Assessment of artificial liver support technology.

0963-6897/92 $5.00 + .00 1992 Pergamon Press Ltd.

Cell Transplantation, Vol. 1, pp. 323-341, 1992 Printed in the USA. All rights reserved.

Review Article ASSESSMENT OF ARTIFICIAL LIVER SUPPORT TECHNOLOGY MARTIN

L.

YARMUSH,*t:\: JAMES

C.

Y. DUNN,:\: AND RONALD

G.

TOMPKINS:\:

*Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ 08854, USA and :j:Surgical Services, Massachusetts General Hospital and the Shriners Burns Institute, Boston, MA 02114, USA

D Abstract - Despite more tban 30 yr of researcb and de-

develop an artificial liver have been published previously (12,29,31,56,81). In this review, we will assess the in vitro and in vivo results of the various approaches to develop an artificial liver support and will discuss their shortcomings and directions for future research.

velopment, an artificial liver bas still not yet become clinical reality. Altbougb previous attempts using a multiplicity of tecbniques including bemodialysis, bemoperfusion, plasma excbange, extracorporeal perfusion, and crossbemodialysis bave sbown minor improvement in patients witb acute hepatic faDure, limited clinical trials bave faDed to demonstrate any survival benefit. Encouraged by tbe progress on techniques tbat maintain long-term cultures of bepatocytes, more recent efforts bave been directed at tbe use of bepatocytes as tbe basis of liver support. Tbis review takes a critical look at past and present concepts in tbe development of artificial liver supports and both qualitatively and quantitatively evaluates tbe advantages and disadvantages of tbe available metbodology.

BIOLOGY OF THE LIVER

A description of normal liver function and structure are presented briefly to illustrate the complexity of the liver and the difficulties one might encounter in planning the development of partial or complete liver replacement. In an idealized 70-kg man, the liver weighs 1500 g. It is a highly vascularized organ (25 to 30 milliliters per 100 grams) receiving one-fifth of the total cardiac output. This large blood flow emanates from two major blood supplies. The hepatic artery, rich in oxygen, supplies about one-quarter of the liver blood flow. The portal vein, which is the collection of venous drainages from the spleen, the pancreas, and the intestines, supplies the rest of the liver blood flow. This special arrangement illustrates the liver's crucial role in maintaining homeostasis of the systeIniccirculation. The highly variable input from the gastrointestinal tract first encounters liver (before entering the systemic circulation) and is subjected to extensive processing. The liver modifies the concentration and composition of the incoming nutrients, detoxifies xenobiotics, secretes transport proteins, synthesizes a variety of other critical proteins, excretes bile, and stores excess products. Microscopically, the liver is comprised of lobules (Fig. 1). Each lobule is made up of multiple hepatic acini. The hepatic acinus is a plate of liver cells that extends from the portal triad (containing the bile duct, the hepatic artery, and the portal vein) to the central

D Keywords - Artificial liver; Hepatocyte transplantation; Liver support device. INTRODUCTION

Artificial organs are designed to replace essential organ functions. In renal failure, dialysis has been adopted as the standard medical treatment as either a temporary or permanent support regimen. However, this is not the case for liver failure. Although efforts to develop a liver support system date back over 30 yr, no standard process currently exists to replace liver function. This is partly due to the complexity of the liver. Unlike the kidney, which functions primarily as a filter, the liver performs a variety of anabolic, catabolic, detoxifying, secretory, and excretory functions. It can be viewed as a complex bioreactor within which hundreds of precisely coordinated chemical reactions occur. The design of an artificial liver needs to capture the essential chemical reactions and to regulate the reactions in accordance to physiological stimuli. Further complicating the design of an artificial liver is the lack of knowledge regarding which liver functions are indeed essential for survival. Historical reviews of efforts to ACCEPTED

[To whom correspondence should be addressed.

6/30/92. 323

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Fig. 1. Schematic drawing of the liver lobules. The portal triads are located at the corners of the lobule. The sinusoids interconnect the portal venules and hepatic arterioles to the central vein.

vein. The plates are mostly comprised of- single-cell layers of hepatocytes, the major parenchymal cells of the liver, lined on either side with sinusoidal cells. Between adjacent hepatocytes lies the bile canaliculus that serves as a channel for bile excretion. The bile canaliculus is considered to be the hepatocyte's apical surface, and the apicolateral surface has typical epithelial specializations, such as intercellular junctions. The bile canaliculus surrounds each hepatocytes and divides the remaining cell surface into two basolateral domains, each in close proximity to the blood supply. Surrounding the plates of cells that form the liver parenchyma are the sinusoids, which are grossly enlarged capillary lumens that interconnect the blood supply and the blood drainage. Lining the sinusoids are fenestrated endothelial cells, phagocytic Kupffer cells, fat-storing Ito cells, and other undefined mesenchymal cells. The space of Disse is a histologic designation that refers to the extracellular space between hepatocytes and sinusoidal cells. It is loosely filled with extracellular matrix products including collagen and fibronectin (51).

Hepatocytes are large multifaceted cells measuring roughly 25 ~m on each side. There is an estimated 250 billion hepatocytes in the adult liver. Their cytoplasm contains an abundance of smooth and rough endoplasmic reticulum, ribosomes, Golgi apparatus, lysosomes, and mitochondria. Hepatocytes are involved in carbohydrate metabolism (e.g., glycogenolysis and gluconeogenesis), in fat and lipid metabolism (e.g., synthesis of lipoproteins and cholesterol), and in amino acid metabolism and protein production (e.g., synthesis and secretion of serum albumin, transferrin, and clotting factors). Hepatocytes are also active in detoxification and transform toxins and hormones into water-soluble compounds for renal and biliary excretion, primarily by cytochrome P-450 oxidation and glucuronyl or sulfate conjugation. Hepatocytes also serve an excretory function by producing bile, which is composed of bile salts and other conjugated products. In addition to these functions, hepatocytes store large pools of many essential nutrients, such as retinol, folic acid, and cobalamin. Selected quantitative and qualitativeaspectsof the liverare summarizedin Table 1.

Artificial liver support. M.L. YARMUSH

Table 1. Selected quantitative aspects and functions of the human liver* Hepatocytes 250 X 109 cells 25 /Lm in diameter 60% of the total hepatic cells 750/0 of the total hepatic volume carbohydrate, amino acid, and lipid metabolism plasma protein and clotting factor syntheis detoxification bile excretion Sinusoidal cells 6.5% of the total hepatic volume endothelial cells with 0.1-0.2 /Lm fenestrations Kupffer cells fat-storing cells Extracellular space 25-30 mL blood/100 g of liver sinusoid volume: 12% of the total hepatic volume sinusoid lumen width: 7-15 /Lm sinusoid pressure: 2-3 mm Hg Dissespace volume: 6% of the total hepatic volume *Data taken from The liver: Biology and pathobiology, Arias, I.M.; Popper, H.; Schachter, D.; Shafritz, D.A., eds. New York: Raven Press; 1982.

