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Artificial LiverArtificial Liver

Nandini DhimanNandini Dhiman

M.Tech - BMEM.Tech - BME

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Biology of the LiverBiology of the Liver

Left lobeRight lobe

KidneysGallbladder

Inferior Vena Cava

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LiverLiver

It is both the largest It is both the largest internalinternal organ (the organ (the skin being the largest organ overall) and skin being the largest organ overall) and the largest gland in the human bodythe largest gland in the human body

The various functions of the liver are The various functions of the liver are carried out by the liver cells or carried out by the liver cells or hepatocyteshepatocytes

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What does the Liver do?What does the Liver do?

Among the most important liver functions are:Among the most important liver functions are: Removing and excreting body wastes and hormones as Removing and excreting body wastes and hormones as

well as drugs and other foreign substanceswell as drugs and other foreign substances Synthesizing plasma proteins, including those necessary Synthesizing plasma proteins, including those necessary

for blood clotting for blood clotting Producing immune factors and removing bacteria, Producing immune factors and removing bacteria,

helping the body fight infection helping the body fight infection

Other important but less immediate functions include:Other important but less immediate functions include: Producing bile to aid in digestion Producing bile to aid in digestion Excretion of bilirubin Excretion of bilirubin Storing certain vitamins, minerals, and sugars Storing certain vitamins, minerals, and sugars Processing nutrients absorbed from digestive tractProcessing nutrients absorbed from digestive tract

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RegenerationRegeneration

The liver is the only internal human organ The liver is the only internal human organ capable of natural regeneration of lost capable of natural regeneration of lost tissue tissue

As little as 25% of a liver can regenerate As little as 25% of a liver can regenerate into a whole liver into a whole liver

This is predominantly due to the This is predominantly due to the hepatocytes re-entering the cell cycle hepatocytes re-entering the cell cycle

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Bioartificial LiverBioartificial Liver

A A bioartificial liver device (BAL)bioartificial liver device (BAL) is an artificial is an artificial extracorporeal supportive device for an individual with liver extracorporeal supportive device for an individual with liver failure, acute or chronicfailure, acute or chronic

Such devices are in principle more complex than dialysis and Such devices are in principle more complex than dialysis and filtration systems, they could provide biochemical and filtration systems, they could provide biochemical and synthetic functions that are not available in the systems synthetic functions that are not available in the systems containing no cells containing no cells

““Tissue Engineering and Artificial Organs - Joseph D. Bronzino”Tissue Engineering and Artificial Organs - Joseph D. Bronzino”

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Why would someone need a Bioartificial Why would someone need a Bioartificial Liver?Liver?

Liver diseasesLiver diseases Infections such as hepatitis A, B, C, E,Infections such as hepatitis A, B, C, E, Alcohol damageAlcohol damage Fatty liver Fatty liver Cirrhosis Cirrhosis CancerCancer Drug damage (especially acetaminophen, cancer Drug damage (especially acetaminophen, cancer

drugs)drugs)

JaundiceJaundice Alpha-1 antitrypsin deficiencyAlpha-1 antitrypsin deficiency

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The path to liver disease and The path to liver disease and failurefailure

04/11/2304/11/23 99Healthy LiverHealthy Liver

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A liver with cirrhosisA liver with cirrhosis

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Liver Transplantation NowLiver Transplantation Now

Patients are in waiting list ranked according to severity of disease and life expectancy among other variables

Can be from a cadaveric donor or from a live donor Involves heavy use of immunosuppressants during and after

surgery The risk of rejection is always present

Liver Transplant Statistics in 2000

02000400060008000

100001200014000160001800020000

Transplants Waiting List

Pa

tie

nts

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Requirements for a liver assist Requirements for a liver assist device/bioartificial liver:device/bioartificial liver:

1)1) Cellular components must be purified and every Cellular components must be purified and every component in it must be clearly identifiedcomponent in it must be clearly identified

2)2) The cellular preparation must be clearly shown to not The cellular preparation must be clearly shown to not transmit any infectious diseases of any kindtransmit any infectious diseases of any kind

3)3) The cellular component must stay viable and activeThe cellular component must stay viable and active

4)4) The synthetic component must be fully biocompatible, The synthetic component must be fully biocompatible, integrity of the material and parts must also be integrity of the material and parts must also be demonstrateddemonstrated

