BIOARTIFICIAL LIVER SYSTEMS

Özgür Genç

Abstract

Bioartificial liver is a device that fills the essential gap between the transplantation and Acute Liver Failure. Since Acute Liver Failure results with sudden death of people, the only way to compete the disease is to revitalise the liver function artificially. Bioartificial liver is a hybrid device in which biologic and non-biologic components come together. Hepatocytes are the single biologic component of BAL devices. Hepatocytes may be derived from several mammalians like porcine. Hepatocyte viability is the key issue of BAL concept. The efforts are concentrated on this topic since the functionality of the device totally depend on the living hepatocytes. Bioreactor designs serve the optimum conditions by means of cell viability, perfusion rate, device size, geometry and so on. There are several approaches for BAL systems like entrapment based system, hollow fiber system and direct perfusion system.

Introduction

Liver : An Essential Part of the Body

Liver has a very critical role in the metabolism. The liver carries out many metabolic functions by the help of hepatocytes those are the biochemically complex cells in the human body. Those homeostatic functions are known as gluconeogenesis, synthesis of blood proteins, amino acid metabolism, urea synthesis, lipid metabolism, drug detoxification, waste removal, and immune and hormonal modulation.[1] Also, liver is the unique internal organ, which can regenerate itself.[2]

Summary of Liver Functions [3]

endocrine functions
clotting functions
synthesis of plasma proteins
digestive functions
organic metabolism
cholesterol metabolism
excretory and degradative functions

Definition of Liver Failure

Liver failure is characterized by the development of hepatic encephalopathy, jaundice and coagulopathy. Acute liver failure is used as a term covering clinical presentations, such as hyperacute, fulminant, and subacute liver failure. If hepatic encephalopathy develops within 10 days of the onset of jaundice, it is defined as hyper-acute liver failure. When hepatic encephalopathy develops 10-30 days after the onset of jaundice, liver failure is defined as fulminant. Few patients develop subacute liver failure, occurring after 4 weeks of illness.[4] Fulminant liver failure has a time course from onset of illness to death of not more than 10 days [5]

Despite the fact that liver is an organ capable of high level of regeneration, a minimum critical mass of hepatocytes has to be alive in order to support homeostasis during the process of regeneration. When this critical mass is absent, liver failure supervenes and regeneration process is impaired, this is because of the build-up of toxic metabolites.

Acute fulminant hepatic failure is a clinical syndrome has origins in virus or drugs, associated with sudden severe impairment or loss of hepatocyte function. This situation generally results in encephalopathy [6], as we noted before.

The survival rate of FHF patients was reported to be less than 10%–15% [7]. This rate is fortified by the shortage of cadaveric organ donors and time constraints in finding a suitable donor.[8] The death of the patient is basically related with the massive necrosis of hepatocytes. Result of this, synthetic and metabolic pathways are destructed.[9]

Therapeutic options for Liver Failure

For a long time, orthotopic liver transplantation was the only way of the definitive treatment for FHF and its neurological complications.[10] However the major drawback is the limitation of organ donors, as a result of this, many patients die before a transplantation occurs or patients are too ill for operation by the time a liver becomes available. Many patients with hepatic failure do not satisfy the needs for liver transplantation, this is related with concomitant infection, metastatic cancer, active alcoholism or concurrent medical problems.[11]

Limitations on the liver transplantation opened a way for the development of an artificial organ. This was a breakthrough development in the history of liver diseases.

Liver assist devices were designed to bridge the time to liver transplantation or to recovery of the patients’ own liver. [12] An artificial liver assist device is needed not only to bridge patients with fulminant hepatic failure to liver transplantation or own liver regeneration, but also to improve the quality of life of patients with chronic liver insufficiency.[13]

Artificial Liver Support

The role of artificial liver support is quite different from a typical kidney hemodialysis machine. This is a predictable situation since the liver has functionality by means of biochemical activities further than physical response. The removal of toxins from body (e.g. aromatic amino acids and ammonia) the synthesis of many essential products (e.g. coagulation factors and albumin), and reversing of the inflammatory process are all taking place in the liver[14]

The origins of the bioartificial liver are at the middle of 20^th century. There are two major approaches those the researcher followed since seventies to develop artificial livers: First group named as Filtration-based devices [15] or non-biological [16] and the second group named as bioreactor designs [17] using living hepatocyte cultures or hybrid biological artificial support. [18]

Those two ways can also be categorized as artificial and bioartificial liver designs.[19] (Fig. 1)

Figure 1: Classification of the different artificial and bioartificial organs for temporary liver support.[20]

A brief history

During 70s and 80s, there were many approaches for tackling with the liver failure. The devices those developed over the logic of detoxification were just useable for end stage renal disease. Also those devices was serving only transient benefit to patients with severe liver failure since those systems were not well equipped for the management of complex metabolic disorders related with liver disease.

