Artificial Liver

Nandini Dhiman M.Tech - BME

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Biology of the Liver

Right lobe Gallbladder Kidneys

Left lobe

Inferior Vena Cava

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Liver

It is both the largest internal organ (the skin being the largest organ overall) and the largest gland in the human body The various functions of the liver are carried out by the liver cells or hepatocytes

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What does the Liver do?

Among the most important liver functions are: Removing and excreting body wastes and hormones as well as drugs and other foreign substances Synthesizing plasma proteins, including those necessary for blood clotting Producing immune factors and removing bacteria, helping the body fight infection
Other important but less immediate functions include: Producing bile to aid in digestion Excretion of bilirubin Storing certain vitamins, minerals, and sugars Processing nutrients absorbed from digestive tract
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Regeneration
The liver is the only internal human organ capable of natural regeneration of lost tissue As little as 25% of a liver can regenerate into a whole liver This is predominantly due to the hepatocytes re-entering the cell cycle

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Bioartificial Liver

A bioartificial liver device (BAL) is an artificial extracorporeal supportive device for an individual with liver failure, acute or chronic Such devices are in principle more complex than dialysis and ltration systems, they could provide biochemical and synthetic functions that are not available in the systems containing no cells
Tissue

Engineering and Artificial Organs - Joseph D. Bronzino

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Why would someone need a Bioartificial Liver?

Liver diseases

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Infections such as hepatitis A, B, C, E, Alcohol damage Fatty liver Cirrhosis Cancer Drug damage (especially acetaminophen, cancer drugs) Jaundice
Alpha-1 antitrypsin deficiency
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The path to liver disease and failure

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Healthy Liver

A liver with cirrhosis

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Liver Transplantation Now

Liver Transplant Statistics in 2000
20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 Transplants Waiting List

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
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Patients

Requirements for a liver assist device/bioartificial liver:

1) 2) 3) 4)

Cellular components must be purified and every component in it must be clearly identified The cellular preparation must be clearly shown to not transmit any infectious diseases of any kind

The cellular component must stay viable and active

The synthetic component must be fully biocompatible, integrity of the material and parts must also be demonstrated 5) The device must be able to introduce the therapeutic and regulatory molecules that a healthy liver provides, and it must also filter substances from the blood the way that the normal liver does 6) Must be immunocompatible 7) Blood must perfuse properly through system 26-03-2012 12

Enabling Technologies for Bioartificial Liver Development:

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

Maintenance of Cell line (hepatocyte cell line) - Cells used for liver therapy must be able to
survive and/or proliferate in the device, and they must also maintain their specific liver function.
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Cell Separation
Hepatocytes are separated from the liver by collagenase digestion, which is a two step process:
1.

2.

The liver is placed in an isotonic solution, in which calcium is removed to disrupt cell-cell tight junctions by the use of a calcium chelating agent A solution containing collagenase is added to separate the hepatocytes from the liver parenchyma

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

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Extracorporeal Device Designs

The components of extracorporeal devices are: - the cellular component,

- membrane component, and - configuration

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Different Types of Cellular Components present in 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 16

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

Table : Anchoring Methods for Cells
Method Description Comments

Microcarriers

Polymeric particles containing Can be combined cells. Alginate microspheres with ECM material to can be used. improve anchorage Envelopment of hepatocytes in a polymeric matrix. Can use polysaccharide hydrogels, collagen, or other materials. Luminal membranes that provide anchorage for hepatocytes. May be supplemented by ECM material (i.e. collagen) Can be helpful in maintaining a threedimensional structure for the hepatocytes May be coiled, bundled or wound to allow maximum length and surface area
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Encapsulation

Hollow fibers

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

Membrane area, pore size, and thickness can also affect biocompatibility and bioadhesion
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Table : Membranes used in Bioartificial Liver Devices

Membrane
Cellulose acetate

Charge Positive

MWCO (Kda) 70-100

Comments - Weak cell attachment - Sufficient metabolic activity - Low cell damage - Low thrombogenicity - No considerable cell attachment - High percentage of damaged cells - Adequate cell adhesion - Sufficient metabolic activity - Adequate cell adhesion - Satisfactory biocompatibility - Adequate cell adhesion

Cuprophan

Neutral

<12

Hemophan

Positive

<10

Polyamide

Not Applicable

<1000

Polypropylene

--

<1000

Polysulfone

Negative

100

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- Increased cell attachment - Low cell damage - Maintenance of metabolic activity 19

Encapsulation Technology

To eliminate the risk of rejection or the need to use problematic antirejection drugs is to encapsulate naked cells in polymeric substances PRINCIPLE: - To develop a capsule with sufficient permeability that
nutrients and oxygen can reach the transplanted cells, and appropriate cellular products (insulin in the case of islet cells, for example) can be released into the bloodstream or to adjacent tissues - The capsular material must be restrictive enough to exclude immune cells and antibodies that would cause rejection and destroy the implant

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Principle

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Several Polymeric Encapsulation Systems Include:

Polysaccharide hydrogels Chitosan Calcium or barium alginate A layered matrix of alginate and polylysine

Novocell, Inc., has developed a photopolymerizable

poly(ethylene glycol) (PEG) polymer to encapsulate individual cells or cell clusters. Various polyacrylates are also being tested, including polymers such as hydroxyethyl methacrylate methyl methacrylate.
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Cell sources

Human liver resections, at least 20% of the liver mass may be required for adequate liver support Immortalized human cell line HepZ Hepatoblastoma cell lines HepG2 Problem is the possible risk in the transmission of potentially tumorigenic cells to patients Xenogeneic hepatocytes Problems are PERV, PCMV,PLHV
Its not possible for self cell lines as they may be infected and also time consuming

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Maintenance of liver- specic functions

Complex extracellular matrix to maintain longterm differentiated hepatocytes in vitro thus a great interest in effective 3D culture systems
In contrast to a single layer of hydrated rat tail tendon collagen gel, the sandwiching of rat hepatocytes between two collagen gel layers preserves a variety of liver-specic functions (Dunn et al. 1991, 1992)

ECM remodeling in response to cytokines induces cell proliferation, an important parameter of liver regeneration
Proliferation of hepatocytes is initiated by cytokine mediated G0/G1-transition of the cells, while the G1/S transition is also controlled by hormones (Costa et al. 2003; Taub 2004)
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Methods

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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Results

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

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Hollow-Fiber

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Flat membrane systems

Hepatocytes are co-cultured with nonparenchymal hepatic cells within an extracellular matrix between oxygen permeable at sheet polymeric membranes as individual plates Micro porous PTFE membrane separate the cell compartment from the medium compartment. Protects against shear and regulates transfer

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Comparison to liver dialysis

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

Molecular Adsorbent Recycling System (MARS) Extracorporeal Liver Assist Device (ELAD)

HepatAssist 2000 System

Excorp Medical, Inc.'s Bioartificial Liver Support System (BLSS) Algenix Inc.'s LIVERX2000 System Modular Extracorporeal Liver System (MELS)

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HepaLife

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Current and Future Challenges

GOAL: To produce a fully implantable bioartificial liver.

Problems:

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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

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References
http://www.hepalife.com/animation.php http://biomed.brown.edu/Courses/BI108/BI10 8_2002_Groups/liver/webpage/LDU_pg.htm http://en.wikipedia.org/wiki/Bioartificial_liver_ device http://en.wikipedia.org/wiki/Liver

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Thank You

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