5)5) The device must be able to introduce the therapeutic The device must be able to introduce the therapeutic and regulatory molecules that a healthy liver provides, and regulatory molecules that a healthy liver provides, and it must also filter substances from the blood the and it must also filter substances from the blood the way that the normal liver doesway that the normal liver does

6)6) Must be immunocompatibleMust be immunocompatible7)7) Blood must perfuse properly through systemBlood must perfuse properly through system

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Enabling Technologies for Bioartificial

Liver Development:

Hemodialysis/hemofiltration hollow fibresHemodialysis/hemofiltration hollow fibres - - Necessary for the controlled interaction of cells and Necessary for the controlled interaction of cells and circulating fluids. Biomaterials technology is also key circulating fluids. Biomaterials technology is also key to this area to this area

Maintenance of Cell line (hepatocyte cell Maintenance of Cell line (hepatocyte cell line) -line) - Cells used for liver therapy must be able to Cells used for liver therapy must be able to survive and/or proliferate in the device, and they must survive and/or proliferate in the device, and they must

also maintain their specific liver function.also maintain their specific liver function.

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Cell SeparationCell Separation

Hepatocytes are separated from the liver Hepatocytes are separated from the liver by collagenase digestion, which is a two step by collagenase digestion, which is a two step process:process:

1.1. The liver is placed in an isotonic solution, in which calcium The liver is placed in an isotonic solution, in which calcium is removed to disrupt cell-cell tight junctions by the use of is removed to disrupt cell-cell tight junctions by the use of a calcium chelating agenta calcium chelating agent

2.2. A solution containing collagenase is added to separate the A solution containing collagenase is added to separate the hepatocytes from the liver parenchymahepatocytes from the liver parenchyma

This process creates a suspension of This process creates a suspension of hepatocytes, which can be culturedhepatocytes, which can be cultured

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Extracorporeal Device DesignsExtracorporeal Device Designs

The components of extracorporeal The components of extracorporeal devices are:devices are:

- - the cellular component, the cellular component,

- membrane component, and - membrane component, and

- configuration- configuration

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Different Types of Cellular Components present in Different Types of Cellular Components present in Bioartificial Livers (BAL) Bioartificial Livers (BAL)

Cellular Components of Bioartificial Liver

  Concept Pros Cons

Primary Hepatocytes Cells isolated directly from the liver exhibit specific functions

If porcine: - proteins produced may cause immunogenic response

-concern with infection, animal handling, and hepatocytes harvest, isolation, and storage procedure

Immortalized Cell Lines Immortalized hepatocytes lines that are obtained from cultures:

 - transforming human hepatocytes

-genetically engineered to proliferate

-cells allow for longer device operating time than primary hepatocytes

-if cultured from humans, it produces human proteins and cells are readily available

-Cells often lose function in vivo

-unclear whether fully differentiated hepatic and metabolic function is maintained

-outperformed by primary hepatocytes

-concern of spontaneous mutations or changes in gene expression during culture

-some cell lines are hepatoblastoma cell lines (tumor derived), which pose a theoretical threat of patient seeding

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Hepatocytes must be anchoredHepatocytes must be anchored to a substrate in order to  to a substrate in order to function properly. function properly.

Table : Anchoring Methods for Cells

Method Description Comments

MicrocarriersPolymeric particles containing cells. Alginate microspheres can be used.

Can be combined with ECM material to improve anchorage

Encapsulation

Envelopment of hepatocytes in a polymeric matrix. Can use polysaccharide hydrogels, collagen, or other materials.

Can be helpful in maintaining a three-dimensional structure for the hepatocytes

Hollow fibers

Luminal membranes that provide anchorage for hepatocytes. May be supplemented by ECM material (i.e. collagen)

May be coiled, bundled or wound to allow maximum length and surface area

 

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Cell adhesion, growth, and function can be Cell adhesion, growth, and function can be greatly affected by the type of membrane greatly affected by the type of membrane used: it’s morphology, membrane used: it’s morphology, membrane hydrophilicity, and molecular-weight cutoff hydrophilicity, and molecular-weight cutoff (MWCO).  (MWCO). 