In haemodialysis, a semipermeable dialysis membrane is used. By this method, fluid and small solutes may pass via diffusion through the membrane. By haemoperfusion many toxins and molecules are removed from the blood according to weight difference by using different adsorbents. Charcoal is an effective adsorbent for wide range of water soluble molecules except ammonia or protein bound compound.[21]

Those extracorporeal therapies such as hemodialysis, hemofiltration, exchange transfusion, plasma exchange and charcoal perfusion have been tried alone or in combination. As a result improvement in biochemical parameter is observed, unfortunately survival was not seen. The major reason for failure is related with applied techniques those are efficient only against the removal of water soluble substances such as ammonia or lactate, whereas protein bounded substances such as bile acids are removed in small amounts. The accumulation of bile acids result with liver failure.[22]

From this point scientists got a new perspective. Since liver performs multiple and complex functions (detoxification, transformation, and synthesis), it was revealed that simple mechanical or chemical forces can not be adapted to the treatment of acute liver diseases. [23] This situation also defined like the inefficiency of mechanical devices such as haemodialysis and haemoperfusion against the metabolic abnormalities that exist in the final stages of liver disease.[24]

In contrast to the kidney, the detoxification activity of the liver is in particular focused at protein-bound and lipid soluble compounds, these newer dialysis techniques have included albumin adsorption in their system. However, it is generally assumed that a real effective artificial liver should be based on the capacity to perform the liver’s multiple synthetic and metabolic functions, including detoxification and excretion. Hybrid bioartificial systems based on the presence of active functioning hepatocytes in an extracorporeal device that can be connected to the circulation of the patient with liver failure are promising to meet these criteria. The heart of such a system consists of a bioreactor with a sufficient amount of well-nourished and oxygenated viable hepatocytes immobilized on a mechanical support. [25]

Essentiality of a biological component to provide adequate support in liver failure was proven more than 30 years ago. [26] The absence of hepatocyte-derived factors and lack of regulation is addressed by efforts to provide cell function with hepatocyte-based systems.[27] Early attempts used cross-circulation of blood from one individual through the liver of another individual or mammal, perfusion of whole animal livers, freeze dried liver homogenates or fresh liver slices.[28]

One of this attempts, cross circulation is important not because of its efficiency but giving the idea of using xenogenic cells in liver devices. In 1959, Kimoto investigated cross dialysis between humans and dogs. Waste products from the human were metabolized by the canine liver and run through a cation exchange filter. Although the patient died after 7 days, his clinical condition was improved. However, reduction in nitrogenous waste products was demonstrated and no antidog antibodies could be detected in the patient’s serum. This suggested that xenogenic hepatocytes could be used without detectable immune activation if a semipermeable membrane was used to separate the circuits. The same trial has been applied on two human using relatives of same blood grouping. Human cross circulation has also been examined with some success.[29] Apart from the valuable ideas derived from those investigations, these techniques were associated with several adverse events and have not been generally accepted. However advanced technology has now made it possible to include biological components in bioartificial (hybrid) liver support systems.[30]

Bioartificial Liver Systems

Recent advances in biotechnology and tissue culture technology have facilitated the importance of incorporating a biological component in artificial liver support systems and, thus, have given rise to a number of different designs of bioartificial liver (BAL) support systems. Here, are presented many BAL systems in pre-clinical state of clinical trials: Vitagen Extracorporeal Liver Assist Device (ELAD), Hepatassist Circe Biomedical HepatAssist system, TECA Hybrid Artificial Liver Support System (TECAHALSS), Excorp. Medical Inc. Bioartificial Liver Support System (BLSS), Radial Flow Bioreactor (RFB), Hybrid Liver Support System (LSS) Bioartificial Hepatic Support (BHS) and Amsterdam Academic Medical Center-Bioartificial Liver (AMC-BAL).[31]

There are different approaches those lead to the occurrence of such kind of devices. Various parameters are known but the most significant ones are hepatocyte type and usage, bioreactor design and extracorporeal components (like plasma separation, oxygenation, etc)

Here, a classification is done based on those parameters. This classification may not be universal; it is formed consistent with major approaches in the field of bioartificial liver studies aiming to get the best fit. Before examining the several approaches, it is important to evaluate the importance of a component: hepatocyte

Hepatocytes

As commonly named “hybrid systems” are designs in which living cells or tissues and inorganic bioreactor vessel is incorporated. A desirable system has to carry the condition where cultured hepatocytes are provided with an environment permitting the expression of major liver functions like protein synthesis, enzyme activity, bilirubin conjugation, and drug metabolism.