In view of the liver's complexity, it is clear that replacement of all liver functions is a formidable task. Yet, it may not be necessary to replace all liver functions to temporarily support patients suffering from liver failure. An analogy can be made to the artificial kidney: in hemodialysis, the filtration process replaces the primary function of the kidney but does not completely replace all kidney functions e.g., the secretion of erythropoietin. Unfortunately it is not clearly known which liver functions are critical for survival, but some clues might be available. THE CLINICAL PROBLEM

The liver is normally capable of rapid and complete regeneration after tissue damage. When the regenerative process is compromised and the residual functional capacity of the diseased liver is barely capable to sustain life, liver failure ensues. In 1988, more than 30,000 people died as a result of liver disease in the United States (Table 2). This occurs principally in two clinical settings: cirrhosis and fulminant hepatic failure. Liver cirrhosis, an irreversible, end-stage liver disease whereby fibrotic tissue gradually replaces liver tissue as a result of chronic injury, accounts for more than half of the mortality from liver disease. Fibrosis physically confines the remaining liver cells such that

325

ET AL.

Table 2. United States mortality for hepatic disease* Etiology

Number

Male

Female

Cirrhosis without alcohol Alcoholic cirrhosis Alcoholic liver damage Alcoholic fatty liver Chronic hepatitis Acute alcoholic hepatitis Hepatorenal syndrome Hepatitis Hepatic coma Biliary cirrhosis Necrosis of liver Portal hypertension Abscess of liver Others Total

12,409 7,883 2,255 915 812 739 630 498 456

7,625 5,707 1,651

4,784 2,176 604 251 435 231 221 247 173 332 180 62 76 795

393

383 163 160 2,005 32,283

664

377 508 409 251 283 61 203 101 84 1,210 20,640

11,643

*Adapted from Vital Statistics of the United States, 1988, Volume II, Part A, Table 1-23. US Department of Health and Human Services.

complete regeneration is no longer possible. Common etiology of cirrhosis includes alcoholism and chronic hepatitis. Fulminant hepatic failure is a highly lethal disease with a mortality rate of greater than 800/0. Common etiologies are chemical and viral hepatitis. The sudden onset and rapid progression of the disease can lead to death in a few weeks. The regenerative process simply cannot keep pace with the massive destruction of liver cells. Clinical evidence of liver dysfunction includes increased serum liver enzymes, ammonia, and bilirubin and decreased serum albumin and clotting factors. Both chronic and acute liver failure result in life-threatening complications including portal hypertension, variceal bleeding, ascites, and hepatic encephalopathy. In cirrhosis, the increased vascular resistance results in increased pressure of the portal system, which contributes toward variceal bleeding and ascites formation. Various palliative therapies such as shunting procedures, variceal sclerosis, and diuretics are employed to minimize complications. Hepatic encephalopathy is thought to occur from the toxin buildup due to the loss of liver's detoxifying function and the diversion of portal blood flow, but its exact cause remains unknown. Hepatic encephalopathy is often precipitated by episodes of bleeding and infection. Progression to deep coma is an ominous sign of impending death. Clinical intensive management of liver failure includes fluid and hemodynamic support, correction of electrolyte and acid-base abnormalities, administration of lactulose, antacids, and cimetidine, respiratory as-

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sist, and treatment of brain edema. Despite such aggressive therapy, the mortality rate of liver failure remains high. Currently, the only satisfactory longterm solution to these liver diseases is transplantation. In 1989, 2160 liver transplant operations were performed in the United States. Liver transplantation today has a high success rate with a 5-yr survival rate averaging around 80070 (50,77). Although widely accepted as the best therapy, liver transplantation suffers from many limitations. For example, there are many contraindications to transplantation, including advanced cardiac disease, severe pulmonary disease, and extrahepatic malignancy and infection. Yet the most serious limitation to transplantation is donor scarcity. In spite of the tremendous growth of liver transplant centers, the establishment of organ-sharing network, and increased public awareness and physician education programs, an estimated 3,000 to 5,000 potential recipients die annually as a result of organ scarcity (88). Moreover, this large deficit is increasing, as the indication for liver transplantation broadens (14). Even if new modalities such as segmental transplants and living-related transplants become acceptable practice, it is unlikely that the additional supply of organs will meet the increasing demand. In contrast to potential kidney recipients, patients in liver failure do not have the viable alternative of dialysis until an organ becomes available. Instead, they must face the progressive, deteriorating nature of their disease. Therefore, there is a critical need for techniques which provide both short-term and long-term liver support. APPROACHES TO LIVER FUNCTION REPLACEMENT

The clinical features of liver failure indicate that all aspects of liver function are compromised. The immediate cause of mortality is often related to complications of portal hypertension and hepatic encephalopathy. Given that replacing all liver function by artificial means would be very difficult if not impossible, early investigators chose to focus on replacing singular liver functions such as the liver's detoxifying ability. Approaches which were investigated with this in mind included hemodialysis, hemoperfusion, and perfusion with immobilized enzymes. The goal of these modalities was primarily to remove toxin buildup, which is thought to be the culprit of hepatic encephalopathy. Other approaches which attempted to replace most or all liver functions have also been tried. These include exchange transfusion, plasma exchange, crosscirculation, extracorporeal perfusion, crosshemodialysis, extracorporeal bioreactor, and cell transplantation. The evaluation of a liver support system should start with the demonstration of its functions in vitro, fol-

lowed by device testing in animal models, and finally clinical trials in human subjects. The in vitro functions should be quantitatively measured to allow for the estimation of the support system's efficiency. The animal model used for in vivo testing of the device should closely resemble the natural disease. The clinical trial should be well-controlled and randomized to avoid the observer bias. The liver support systemsthat have been clinicallyevaluated are summarized in Table 3. Unfortunately, the results of most efforts have been inconclusive due to unsatisfactory study design. In addition, these approaches have potential technical limitations. A listing of these shortcomings is provided in Table 4. Hemodialysis Hemodialysis was employed to remove toxins thought to cause hepatic encephalopathy in fulminant hepatic failure as early as 1958 (39). The putative toxins include ammonia, false neurotransmitters, phenols, aromatic amino acids, and undefined substances with molecular weight less than 5,000 (10). These toxins are often water insoluble and are primarily protein bound. Since traditional hemodialysis utilizes cellulosic membranes that are impervious to large-size molecules, polyacrylonitrile membranes permeable to molecules up to 15,000daltons have been used for the treatment of hepatic encephalopathy (64,76). Many anecdotal reports of successful treatment of a few patients by hemodialysis exist in the literature; however, well-controlled studies are rare. In one study with successive patients with fulminant hepatic failure, 20 out of 65 (31 %) patients survived after receiving repeated polyacrylonitrile membrane dialysis, compared to the survival of 8 out of 53 (15%) patients who received conventional supportive therapy. Although the in vitro capability of hemodialysis is well characterized, there have been few studies that demonstrate its effectiveness in animal models of liver failure ..The efficacy of hemodialysis as an artificial liver support needs to be better documented by a careful evaluation of its use in animal models of liver failure. Results from the limited clinical trials showed some beneficial effect of polyacrylonitrile membrane dialysis in the treatment of fulminant hepatic failure. It is unclear whether the benefit was the result of the removal of larger molecules allowed by the polyacrylonitrile membrane or the connection of the electrolyte balance by dialysis, since the effectiveness of conventional hemodialysis in treating these patients was not evaluated. Future clinical trials with polyacrylonitrile membrane dialysis need to incorporate into the study design an additional control group who are treated by conventional hemodialysis alone. The overall survival rate in patients with fulminant

Artificial liver support. M.L.