Membrane area, pore size, and thickness can Membrane area, pore size, and thickness can also affect biocompatibility and bioadhesionalso affect biocompatibility and bioadhesion

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Table : Membranes used in Bioartificial Liver Devices

Membrane Charge MWCO (Kda) Comments

Cellulose acetate Positive 70-100 - Weak cell attachment- Sufficient metabolic activity- Low cell damage

Cuprophan Neutral <12 - Low thrombogenicity- No considerable cell attachment- High percentage of damaged cells

Hemophan Positive <10 - Adequate cell adhesion- Sufficient metabolic activity

Polyamide Not Applicable <1000 - Adequate cell adhesion- Satisfactory biocompatibility

Polypropylene -- <1000 - Adequate cell adhesion

Polysulfone Negative 100 - Increased cell attachment- Low cell damage- Maintenance of metabolic activity

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Encapsulation TechnologyEncapsulation Technology

To eliminate the risk of rejection or the need to use To eliminate the risk of rejection or the need to use problematic antirejection drugs is to encapsulate “naked” problematic antirejection drugs is to encapsulate “naked” cells in polymeric substancescells in polymeric substances

PRINCIPLE: PRINCIPLE: - - To develop a capsule with sufficient permeability that To develop a capsule with sufficient permeability that

nutrients and oxygen can reach the transplanted cells, and nutrients and oxygen can reach the transplanted cells, and appropriate cellular products (insulin in the case of islet cells, for appropriate cellular products (insulin in the case of islet cells, for example) can be released into the bloodstream or to adjacent example) can be released into the bloodstream or to adjacent tissues tissues

- The capsular material must be restrictive enough to - The capsular material must be restrictive enough to exclude immune cells and antibodies that would cause rejection and exclude immune cells and antibodies that would cause rejection and destroy the implantdestroy the implant

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PrinciplePrinciple

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Several Polymeric Encapsulation Several Polymeric Encapsulation Systems Include:Systems Include:

Polysaccharide hydrogelsPolysaccharide hydrogels ChitosanChitosan Calcium or barium alginateCalcium or barium alginate A layered matrix of alginate and polylysineA layered matrix of alginate and polylysine

“ “Novocell, Inc., has developed a photopolymerizable Novocell, Inc., has developed a photopolymerizable poly(ethylene glycol) (PEG) polymer to encapsulate poly(ethylene glycol) (PEG) polymer to encapsulate individual cells or cell clusters. Various polyacrylates are individual cells or cell clusters. Various polyacrylates are also being tested, including polymers such as also being tested, including polymers such as hydroxyethyl methacrylate methyl methacrylate.”hydroxyethyl methacrylate methyl methacrylate.”

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Cell sources

Human liver resections, at least 20% of the liver Human liver resections, at least 20% of the liver mass may be required for adequate liver support mass may be required for adequate liver support

Immortalized human cell line HepZImmortalized human cell line HepZ Hepatoblastoma cell lines HepG2 – Hepatoblastoma cell lines HepG2 – Problem is the Problem is the

possible risk in the transmission of potentially possible risk in the transmission of potentially tumorigenic cells to patientstumorigenic cells to patients

Xenogeneic hepatocytes – Xenogeneic hepatocytes – Problems are PERV, Problems are PERV, PCMV,PLHVPCMV,PLHV

Its Its notnot possible for self cell lines as they may be infected and possible for self cell lines as they may be infected and also time consumingalso time consuming

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Maintenance of liver- specific functionsMaintenance of liver- specific functions

Complex extracellular matrix to maintain long-Complex extracellular matrix to maintain long-term differentiated hepatocytes in vitro thus a term differentiated hepatocytes in vitro thus a great interest in effective 3D culture systemsgreat interest in effective 3D culture systems

In contrast to a single layer of hydrated rat tail tendon collagen gel, In contrast to a single layer of hydrated rat tail tendon collagen gel, the sandwiching of rat hepatocytes between two collagen gel the sandwiching of rat hepatocytes between two collagen gel layers preserves a variety of liver-specific functions (Dunn et al. layers preserves a variety of liver-specific functions (Dunn et al. 1991, 1992)1991, 1992)

ECM remodeling in response to ECM remodeling in response to cytokinescytokines induces cell proliferation, an important induces cell proliferation, an important parameter of liver regenerationparameter of liver regeneration

Proliferation of hepatocytes is initiated by cytokine mediated Proliferation of hepatocytes is initiated by cytokine mediated G0/G1-transition of the cells, while the G1/S transition is also G0/G1-transition of the cells, while the G1/S transition is also controlled by hormones (Costa et al. 2003; Taub 2004)controlled by hormones (Costa et al. 2003; Taub 2004)

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MethodsMethods

HCs and nonparenchymal liver cells from Lewis rats were seeded onto 3D biodegradable polymer scaffolds

Microporous 3D polymers were created using 3D printing on copolymers of polylactide-coglycolide (PLGA)

The cell/polymer constructs were placed in static culture or continuous flow conditions

The devices were retrieved after 2 days and examined by scanning electron microscopy and histology. Culture medium was analyzed for albumin by enzyme-linked immunosorbent assay (ELISA)

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ResultsResults Scanning electron microscopy revealed

successful attachment of HCs on the 3D poymer in both static and flow conditions.

