitytesting

• Chemicals

• Drugs

• Cosmetics

Science

• Physiologicalmodels

• Disease models

Problems

• Hepatocytes do not proliferate

• Have high oxygen requirements, but cannottolerate shear

• Loose metabolic function in vitro

Implies that liver function is inextricably linkedwith its function

Liver regeneration

The three main approaches that have been proposed are:

• transplantation of isolated hepatocytes

• implantable tissue-engineered constructs

• perfusion of blood through an extracorporeal bioartifical liver device (BAL)

Hepatocyte transplantation

• http://www.cytonetllc.com/?page_id=135

• For urea cycle defects in infants

• Stem cells have still a long way to go

• For hemophilia (Reliver)

Tissue Engineered Liver

• Hepatocytes on porous mesh

• Encapsulated hepatocytes

Materials?Architecture?Cells?Cell source?

Ex-vivo liver assist devices

Mass Transfer Models for basic replacement of liver function:

• Hemodialysis: Mechanism exactly parallel to kidney dialyzer that clears only ammonia and small molecules. Relies upon convective pressure or diffusion models

• Hemofiltration: Has larger molecular pores that allows for the convective clearance of larger toxins. Relies upon the same mechanics of hemodialysis.

• Hemoperfusion: Passing blood component over particular absorbent compounds that bind toxins. A hemoperfusion device using activated charcoal is often used in biohybrid LADs as an additional part of the blood pathway.

• Exchange Transfusion or Plasmapheresis: Operates on the principle that either whole blood or toxin-bearing plasma can be replaced entirely over time to reduce toxin-content. Though there are thoughts about combining plasma replacement with hemoperfusion, these procedures are not yet functional long-term solutions to liver insufficiency.

• . The principle problem with these devices is they are metabolically inert, while the liver's principle functions are largely biochemical and excretory - functions that mechanical models cannot duplicate.

BAL/LADGlobal replacement of liver function:* Cross-Circulation: Links a biocompatible individual's circulation to the circulation of the patient with liver failure in a prolonged exchange transfusion that allow the patient's blood to be processed "globally" by the healthy individual's hepatocytes.* Hemoperfusion over Liver Slices: Similar to the mechanical model of hemoperfusion, except that the absorbent compound is active, biocompatible hepatocytes.* Organ Transplant: The procedure of choice, though severely limited by donor organ supply. Recently segments of living-related donor liver tissue have proven effective in treatment of liver failure.* Heterotopic Hepatocyte Transplant: Direct transplantation of hepatocytessuspended in a matrix to provide temporary relief to a failing liver. There is much disagreement on how and where to deliver the hepatocytes, how many to deliver and what kind of matrix to place them in.* Bio-Artificial/Bio-Hybrid Replacement: A device that fixes and sustains functional, harvested hepatocytes within a matrix that allows for biochemical interactions with patient's blood or plasma. These mechanisms are the models for LADs that are currently in clinical trials.

BAL

Allen et al. 2001. “Advances in BioartificialLiver Technology.” Hepatology. 34(3): 447-455

Which cells?

• Human or Porcine? Human hepatocytes are preferable because the biochemicals they manufacture are same products that native hepatocytes produce. Also, they are less immunogenic and there is no risk of porcine endogenous retroviruses (PERV). However, human hepatocytes grow poorly in culture media and there is a problem with accumulating enough human hepatocytes considering the enormous existing liver support burden. Furthermore, it appears that human C3A cells (the most common human hepatoma cell line) have less ammonia clearing ability than porcine cells. New cell lines such as HepaRG and engineered hepatocytes are also being explored.

Other questions

What should be the particular geometry of the device?• * Should hepatocytes be cultured or seeded in the fibers or outside? • * How can the hepatocytes be evenly distributed and how can this distribution be maintained with

time? This question is one of the biggest problems with hollow-fiber bioreactors today and is the subject of a number of investigations.

Should the perfusate be blood or plasma?• * Blood - Easy uncomplicated circuit design, sufficient oxygen and nutrient support for hepatocytes,

coagulation problems.• * Plasma - Less coagulation problems, can be filtered before returning it to the blood stream, circuit

is complicated and plasma has a lower oxygen-carrying capacity.What should the nature of the membrane be?• * Open-membrane - Allows toxin-bearing albumin to cross and interact with hepatocytes allowing

for increased overall effectiveness of the BAL. However, this also increases the immunogenicity of the device.

• * Closed-membrane - Allows for greater immunoisolation, prevents toxin-bearing albumin from interacting with hepatocytes and therefore decreases the effectiveness of the LAD.

• Most devices have switched over to an open-membrane BAL and have generated other solutions to the immune comprise that this concession creates (drugs)

Whole organ engineering of liver

• The first example of a whole organ was the heart (2008, Ott etal..Perfusion-decellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14:213-221.)

• Uygun et al, Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med 2010;16:814-820.

Perfusion with detergents

Intact vascular network

Results

Problems with whole organengineering

• Whose organ? The pig liver has 5 lobes, humanhas 4.

• Whose cells? • The matrix is highly compromised after

processing.

«Structural collapse» issues

Untreated IonicControl Non-ionic

change in lobule dimensions and shape due to removal of cells

GTA-fixed sample

In-vitro models of liver

• Probably the most promising and realistic applications is that of models for personalized therapy, drug testing and disease models.

• The liver metabolizes all exogenous compounds• Drugs and chemicals are tested on healthy,

young animals• Many drugs have passed animal tests and

preliminary trails and then been found to be toxic (vioxx, diclofenac, rosiglitazione see http://en.wikipedia.org/wiki/List_of_withdrawn_drugs)

3D PLGA and PLLA Hexagonal structures

Hepatocytes

Unit side length

Line width

500 micron

50 micron

control 0

1

2

3

4

5

6

albumin

production,

pg/cell.day

1 2 3 4 5 6 7

days

dynamic 3D

static 3D

Data from various papers by Vinci et al. Biotech. J

Good Results

• Hepatocytes on 3D matrices

• In the presence of flow

Really good when

• Multiple cell types

0

2

4

6

8

10

12

14

albumin,

pg/cell.day

15 h 24 h 48 h

time

static 3D

dynamic 3D

3 tissue 3D

Conclusions

Nutrients (bioreactor)

Biochemical signals

Increased cell-cell contactEnhanced

metabolic function

Shear stress damage

Co

mp

lexi

ty

Between a network of thin hollow fiber membranes, human hepatocytes or liver cells are kept alive by a constant supply of oxygen and a "culture medium" to feed on. The modular design is based on a parallel plate geometry. Each functional element consists of a rectangular cross-section flow channel formed by two polycarbonate plates. The lower plate supports a semi-permeable membrane to which the liver cells are attached. A parallel array of gas permeable hollow fibres (200 micron ID x 280 micron OD) are mounted on the upper plate. Blood plasma from the patient flows along the channel and is therefore in direct contact with the liver cells. The modular design allows for scale-up of the device; a 10 channel stack contains 2.5 x 108 cells (500 grams). The cells can survive for up to two months in these conditions. The flow rate through the bioreactor is 100 mL/minThe Process:Patients' plasma is separated from their blood in a plasma separator before the plasma is allowed to pass into the bioreactor (filtration plasmapheresis). As it is flowing over the capillary-like network of fibers, the liver cells function normally, drawing toxins out of the plasma, effectively cleaning and refreshing the patient's blood without actually mixing with it. Heparin, an anticoagulation drug, is delivered to patients.Question: scale a liver Bioreactor based on these considerations