Bioartificial support systems utilize living hepatocytes. In clinical applications of BAL devices, there is a wide range of hepatocyte mass used in the culture changing from 2% to 33% which represents the mass of 30g to 500g.[32] This quantity changes according to the utilization mode of hepatocytes in the device.

Selection of hepatocyte type is a critical step in the establishment of the BAL system. Those lines can be derived from human or animal livers, or other cells manipulated through genetic modification.

It is suitable to divide the hepatocyte sources into two as obtaining from human or animal[33].

Animal hepatocytes are commonly used and has subcategories:

i) porcine hepatocytes

ii) simian hepatocytes

iii) implantation of human hepatic genes into animal cells

Porcine hepatocytes are commonly used. The main advantage of using porcine hepatocytes is the availability and low price. However, there are many potential problems including interspecies differences in enzyme activities, production of xenogenic antibodies and transfer or infections (zoonosis).

The last point can be clarified. The presence of viruses can not be completely excluded, and in particular retroviruses, present in all porcine cells, are a potential threat to humans. Porcine hepatocytes produce xenogeneic proteins that will induce an immune response: repeated application may provoke an anaphylactic reaction. Up to now, compatibility of porcine regulation processes and synthesis products are not proven.

There are three types of applications in order to obtain fresh human hepatocytes,

i) surgical removal of liver from a person whose brain death is occurred

ii) cell strains derived from human liver cancer cells

iii) artificially immortalised cells

The use of hepatocyte tumor cell strains has the advantage of easier logistics and availability in large quantities; the disadvantages are the risk of metastases and a lower biochemical performance compared with primary hepatocytes. For the former reason, there are some serious concerns regarding the possibility of tumour seeding with the use of hepatoma derived liver cell lines if the cells were to escape the bioreactor. The risk of developing a tumour because of the use of these cells is thought to be low as the accidental rupture of hollow fibres is rare and the pressure across the hollow fibre membrane would prevent the cells from entering the systemic circulation should an accident occur.[34]

Clinical use of human gene-technologically immortalized cell lines is not yet established. In order to avoid the drawbacks involved in using porcine cells and human liver tumor cell lines, the use of primary human liver cells is desirable for both extracorporeal liver support and cell transplantation.[35]

Since hepatocytes are the universal source for biological component of assist devices, it is essential to organize the condition compensating the needs for proper functioning of hepatocytes.

Determining the hepatocyte volume and quantity

As it is mentioned before hepatic encephalopathy occurs when liver function falls below 25–30% of normal (i.e. when <300–420 g of viable hepatocytes remain).Therefore, it is thougth that in order to provide adequate liver support for humans in acute liver failure, approximately 400 g of hepatocytes are required. Also animal studies have confirmed that 20–40% of host liver mass is required for effective support.[36]

Maintenance of hepatocyte viability

There are many ways to prolong the hepatocyte survival. Addition of growth factors and hormones to the culture medium is effective. [37] Those are also common, like culturing the cells in the presence of attachment factors and extracellular matrix constituents and in many cases with non parenchymal cells.

We can summarize these techniques below:

i) immobilization of hepatocytes within a collagen gel prior to monolayer culture or the use of an overlying layer of type I collagen forming a collagen gel sandwich

ii) addition of extracellular matrix

iii) application of techniques promoting three dimensional cell-cell adhesion, such as the use of a small number of collagen-coated dextran, polystrene or glass microcarriers

In bioreactor designs, the hepatocytes are utilized according to those criterias.

These methods give the optimal conditions in where hepatocytes are located:

Immobilization on Collagen Coated Flat Plates

In 1988 Uchino et al. published their BAL based on freshly isolated canine hepatocytes attached to collagen coated glassplates. As we know now, a too small number of animals to obtain significancy due to the large biological variation of such a model.

Culture within a Porous Three-Dimensional Matrix

In this model there are different ways of cell attachment in the bioreactor. Cells are attached to a nonwoven polyester fabric as a matrix. This matrix is either enclosed as different pieces in a cartridge or as a spirally wound tissue.