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YARMUSH ET AL.

Table 3. Representative clinical trials of artificial liver support Survival rate] Method Polyacrylonitrile dialysis Charcoal hemoperfusion

Plasma exchange Extracorporeal perfusion Cross hemodialysis Hepatocyte hemoperfusion

Patient* population

Treatment

FHF4 FHF4 FHF4 FHF3 FHF4 FHF3 FHF4 FHF4 FHF FHF, Cirrhosis FHF FHF 4, Cirrhosis FHF, Cirrhosis

21 (5/24) 31 (20/65) 24 (17/71) 65 (20/31) 20 (9/45) 51 (38/75) 35 (10/29) 50 (5/10) 34 (15/45) 23 (3/13) 57 (13/23) 27 (3/11) 63 (27/59)

Control

Neurologict improvement 71 (17/24)

15 (8/53) 15 (8/53)

Reference Opolon, 1976 Silk, 1978 Silk, 1978 Gimson, 1982 O'Grady, 1988

39 (13/33) 14 (5/35) 69 (9/13) 45 (5/11) 41 (27/67)

Inoue, 1981 Yamazaki, 1988 Tung, 1980 Lie, 1990 Ozawa, 1982 Margulis, 1989

*FHF = fulminant hepatic failure. The number refers to the grade of hepatic coma. tSurvival rates are expressed in percentages. Actual numbers are given in parentheses. tlmprovement rates are expressed in percentages of the treatment group. Actual numbers are given in parentheses.

hepatic failure treated by polyacrylonitrile membrane dialysis was still quite low. Two possible explanations for these poor results are: 1. the liver detoxifying functions are not adequately replaced, and 2. other essential liver functions are not replaced. Several investigators have speculated that better results might be achieved with more aggressive removal of molecules that are protein bound. This notion led to the advance of the other major form of mechanical liver support, hemoperfusion.

Hemoperfusion In hemoperfusion, a patient's blood is perfused over a solid support such as activated charcoal or anion exchange resin (32,36). Early studies with charcoal hemoperfusion demonstrated the ability of charcoal hemoperfusion to reverse hepatic encephalopathy (10). However, these studies were complicated by problems of biocompatibility: activated charcoal perfusion resulted in platelet loss and induced hypotensive reactions. These problems have been resolved by the application of coatings to the adsorbent and the administration of prostacyclin as a protective agent for platelets (25,38). Furthermore, it was thought that hemoperfusion should begin before the development of severe (grade 4) hepatic encephalopathy. In 1982, a clinical trial of 76 fulminant hepatic failure patients who received5 h of hemoperfusion daily demonstrated a higher overall survival rate for patients who received

charcoal hemoperfusion earlier in their illness (65% for grade 3 and 20070 for grade 4) (26). Unfortunately, like many other clinical reports of hemoperfusion, this study lacked a control group with no treatment. The difference in the survival rates of the two groups may simply reflect the difference in disease severity. In 1988, a clinical trial from the same center reported that: Table 4. Disadvantages of artificial liver support systems Method Polyacrylonitrile dialysis

Charcoal hemoperfusion

Immobilized enzymes Plasma exchange

Cross circulation Extracorporeal perfusion

Cross hemodialysis

Hepatocyte hemoperfusion

Associated problems Ineffective removal of large molecules, no replacement of synthetic functions Uncontrolled removal of molecules, no replacement of synthetic function Availability of only a subset of enzymes Large amount of plasma necessary, certain functions still not replaced Donor toxicity Deterioration of donor liver, limitation of animal source Complex procedure, ineffective in those with preexisting liver disease Unstable hepatocytes

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1. in 75 patients with grade 3 encephalopathy, the

overall survival rates were 51% and 50070 for patients who received 5 and 10 h of hemoperfusion daily, respectively, and 2. in 62 patients with grade 4 encephalopathy, the overall survival rates were 39% and 35% for patients who received 0 and 10 h of hemoperfusion daily (63). This demonstrates that no survival benefit results from hemoperfusion for grade 4 encephalopathy, and that no additional survival benefit occurs from prolonged hemoperfusion for grade 3 encephalopathy. However, due to the lack of a no treatment group, it was still unclear whether any hemoperfusion was of any survival benefit for grade 3 encephalopathy, especially in view of the reported high survival rate for the untreated patients with grade 4 encephalopathy. Both in vitro and animal studies have demonstrated the efficacy of charcoal hemoperfusion. Clinical trials of charcoal hemoperfusion demonstrated consistent temporary improvement of hepatic encephalopathy in patient with fulminant hepatic failure; however, an increased survival rate could not be clearly demonstrated. Reports which suggested survival benefit as a result of hemoperfusion were mostly anecdotal or uncontrolled. The interpretation of these clinical reports was further complicated by evolving supportive management, which had improved the survival rate of certain subgroups. For example, with only intensive care support, the survival rate for acetaminophen-induced fulminant hepatic failure was 56% compared to 25% for viral-induced failure (63). These results suggest that the survival rate of fulminant hepatic failure varied according to the underlying etiology. Future clinical trials need to report the results segregated by the etiology of hepatic failure, and, most importantly, the study design must include a parallel control group to allow proper interpretation. The more aggressiveremoval of the larger molecules by charcoal hemoperfusion did not result in additional benefits. The limited success of charcoal hemoperfusion may be the result of its inherently nonspecific approach. Charcoal hemoperfusion removes toxins without clearly identifying what is indeed removed. Consequently, it is possible that major toxins are not sufficiently removed (or not removed at all), while salutary blood factors may be adsorbed along with the toxins. Ideally, one would like to detoxify in the same manner as that performed by normal liver. The development of immobilized enzyme systems that more closely mimic the natural detoxifying functions of hepatocytes is the next logical step and is described in the following section.