Histology demonstrated viable HCs in both conditions.

ELISA demonstrated a significantly higher mean concentration of albumin in flow conditions than in static conditions.

Culture parameter analysis revealed a significantly higher P02 and glucose level, and a more physiologic pH in flow conditions than in static conditions.

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ConclusionConclusion

HCs cocultured with nonparenchymal cells can attach to and survive on the 3D polymer scaffolds in both static and flow conditions in the size and configuration used in this study.

Flow conditions may provide a more conducive environment for HC metabolism and albumin synthesis than static conditions

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Bioreactor DesignsBioreactor Designs

A bioreactor refers to a device or A bioreactor refers to a device or system that supports a biologically system that supports a biologically active environment.active environment.

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Hollow-FiberHollow-Fiber

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Flat membrane systemsFlat membrane systems

Hepatocytes are co-cultured with non-Hepatocytes are co-cultured with non-parenchymal hepatic cells within an extracellular parenchymal hepatic cells within an extracellular matrix between oxygen permeable flat sheet matrix between oxygen permeable flat sheet polymeric membranes as individual platespolymeric membranes as individual plates

Micro porous PTFE membrane separate the cell Micro porous PTFE membrane separate the cell compartment from the medium compartment. compartment from the medium compartment. Protects against shear and regulates transferProtects against shear and regulates transfer

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Comparison to liver dialysisComparison to liver dialysis

The advantages of using a BAL, over The advantages of using a BAL, over other dialysis-type devices (e.g. liver other dialysis-type devices (e.g. liver dialysis), is that metabolic functions (such dialysis), is that metabolic functions (such as lipid and plasma lipoprotein synthesis, as lipid and plasma lipoprotein synthesis, regulation of carbohydrate homeostasis, regulation of carbohydrate homeostasis, production of serum albumin and clotting production of serum albumin and clotting factors, etc.), in addition to detoxification, factors, etc.), in addition to detoxification, can be replicated without the use of can be replicated without the use of multiple devices multiple devices

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Current Work on the Bioartificial Current Work on the Bioartificial LiverLiver

Molecular Adsorbent Recycling System Molecular Adsorbent Recycling System (MARS)(MARS)

Extracorporeal Liver Assist Device (ELAD)Extracorporeal Liver Assist Device (ELAD) HepatAssist 2000 SystemHepatAssist 2000 SystemExcorp Medical, Inc.'s Bioartificial Liver Excorp Medical, Inc.'s Bioartificial Liver

Support System (BLSS)Support System (BLSS)   Algenix Inc.'s LIVERX2000 SystemAlgenix Inc.'s LIVERX2000 SystemModular Extracorporeal Liver System Modular Extracorporeal Liver System

(MELS) (MELS)

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HepaLifeHepaLife

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Current and Future ChallengesCurrent and Future Challenges

GOAL: To produce a fully implantable GOAL: To produce a fully implantable bioartificial liver.bioartificial liver.

Cell viability Fibrosis around implanted

capsules Proteins greater than pore

size cannot be released To achieve density of cells

needed to replace liver, an estimated 1000m of hollow fibers would be needed

Problems:

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ReferencesReferences

http://www.hepalife.com/animation.phphttp://www.hepalife.com/animation.phphttp://biomed.brown.edu/Courses/BI108/http://biomed.brown.edu/Courses/BI108/

BI108_2002_Groups/liver/webpage/BI108_2002_Groups/liver/webpage/LDU_pg.htmLDU_pg.htm

http://en.wikipedia.org/wiki/http://en.wikipedia.org/wiki/Bioartificial_liver_deviceBioartificial_liver_device

http://en.wikipedia.org/wiki/Liverhttp://en.wikipedia.org/wiki/Liver

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Thank YouThank You