Encapsulation in Gels.

Encapsulation of liver cells has been successful by the use of alginate. This development is still in the experimental phase, and proof of principle in experimental acute liver failure has to be obtained before clinical application will be justified.

Appropriate membrane permeability can allow capsules to prevent immune response. Therefore, the pore size manipulation of membranes by varying solution conditions such as pH, and polymer concentration is a critical parameter for its application. The long-term culture with enhanced liver-specific activity is essential for the development of artificial liver. Hepatocytes cultured as spheroids have been observed to exhibit enhanced liver-specific function and differentiated morphology compared to hepatocyte maintained as a monolayer. Therefore, the encapsulated hepatocyte spheroid with pore size modulation can be a good candidate for the artificial liver.[38]

Based on the optimisations for hepatocyte survival and activity, several bioreactor designs made.

Capillary Hollow Fiber Systems

In this design, blood or plasma flow through the intracapillary lumen whereas hepatocytes are attached to the outside of the semipermeable fiber membrane. Those hepatocytes are bundled together with a synthetic shell. Semipermeable membrane enables the interaction of the cells and the plasma. By using collagen gel, cells can be cultured inside the lumen besides the attachment outside the hollow fibers. In theory, the cells resting on the capillaries’ exterior surface and within the plastic shell should provide hepatic metabolic function and a symbiotic relationship should be established between the host and the cultured hepatocytes through the capillary membrane. In such a system the cultured hepatocytes are protected from the body’s immune system by the semipermeable capillary membrane. However, all these hollow fiber devices have the disadvantage of indirect contact between plasma and liver cells.

Additionally, cells affix to the surface of the fibers or beads and aggregate in layers with a drawback of shearing stress and disruption of the equal distribution of oxygen and nutrients to all cell layers. Also uneven distribution in the flow rate of the blood leads to the building of waste products, which have a direct effect on pH values and gas concentrations. As a result of this, quality is affected negatively and cell viability is reduced significantly. Those are the major drawbacks of this system, which are limiting the use of this system for long-term applications.

In order to compete with those problems, by another model with a three dimensional culture is the basis of the utilization mode of hepatocytes. In a representative model, a radial flow bioreactor (RFB) overcomes the problems occur in hollow fiber models. The RFB column consist of vertically extended cylindrical matrix composed of porous glass bead microcarriers, through which liquid medium flows from the periphery toward the central axis, generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or build-up of waste products. The highly porous microcarrier bead structures are suitable for adhesion and the proliferation of cell colonies in three dimensions over a vast surface area. As a result system carries high-density capacity. [39] This model is told in the next part as direct perfusion systems

It is generally agreed that the in vitro immobilization of hepatocytes requires a material suitable for cell attachment. This material has a strong influence on the construction of a bioreactor; for example, the use of micro-carriers for hepatocyte attachment introduces a large volume of material which contributes nothing to the efficiency of the bioreactor. Different methods of immobilization have been used, such as microcarriers and membranes.[40]

Table1. adapted from Legallais et. al.

Fig 2. adapted from Legallais et. al.

Fig 3. adapted from Dixit et. al.

Direct Perfusion Systems

The system works simply, direct perfusion of attached cells by plasma or blood gives satisfactory results. One of the advances of the system is mass transfer efficiency. There are many attempts one of, which forms a 3D environment for the liver cells, mimicking the native organ. In this model, the cells could form small aggregates, or be directly attached to a porous support. They were thus almost individually perfused, under a low diffusion gradient as in a normal liver.

Table2. adapted from Legallais et. al.

Entrapment based systems

A better approach is the inclusion of hepatocytes within a semi-permeable spherical structure usually called “bead” or “capsule”. The polymer bead matrix offered anchorage facilities to hepatocytes and its porous structure could act as an immunological barrier. Hepatocytes viability was found to be maintained in such a tridimensional structure, even after cryopreservation, a form of hepatocyte storage. The beads protect the cells from shear stress damage during the flow. There are several materials applicable for encapsulation. HEMA-MMA copolymer, chitosan-dextrose, and calcium alginate are major substances used. Calcium alginate is the most popular material because of its porosity, its mechanical properties and its biocompatibility. The alginate bead external structure might be strengthened by the addition of chelating components like lysine. The bead diameter ranged about 1mm or less, allowing for sufficient mass transfer and oxygenation of all the hepatocytes.