Immobilized Enzyme Systems In immobilized enzyme systems, blood is perfused over liver enzymes that are either linked to an insoluble substrate or encapsulated in artificial cells. Presumably this method removes toxins more efficiently and specifically than hemoperfusion. Many liver enzymes have been successfully immobilized, including urease, tyrosinase, L-asparaginase, glutaminase, and UDP-glucuronyl transferase (7,11,73). Much in vitro evidence supports the efficacy of such systems. These systems now await in vivo experimentation to demonstrate their usefulness. There are several potential limitations of the immobilized enzyme systems. These include the difficulty with inactivation of enzymes in vivo, the problem of host protein and platelet depositions on the contacting surface, and the lack of a complete set of functional liver enzymes. It is generally accepted that efforts to remove toxins have some beneficial effect in temporarily improving mental function of patients in hepatic encephalopathy. Although this represents a step forward in the treatment of fulminant hepatic failure, the overall success is extremely limited. While this may be due to the inability of the current systems to effectively remove the offending toxins, the major barrier of this approach may be the inability of the system to perform other aspects of liver function. Although toxin buildup is an important determinant of complications associated with fulminant hepatic failure, other aspects of liver function such as metabolism and synthesis are likely involved as well. Furthermore, different etiologic groups of acute fulminant failure are associated with different survival rates, thereby suggesting that varying degree of support beyond simple detoxification may be needed depending upon the extent of hepatic failure. Since it is unknown which functions are essential in a particular case, and that detoxifying supports appear to be inadequate, many have begun to focus their approach of liver support to methods which encompass a wider range of the liver's functional repertoire. Exchange Transfusion and Plasma Exchange The majority of liver function is ultimately manifested in the composition of the systemic circulation. Potentially by exchanging the blood volume of a patient in liver failure, one could reduce the levelof toxins and replace the deficient factors in patient's circulation. Anecdotal reports on the use of exchange transfusion in hepatic encephalopathyshowedthat temporary improvement of neurological status and biochemical markers could be achieved (8), but increased survival was not consistently observed. With the advent of blood component therapy, in-


vestigators have replaced exchange transfusion with plasma exchange so that formed elements in the blood are not needlessly discarded. Initially plasma exchange was performed in batch where patient's blood was repeatedly withdrawn and the separated cells, together with donor fresh frozen plasma, were infused after each phlebotomy (70). This laborious technique was replaced by plasmapheresis so that a continuous exchange became possible. Similar to exchange transfusion, clinical reports of plasmapheresis demonstrated biochemical and neurological improvement in patients with fulminant hepatic failure; however, increased survival was, at best, marginal. In a recent study of 80 patients with fulminant hepatic failure, 45 were treated with plasma exchange in the amount of 5000 mL daily until they fully recovered or died (89). The survival rate for the treated group was 34010, while that for the untreated group was 14%. Unfortunately, this study was not randomized, and the patient population was not controlled with respect to the underlying etiology or disease severity. Future clinical trials need to incorporate these considerations into the study design. The overall survival rate of plasma exchange therapy for the treatment of fulminant hepatic failure is still low. This may be attributed to insufficient exchange and procedure toxicity. The most important parameter in plasma exchange is the amount of plasma exchanged in a given period. Although a continuous plasma exchange will most closely replace liver function, the large amount of donor plasma is associated with increased risk of chemical toxicity and viral contamination. The balance of these opposing factors results in a compromise that leaves patients partially treated by plasma exchange. Practically speaking, the large amount of plasma required severely limits the usefulness of this approach as an artificial liver support. It has been shown, however, that plasma exchange may be beneficial in the perioperative period before liver transplantation to correct severe coagulopathy (60). Cross Circulation In this technique, a patient's circulation is tied to that of a human donor. Human cross circulation was reported to be successful in the treatment of one out of three patients with acute liver failure (8). In every case, the donor suffered from significant adverse reaction during the procedure. The potential toxicity to the donor severely limits this approach. It is doubtful that any additional clinical trial is warranted at this time. Extracorporeal Perfusion and Crosshemodialysis In extracorporeal perfusion, patient's blood is perfused through a xenogeneic liver (58). Porcine livers

YARMUSH ET AL.

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were first used in clinical trials for the treatment of grade 4 encephalopathy from fulminant hepatic failure. The results demonstrated improvement of encephalopathy after 6 h perfusion, but patient survival was unchanged (23,72). More recently, a clinical trial of 99 baboon liver perfusions for the treatment of 23 patients in fulminant hepatic failure showed long-term survival in 13 of these patients (45). The improved survival rate was thought to be secondary to the longer perfusion time possible with baboon livers, each lasting approximately a day. The study also suggests that a xenogeneic source of liver cells might improve liver function on a short term basis. Although the result is encouraging, the lack of a concurrent control group makes its interpretation difficult. The procedure is far from ideal because the perfused liver is unstable and degrades significantly over a relative short period of time. Whether this is due to technical failure of the procedure or the inflammatory reaction to the xenogeneic liver remains unclear. This technique, however, is not very feasible because baboon livers have become increasingly scarce. As an attempt to better preserve the xenogeneic liver, extracorporeal perfusion was combined with hemodialysis (65). The extracorporealliver was perfused with blood that was dialyzed against recipient's blood across a Cuprophan membrane. The perfusion pressure, oxygenation, temperature, pH, and insulin level were controlled in the extracorporeal circuit. Experimental study employing pig livers and partial hepatectomized rabbits as recipients demonstrated that the adenylate energy charge of the extracorporealliver [defined as (ATP+0.5ADP)/(ATP+ADP+AMP)], was better preserved by crosshemodialysis (66). The adenylate energy charge is a reflection of the state of the oxidative phosphorylation pathway. A clinical trial utilizing this technique for 11 patients in grade 4 hepatic coma reported a mental status improvement rate of 45% and an overall survival rate of 27%. The duration of each crosshemodialysis was, at most, 8 h, and the adenylate energy charge levelin the liver circuit remained high during the procedure. These investigators also found that the response to crosshemodialysis correlated with an increase in the adenylate energy charge level in the recipient's circulation. The level of adenylate energy charge appeared to correlate with metabolic function of the liver. These results suggest that adequate oxygenation of the extracorporealliver is of critical importance. Although the extracorporeal liver could be better maintained by crosshemodialysis, the membrane that separates the two circulations may pose transport limitations. In particular, the Cuprophan membrane employed in crosshemodialysis would prevent the transport


330

of molecules with molecular weight greater than 5,000. To take full advantage of the functions of the liver, one needs to use a membrane with sufficiently large pores to allow the passage of large molecules such as lipoproteins and clotting factors.

Liver Tissue Hemoperfusion To simplify extracorporeal perfusion, attempts were made to use liver slices or cubes that were fresh, frozen, or freeze-dried (37,40). The liver pieces were enclosed within a stirred-tank reactor and were in direct contact with recipient's blood. These systems were shown to be effective in lowering toxin concentrations and in producing synthetic factors in vitro and in animals for a few hours. Frozen or freeze-dried liver pieces were functionally comparable to fresh liver pieces. Therefore, no viable hepatocytes were needed to produce the observed effect. The frozen or freezedried liver pieces probably contained some active enzymes that functioned for a short period of time. The result further established that the fresh liver pieces were most likely functioning inefficiently, since they performed no better than the dead liver cells. This is not surprising, given that the normal vasculature of the liver is not utilized so that only the cells near the surface are adequately oxygenated. Within the liver pieces, mass transfer occurs only by diffusion, which significantly impairs oxygen delivery. This approach, at best, could provide only limited functional support on a short-term basis. HEPATOCYTE SYSTEMS

Since a full complement of cellular function may be necessary for treatment of liver failure, many investigators have focused on the use of isolated hepatocytes as the basis for artificial liver support. Isolated cells are more amenable to in vitro manipulations; thus, issues such as mass transfer limitation, cryopreservation, and immunological protection can be more easily addressed. Isolated liver cells have been used in a variety of configurations: suspended, substrate-attached,

and encapsulated. These applications of hepatocytes in liver support schemes can be divided into two categories: extracorporeal hepatocyte bioreactors and implantable hepatocyte systems. There have been few clinical experiences with hepatocyte systems. Most of the experimental results are limited to in vitro testing of cell functions for hepatocyte bioreactors (Table 5) and animal testing for implantable hepatocyte systems (Table 6). Insoluble substrates provide a surface for the attachment of hepatocytes, Microcarriers are often employed in mammalian cultures because of their easein handling and capability for scaleup. Typically microcarriers are spheres with diameter of about 200 J.tm, or roughly 10cell diameters. Because of their size, rnicrocarriers provide a large surface area for cellular attachment within a small overall volume. Types of microcarriers available include dextran linked to Type I collagen (Cytodex 3) and fibronectin-coated Biosilon beads (1,74). Under adequate oxygenation and gentle agitation, a concentrated suspension of hepatocytes will form a nearly confluent monolayer of cells on the surface of the microcarriers. However, the microcarrier approach suffers from several drawbacks, including gradients in nutrients and the space between the beads, which represents a significant dead volume. Alginate is commonly employed for encapsulation because it involves a gentle entrapment procedure. Spheres from 300 J.tm are formed by dropping a mixture of hepatocytes and sodium alginate at roughly 107 cells/mL into a solution of calcium chloride. Hepatocytes entrapped in alginate were shown to detoxify and to synthesize proteins in vitro (57,83). The encapsulated cells were immobilized by the alginate such that there was little cell-cell contact. A consequence of the encapsulation methodology is that cells near the center of the capsule may be under hypoxic conditions. A variation that allows for the entrapped hepatocytes to freely float within the encapsulation is the formation of an alginate-polylysine-alginate outer membrane and subsequent dissolution of the inner

Table 5. Extracorporeal hepatocyte systems Method

In Vitro functions

Hepatocytes suspension Microcarrier-attached hepatocytes Encapsulated hepatocytes Hollow fiber Flat plate

Ureogenesis gluconeogenesis Albumin, fibronectin, bilirubin conjugation Ureogenesis, albumin Ureogenesis, cytidine deamination Albumin, ureogenesis, gluconeogenesis

Days of observation

Reference

1 10 4 20 14

Matsumura, 1987 Shnyra, 1990 Sun, 1986 Hager, 1978 Uchino, 1988


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YARMUSH ET AL.

Table 6. Implantable hepatocyte systems Survival rate] Method Hepatocyte suspension

Microcarrier-attached hepatocytes Biomatrix Encapsulated hepatocytes

Animal! AHF model*

Number/ site

Rats Toxic Rats Toxic Rats Anoxic Rats Toxic Rats Surgical Rats Toxic Rats Toxic

2 grams syngeneic Intraperitoneal 1.5 grams syngeneic Portal venous 40 x 106 syngeneic Intraperitoneal 40 x 106 xenogeneic Intraperitoneal 10 x 106 syngeneic Intraperitoneal 20 x 106 syngeneic Intraperitoneal 20 x 106 syngeneic Intraperitoneal

Treatment

Control

Reference

63

6

Sutherland, 1977

71

17

79

38

71

14

40

o

63

35

Saito, 1987

62

29

Cai,1988

Makowka, 1980

Demetriou, 1988

*AHF = acute hepatic failure. tSurvival rate is expressed in percentages.

core of alginate (79). After dissolution of the inner core of alginate, the freely floating hepatocytes encapsulated in the alginate-polylysine-alginate membrane formed aggregates that were generally attached to the inner surface of the membrane. The formation of aggregates has been observed in other configurations of long-term hepatocyte cultures (e.g., cells cultured on Matrigel). This may be the basis for the improved cellular function. The additional diffusional barrier of the encapsulating membrane can be a potential problem; depending on the chosen pore size, transport limitations of large molecular weight molecules may be critically compromised. The disposition of products normally excreted through the bile canaliculus is often not addressed in the development of a bioartificialliver, in part because of the difficulty in reconstructing the biliary tree from a suspension of hepatocytes. Bile products will most likely accumulate in the circulation. This is not a lifethreatening problem, but the accumulation does cause jaundice, pruritus, and decreased ability to digest fat. Conjugated products by the bioartificialliver can be partly excreted through the kidney and partly excreted through the existing biliary tree via uptake by the host hepatocytes. If this proves to be insufficient, resins that bind anions effectively can be incorporated into the liver support device.

Extracorporeal hepatocyte bioreactors One approach to a hepatocyte-based support is to design extracorporeal bioreactors that contain func-

tional hepatocytes. This bioartificial device would support liver functions through an external circuit that exchanges plasma components with the patient. Several variations of hepatocyte bioreactors have been developed. The hepatocytes may be: 1. 2. 3. 4. 5.

in cell suspensions, attached to substrates such as microcarriers, packed within or surrounding hollow fibers, applied to flat plates, or encapsulated in hydrogels.