Table 3. adapted from Legallais et. al.

An experiment performed by Sun-hee et.al.[41] focused on rat hepatocyte encapsulation and significant results obtained. In that experiment, hepatocyte spheroids and hepatocyte were immobilized in chitosan/alginate capsules formed by the electrostatic interactions between chitosan and alginate. After encapsulation, it was seen that a 10% decrease in the viability of spheroids due to the exposure of the cells to a pH 6 during the encapsulation process. However, the encapsulated hepatocyte spheroids maintained over 50% viability and liver specific functions for 2 weeks while the encapsulated hepatocytes, free hepatocytes and free hepatocyte spheroids showed low viability and liver specific functions. Therefore, encapsulated hepatocyte spheroid application to the development of a bioartificial liver is supported by Sun Hee et al.

Pros and cons for bioreactor designs

	Hollow fiber system	Flat plate and monolayer	Perfused bed	Encapsulation based
Pros	high attachment surface, potential for immunoisolation, prevention of cells from shear stress.	a uniform cell distributions and microenvironment	Ease of scale up, promotes 3D architecture, minimal transport barrier	Ease of scale up, uniform microenvironment
cons	Nonuniform cell distribution, transport barrier with membranes or gels	Complex scale up, potential dead volume, cells exposure to shear forces, low surface area to volume ratio	Nonuniform perfusion, clogging, cells exposure to shear forces	Poor cell stability to suspension, transport barrier due to encapsulation, degradation of microcapsules over time, exposure of cells to shear stress

Table 4. adapted from Allen et. al.

Based on many requirements presented here, the needs of BAL systems can be summarized as below.

Essential Requirements[42] for an effective BAL system are

1) Establishment of a viable and highly functional hepatocyte cell line.

2) Development of a suitable bioreactor environment and peripheral control systems

3) Incorporation within an effective extracorporeal circulatory system.

Bioreactor designs and major types of BAL devices

In addition to facilitating viability and function of hepatocytes in culture by promoting normal cellular polarity, bioreactor design must take into account the requirement for adequate perfusion of the hepatocyte component with the patient’s blood and efficient removal of waste products of cellular metabolism. There are two prerequisites to build up a functioning bioreactor device incorporating in the bioartificial liver system. Hepatocytes in culture within the bioreactor module must maintain both viability and expression of differentiated function if any value is to be obtained.

Until now, there are many clinical trials as mentioned before. Since there is not a unique and a best model of bioreactor design, it will be evaluated here comparatively between several studies.

Hepatassist device

The HepatAssist (Circe Biomedical, Lexington, MA, USA) device is composed of a hollow fibre bioreactor, with a cellulose-coated activated charcoal column that is functional to remove inorganic toxins, and an oxygenator used to oxygenate the hepatocytes. Sodium citrate is used as an anticoagulant to prevent thrombosis within the circuit. Hepatocytes (5–7 x10² cells) are contained within the extra-fibre space of a hollow fibre module and attached to collagen coated dextran microcarriers. Plasma is allowed to circulate through the hollow fibres. Since there is not am membrane based structure, an immunologic barrier is omitted.

UCLA-BAL systems

There are many limitations of total diffusion surface area and capacity for hepatocyte mass for most capillary hollow-fiber based BAL designs. It is proposed that a BAL design using microencapsulated hepatocytes to overcome these physical limitations. This new BAL design (UCLA-BAL) involves the direct haemoperfusion of a packed-bed column of microencapsulated porcine hepatocytes within an extracorporeal chamber. Encapsulation takes place within biocompatible alginate-polysine-alginate composite membranes. In extensive animal studies using a well-characterized animal model fulminant hepatic failure (FHF), it is demonstrated that the UCLA-BAL system had superior diffusion surface area and a higher capacity for hepatocytes compared to conventional capillary hollow-fiber based BAL devices. UCLA-BAL treatment significantly improved the survival rate of FHF animals and significantly prolonged the survival time of similar animals with very severe liver injury. BAL treatment was convenient, easy to operate and well tolerated, and did not adversely affect the animal’s hemodynamics during treatment. It is therefore suggested that the UCLA-BAL is a significant improvement over conventional, first-generation, capillary hollow-fiber BAL systems.

fig 4. adapted from Dixit et. alalal)

Modular Extracorporeal Liver Support (MELS) [43]