These devices might be used in a hemodialysis format where patient blood would be separated from the hepatocytes by a semipermeable membrane, or they might be directly perfused with patient plasma or blood. In comparison to implantable systems, extracorporeal bioreactors have some distinct advantages. First, bioreactors allow more control of the cellular environment, particularly with respect to transport considerations. For example, oxygen delivery and macromolecular exchange can be engineered rather than relying on the characteristics of the peritoneal membrane and the surrounding milieu. Second, the timing and the duration of support can be decided by the clinicians. Unlike implantable systems which may require time for vascularization and become intricately intertwined with the patient, bioreactor systems can be started at full functional capacity and be stopped any time. Third, bioreactors can potentially be stored by a wide variety of techniques including

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1. cryopreservation and 2. hypothermic or normothermic perfusion. Fourth, immunological rejection is less problematic because of the potential to separate recipient's lymphocytes away from the exogenous hepatocytes by plasmapheresis. Disadvantages of an extracorporeal system include the need for vascular access and the associated potential for thromboembolic complications. Moreover, because hepatocytes may require certain factors present in the portal system, established methods of vascular access for hemodialysis may be less effective (i.e., portal-systemic shunting operations may be necessary in some patients). Membrane bioreactors. Early efforts to develop hepatocyte bioreactors employed hollow fibers (29). In these units, hepatocytes were attached to the outer surface of the fibers, and blood or plasma perfused the fiber lumen. In vitro demonstration of such units has existed for more than 10 yr, but there are very few reports of its use on animal models. More recently, preliminary reports have described the use of cryopreserved, microcarrier-attached hepatocytes placed outside of the hollow fibers. This device was shown to produce conjugated bilirubin in Gunn rats on a shortterm basis (2). The usefulness of such a bioreactor in the treatment of liver failure remains to be more thoroughly evaluated. A clinical study of bioreactors based on hepatocyte suspension in a modified commercial hemodialysis unit with a replaced dialysis membrane that allowed the passage of larger molecules was reported (52). A patient in hepatic failure secondary to bile ductal carcinoma underwent hepadialysis, whereby recipient blood was dialyzed against a flowing suspension of cryopreserved rabbit hepatocytes. A reduction in serum level of bilirubin and an improvement of mental status were observed in this patient after hepadialysis. However, because this represented an isolated case, no definite conclusions can be drawn from this experience. Perfusion bioreactors. Hemoperfusion with encapsulated hepatocytes and microcarrier-attached hepatocytes is also under development. Alginate films with encapsulated hepatocytes have been incorporated into a rotating disk reactor (90). Short-term hemoperfusion of this bioreactor in cats with surgically induced liver ischemia demonstrated the reversal of ammonia accumulation. This device, however, was unable to provide sustained functional support. Hemoperfusion of microcarrier-attached hepatocytes was also tested in animal models of acute liver failure (75). A single 3-h

hemoperfusion of a column containing microcarrierattached hepatocytes was performed 1 day after the administration of carbon tetrachloride and D-galactosamine. Both the biochemical markers and the survival rate were significantly improved in the treatment group compared to control group with hemoperfusion of empty microcarriers. Given the problems encountered with hepatocytes transplanted into similar animal models of liver failure, it is difficult to interpret these results without controls using dead hepatocytes or splenocytes. It remains unclear whether this device provided functional support or acted by some unknown mechanism. Further experiments with additional controls and surgically induced models of liver failure are necessary to address these questions. Stacked flat plates represent another potential bioreactor configuration. Hepatocytes attached to flat plates form a monolayer of cells similar to classical in vitro cultures of hepatocytes. A large-scale bioreactor based on this design was tested in dogs with total hepatectomy (85). Canine hepatocytes attached to collagencoated borosilicated glass were perfused with recipient plasma continuously. Compared to the controls with no treatment or with plasma exchange, anhepatic dogs treated by multi plated bioreactors survived significantly longer and maintained a low level of serum ammonia. The hepatocyte monolayers appeared to be intact after more than 2 days of continuous in vivo support. Although all animals in this study died in the experiment, this study provides some encouragement for the use of hepatocytes in monolayer for functional support. A clinical study of a hemoperfusion of a capsule containing swine hepatocytes and activated charcoal was reported (49). A group of 59 patients with acute hepatic insufficiency underwent hemoperfusion of this device through an arteriovenous fistula. The mortality rate in this group was 370/0 compared to 59% in the control group, with 67 patients who received only intensive supportive care. Unfortunately, the results are difficult to interpret because the study appears not to have been randomized (i.e., the distributions of hepatic failure etiologies were considerably different in the two groups). In addition, the relative contribution of hepatocytes to the overall outcome is hard to evaluate because the device also contained activated charcoal. There was no treatment group that received only the activated charcoal hemoperfusion in this study. Implantable Hepatocyte Systems An alternative approach to an ex vivo hepatocytebased support is to implant functional hepatocytes into the patient. Similar to the bioreactors, the implanted hepatocytes may be:


1. in cell suspension, 2. attached to substrates such as microcarriers, and 3. encapsulated in hydrogels. An implantable system offers several advantages. First, the implantation procedure is simple if one can inject hepatocytes into the recipient. This potentially spares critically ill patients from major operations. Second, transplanted cells can take advantage of the natural environment provided by the host. For example, transplanted cells may be able to increase cell mass by responding to the regenerative signals in vivo. Third, recipients of cell transplantation may need no chronic care if initial implants successfully support liver functions long term. The disadvantages of cellular transplantation may include significant transport limitation from the lack of vascular supply and the lack of control on the cellular microenvironment after implantation. It should be noted that efforts to vascularize implants may take excessive time for acutely ill patients. Hepatocyte transplantation. Initial effort with hepatocyte transplantation employed a suspension of hepatocytes injected into the peritoneal cavity, the spleen, and the liver (55,80). The majority of work involved the treatment of animal models of acute liver failure with intraperitoneal or intrasplenic injection of hepatocytes. Although intraperitoneal injection of syngeneic, allogeneic, and xenogeneic hepatocytes improved survival rates in toxin-induced liver failure, the significance of the results was questioned when it became clear that the transplanted hepatocytes in each case could not be located in the recipient afterwards (48). In fact, when dead hepatocytes or bone marrows cells were transplanted into the same animal model of liver failure, similar improvement in survival rate was observed (47). Intrasplenic transplantation of hepatocytes also suffered from the same problem of nonspecificity. Splenocytes were found to be equally effective as hepatocytes when injected into the spleen for the treatment of acute liver failure, and only a small fraction of the transplanted hepatocytes remained in the spleen after a few days (18). Although the remaining transplanted hepatocytes were observed to proliferate in the spleen, this process took months, and was unlikely to be responsible for the observed increase in survival. The experience with hepatocyte transplantation yields valuable lessons. A significant portion of the improvement in survival of animals with induced liver failure appears to be related to nonspecific effects of cell transplantation rather than additional metabolic support provided by the transplanted hepatocytes. The

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underlying mechanism of the observed improvement is unclear, although some speculate that the effect is related to the stimulation of liver regeneration by the injected cells. In addition to demonstrating the importance of using extensive controls in such animal experiments, the results also show the inadequacy of the current animal models of liver failure. A convincing demonstration of a successful hepatocyte system must examine the fate and function of the hepatocytes in addition to the overall mortality. An ideal animal model should demonstrate that the survival of the animal is entirely dependent on the liver support device; i.e., the liver function of the animal is essentially being carried out by the exogenous source of functional hepatocytes. In an effort to improve the survival of transplanted hepatocytes, investigators have used attachment substrates. Since hepatocytes are anchorage-dependent cells, it is believed that the survival of transplanted hepatocytes could be improved if they are first attached to an insoluble substrate. The configurations used include microcarriers, fiber networks, and encapsulation. A brief review of each approach will be discussed below highlighting their merits and shortcomings. Substrate-attached implants. Microcarriers were initially developed for the needs of anchorage-dependent cells. Investigators have used that when microcarrierattached hepatocytes are injected intraperitoneally, genetic abnormalities such as glucuronyl transferase deficiency (Gunn rat) and analburninernia (Nagase rat) can be reversed (16). Although these results demonstrate exogenous function presumably performed by the transplanted cell mass, histological examination of the intraperitoneal microcarrier aggregate shows that few, if any, transplanted hepatocytes actually survive (30). The use of transplanting microcarrier-attached hepatocytes in the treatment of liver failure was also evaluated. When microcarrier-attached hepatocytes were transplanted into rats receiving 90070 partial hepatectomy, no improvement in survival was observed if the microcarriers were implanted after the partial hepatectomy was performed (17). Interestingly, some survival benefit was observed if the microcarriers were implanted 3 days prior to the partial hepatectomy. The investigators hypothesized that it was necessary to allow time for the vascularization of the implanted microcarriers to occur. These studies, however, are somewhat problematic from several viewpoints. First, it was reported that 90% partial hepatectomy resulted in 100% mortality. This contradicts demonstrations by earlier studies that reversible liver regeneration is possible after similar procedures (91). Second, the amount of transplanted cell mass on the microcarriers was less

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than 2070 of the normal liver cell mass. It seems unlikely that this small increment could drastically change the course of liver failure. The margin of error in the amount of liver resected is at least as much as the transplanted cell mass. Third, there is no convincing evidence that the transplanted hepatocytes survived long term. Thus, it remains unclear whether this technique provides sufficient functional cell mass for the support of liver failure. The fate of the microcarrier-attached hepatocytes raises further questions. Microcarriers are meant to provide surfaces for cellular attachment, which is thought to be essential for hepatocyte function. To date, there is lack of experimental evidence that suggests intraperitoneal microcarrier-attached hepatocytes survive. There are at least two possible explanations for this outcome. First, intraperitoneal micro carrierattached hepatocytes may not receivesufficient oxygen and/or nutrient transport. This will be discussed in more detail later , as it appears to be a recurrent theme in intraperitoneal transplantation of other hepatocyte systems. Second, microcarrier-attached hepatocytes are, in general, unstable and are presumably destined to perish. While it seems necessary to provide an attachment vehicle for hepatocytes, the microcarrier format appears far from sufficient if one desires longterm functional hepatocytes. Microcarrier-attached hepatocytes invariably showed functional degradation in vitro over the course of 1 week, a behavior much similar to trends observed when hepatocytes are simply attached to culture dishes. Potential methods to improve this inherently unstable configuration exist and will be discussed in the section on hepatocyte culture. In addition to microcarriers, investigators have employed other synthetic and extracellular matrix materials as the vehicle for intraperitoneal hepatocyte transplantation. These include collagen-coated polytetrafluoroethylene fibers, Type I collagen sponges, biodegradable polymers, and a heterogeneous substrate called biomatrix (71,82,86). In all of these cases, the death of a large portion of the transplanted hepatocytes is a common finding. This has been attributed to the high oxygen uptake rate of the transplanted hepatocytes and the relative low oxygen tension in the peritoneum. Attempts were made to first implant the vehicle without the hepatocytes to allow for neovascularization through either a growth factor induced response or a nonspecific inflammatory response (82). Seeding of hepatocytes into such vascularized vehicle was shown to support viable hepatocytes that reverse the glucuronide deficiency of Gunn rats. In some ways, this approach is similar to splenic transplantation of the hepatocytes; i.e., the injection of hepato-

cytes into a vascularized bed. Whether this approach will allow for sufficient quantity of functional hepatocytes to support function in liver failure remains to be tested.

Encapsulated implants. Encapsulation of hepatocytes is another approach to transplant hepatocytes. Investigators who encapsulated hepatocytes with collagen and epithelioid cells in the alginate-polylysinealginate system were able to demonstrate some longterm function in vitro (e.g., urea production and liver-specificprotein synthesis). When hepatocytes encapsulated without the epithelioid cells were implanted intraperitoneally, some improvement in the survival of rats with D-galactosamine induced liver failure was observed (9). The investigators reported that half of the transplanted hepatocytes remained viable after 35 days, although some exhibited atypical histological features. Although the alginate-polylysine-alginate membrane may offer some protection from the massive necrosis usually observed in intraperitoneal hepatocyte transplantation, the mechanism of this protection is unknown. The result unfortunately is difficult to interpret, given the previous ambiguity encountered with transplanting suspensions of hepatocytes in this animal model of liver failure. Since it has been known that transplanting dead hepatocytes or nonliver cells could produce improved survival in rats with D-galactosamine-induced liver failure, it remains unclear whether the encapsulated hepatocytes actually provided the functional support. A major theme that emerges from studies of implantable and extracorporeal systems is the difficulty in maintaining functional hepatocytes. In most cases, the hepatocyte system follows a rapid course of functional degradation. One central issue is how to reconstruct the cellular elements such that they remain functional for long periods of time. To address the issue of functional maintenance, or differentiation, it is instructive to examine ways by which hepatocytes have been cultured. HEPATOCYTE CULTURE

Before cultured liver cells can be used effectively for artificial liver support, one must better understand the key parameters involved in keeping cultured cells functional on a long-term basis. As mentioned previously, hepatocytes are anchorage dependent, highly differentiated cells that are difficult to maintain in vitro (46). Early attempts to culture liver cells from organ explants invariably led to overgrowth of fibroblasts and undefined epithelial cell lines (87). Short-term cultures of hepatocytes became possible with the introduction


of enzymatic dissociation of the liver (3). The standard culture configurations were cell suspensions in stirred flasks and cell monolayers on plastic dishes (4,44,67). Hepatocytes in suspension cultures clustered into large clumps of cells and rapidly lost functions within one day of incubation. Hepatocytes in monolayer cultures dedifferentiated and lost adult liver phenotype within a week of incubation. These hepatocytes gradually died and eventually detached from the culture dish. Concomitantly, other contaminating cell types grew to ultimately overtake the culture. Faced with these difficulties, some investigators turned to use hepatoma cell lines or liver-derived cell lines that grow well in vitro. However, it was found that such cell lines often lacked many liver-specific functions (13), and the tumorigenic nature of these cells limited their application in clinical situations, especially in the absence of a protective membrane. Attempts to better maintain morphology and function of cultured hepatocytes include the addition of extracellular matrix products, the addition of other cell types, and the use of specially formulated media (Table 7). Although the mechanism remains largely unresolved, the effects of extracellular matrix components on cultured hepatocyte have been clearly demonstrated in numerous studies (5,54). Long-term maintenance of functions has been demonstrated for hepatocytes cultured on complex extracellular matrix extracts (68) or in between two layers of simple matrices such as collagen gels (20). Matrigel, a basement membrane extracted from a mouse sarcoma cell line, induces the formation of spherical cell aggregates that maintained long-term liver functions (6). Other methods that induced the formation of spherical hepatocyte aggregates appeared to have similar effects (42). The other alternative long-term culture systems include the cocul-