In this concept different extracorporeal therapy units are combined: The cell module is a bioreactor charged with primary human liver cells, they are isolated from explanted livers. These livers are unsuitable for transplantation. A detoxification module enables single-pass albumin-dialysis for the removal of albumin bound toxins. In this set-up an additional dialysis module performing continuous venovenous hemodiafiltration (CVVHDF) is also employed. The bioreactor consists of hollow fiber membranes with a molecular weight cut-off of 400,000 and hydrophobic membranes with a total surface of 4.3 m2, interwoven to four independent compartments. Two compartments are utilized for perfusion of the cells with the patient’s plasma and one is used for integrated oxygenation/carbon dioxide removal. After liver cells are seeded into the fourth compartment between the hollow fibers, they attach to the surface of the capillaries and form aggregates between them. The hollow fibres are interwoven into a three-dimensional capillary network with numerous sub-units enabling decentralized mass exchange with high performance nutrient supply and metabolite removal.

Fig.5 Adapted from Sauer et. al

Clinical applications

The study of Pazzi et. al on reduction of bile acids in fulminant and acute liver failure by bioartificial liver treatment.

Serum bile acids are increased in liver failure, but the composition of the bile acid pool in this condition has not been studied in detail. This information is of interest because of dihydroxy bile acid toxicity. In a study by Pazzi et al. They measured serum bile acids by gas chromatography–mass spectrometry in 13 patients with fulminant liver failure and five patients with acute-onchronic liver failure. Furthermore, they were analysed serum bile acids in the same patients after 6 h of treatment with a bioartificial liver, consisting of a hollow-fibre cartridge with microcarrier-attached porcine hepatocytes and a charcoal column.

They observed that pre-bioartificial liver serum bile acids demonstrated a high dihydroxy / trihydroxy ratio and were higher in patients with acute-on-chronic liver failure than in those with fulminant liver failure (452.8 ± 98.6 vs. 182.1 ± 39.7 lmol / L; P < 0.05). Bioartificial liver treatment decreased significantly serum bile acids in patients with fulminant liver failure (38.8%) and acute-on-chronic liver failure (35.8%), with a decreased dihydroxy / trihydroxy ratio. In vitro, porcine hepatocytes in the bioreactor cleared most conjugated bile acid species from pooled patient plasma. In conclusion, acute liver failure is associated with very high serum levels of toxic bile acids that could contribute to the pathogenesis of the syndrome. Pazzi et al. showed that bioartificial liver treatment reduces both serum bile acid concentrations and the hydrophobicity of the bile acid pool.[44]

BAL evaluation in a swine FHF patients

In a study, Sgeyuki et al. [45] used porcine hepatocytes entrapped with collagen gel for a BAL device. After 12 h. of treatment for acute liver failure in animals, the porcine hepatocytes in the BAL module were still viable and thus the period of BAL treatment can be extended. As for immunological problems, adverse effects upon liver failure patients from xeno antibodies, such as human anti porcine IgG and IgM have not been detected in patients.

Reduction effect on intracranial pressure by BAL treatment in pigs with FHF

Khalili et al. showed that FHF pigs treated with a BAL using charcoal and hepatocytes were able to maintain lower ICP (intracranial pressure) for a longer period of time when compared with an empty system or charcoal alone. Lower ICP resultw with longer survival time of the patient. ICP is critical since the major cause of death in patients with FHF is intracranial hypertension resulting brainstem damage. [46]

Future projections

There are many clinical trials going on with the utilization of several BAL devices. According to those studies, an improved model of BAL device will be public use. To make closer the time for the patients to benefit from the devices, most of the researches necessitate the more common and universal results applicable for hepatic failure.

Up to now, there are many concerns on development of optimum conditions to prolong the viability of hepatocytes. According to the studies, successful trials performed, however a living tissue possibly should have many drawbacks.

The hepatocyte source is still under investigation. The investigations on the immortalised cell lines would be a better way to compete with the drawbacks resulting with xenogenic cells and tumorogenic cells. Genetic engineering may open up a door on the studies for formation of new cell lines.

The efficiency of the bioreactor is a critical concept. The perfusion rate between the plasma and the cells changes according to the attachment style of hepatocytes. A more simple and direct contact for cells to the plasma has to be generated without allowing the immunologic reactions.

The co-factors that enhance the 3D formation of hepatocytes can be studied in vitro. An advance in proper 3D structure keeps the viability quite long.

A newer approach is the transfer of cell lines with a suitable environment in the body. This application is a promising model since it is not only improving the patient metabolism but also recovers the liver completely. However this model necessitates a good model of hepatocyte carrying conditions.