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ture of hepatocytes with a liver-derived epithelial cell line (28), the culture of hepatocytes in a hormonally defined medium (19), and the culture of hepatocytes in a medium supplemented with dimethyl sulfoxide (DMSO) (34). These latter methods face serious limitations with respect to device application for clinical reasons. For example, DMSO toxicity limits its use in patients with liver failure, and the introduction of an undefined epithelial cell line or tumor-derived materials into patients is clinically unacceptable. Most of the hepatocyte systems employed in bioartificial liver support are based on culture techniques that only function short term in vitro. The function of these systems within the animal models is largely unknown. There is also evidence that freshly isolated hepatocytes do not function optimally (20). Given that the isolated cells are further subjected to an unnatural microenvironment, coupled with the added stress of liver failure, such systems may not be able to produce the desired effect on the basis of the unstable cell functions. With the recent success in maintaining longterm hepatocyte cultures, efforts should be made to incorporate these techniques into the design of future systems. ANIMAL MODELS

Animal models employed for liversupport evaluation include those with genetic deficiencies, toxin-induced liver failure, and surgically induced liver failure. Animals with a genetic deficiency are best used to demonstrate the appearance of a previously missing function provided by the liver support device. While the results confirm the presence of functional hepatocytes, it cannot be assessed whether the technique is capable of sufficient liver support for the treatment of liver failure. Toxins employed to induce acute hepatic failure

Table 7. Long-term hepatocyte cultures Functions

Length of culture

Tyrosine aminotransferase Ligandin, albumin Albumin Albumin, transferrin, fibrinogen, bile salts, urea Albumin, tyrosine aminotransferase

3 weeks 5 months 3 weeks 6 weeks

Michalopoulos, 1975 Rojkind, 1980 Bissell, 1987 Dunn, 1991

2 months

Landry, 1985

Albumin, urea, P450 Albumin, transferrin

3 weeks 6 weeks

Dich, 1988 Isom, 1985

Albumin

6 weeks

Guguen-Guillouzo, 1983

Method Extracellular matrix Floating collagen membrane Liver-derived biomatrix Matrigel Collagen sandwich Methacrylate Culture medium Hormonally defined DMSO Co culture Liver-derived epithelial cells

Reference

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include D-galactosamine, dimethylnitrosamine, and carbon tetrachloride. Although these animal models are frequently used for testing, there are serious concerns regarding their validity. While much data suggest that techniques of liver support increase survival in the treatment group, there is little evidence that viable, functional hepatocytes are necessary. To conclusively demonstrate sufficient functional replacement by the . liver support, one needs to employ surgically induced animal models. These include partial hepatectomy, ischemia from ligation or diversion, and total hepatectomy. Anhepatic models provide the most conclusive evidence that any liver function must derive from the exogenous hepatocytes. The survival of the animal is completely dependent on the sufficient functional support provided by the technique. Despite these advantages, these types of animal models are seldom employed. On the other hand, it is felt that anhepatic animals do not represent realistic models, because most of the work has been focused on temporary support in the treatment of acute hepatic failure. For example, the trauma and stress from the operation may introduce complications not present in liver failure. Nevertheless, a liver support system that could maintain an hepatic animals long term would definitely provide the most convincing evidence of functional replacement. ESTIMATION OF CELL MASS AND OXYGEN REQUIREMENT

If one accepts the idea that functional hepatocytes are available for liver support, then the number of hepatocytes required should to be estimated. The quantity of cell mass previously employed has ranged from 1 to 30% of the normal liver . Unfortunately, it is unknown which liver function is limiting so that one cannot estimate the necessary quantity from considerations of animal demands and cellular output. It is generally accepted that patients can survive after 900/0 partial hepatectomy (59). This has been demonstrated in animals as well. While this provides an estimate on the upper limit on the number of cells necessary, it is unclear what the lower limit might be. The liver may have tremendous reserves. Normally, the majority of cells may not be functioning at full capacity. It has been shown that under stress hepatocytes are capable of increasing specific functions at least tenfold. To make a conservative estimate, we will assume that 10% of the liver cell mass needs to be replaced. This translates into roughly 25 billion cells for humans. The large cell mass raises the practical concern regarding the physical size of the bioartificialliver support. In general, a bioartificial liver device has three components: cells, supporting materials, and flow

channels. The volume of 25 billion hepatocytes is roughly 10% of the volume of the liver excluding extracellular space, or about 100 ml. The volume occupied by the supporting materials can vary considerably depending on the device structure. For the microcarrier system, a 200 ",m bead has enough surface area for 250 hepatocytes, but each bead occupies 4 x 10-6 mL, or 400 mL of additional volume is needed for 25 billion hepatocytes. For the encapsulation system, at 107 cells/mL, the volume necessary for 25 billion cells is 2500 mL. For the hollow fiber system, a 100 em fiber with 400 ",m outer diameter and 200 ",m inner diameter has enough outer surface area for 2.5 X 106 cells, but the fiber wall occupies 0.1 mL, or 1000 mL of additional volume is needed for 25 billion hepatocytes, The volume of the flow channels can vary tremendously depending on their widths and the packing density. For the above hollow fibers, the flow channels would require an additional 300 mL of volume. These rough estimates demonstrate that the bulk of the volume will be taken up by structures other than the cells. Methods for miniaturizing the supporting structures will be particularly important for the development of implantable systems. Both the extracorporeal and implantable systems have confirmed that oxygen appears to be a critical parameter in the design of bioartificial support. This is partly due to the high oxygen uptake rate of hepatocytes and partly due to the large number of hepatocytes necessary to support liver function. The maximal oxygen uptake rate is estimated to be 20 femtomoles per min per hepatocyte (15). For 25 billion hepatocytes this translates to 0.5 millimole of oxygen per min. For the extracorporeal reactor, if plasma with an inlet oxygen partial pressure of 140 mmHg and an outlet oxygen partial pressure of 40 mmHg is used as the perfusate, then an estimated 5 L/min flow of plasma would be necessary to meet the oxygen requirement of 25 billion hepatocytes. This is not feasible, as the flow exceeds the normal total cardiac output. Methodologies to increase oxygen content of the plasma will need to be addressed. The oxygen content can be increased fivefold by incorporating a membrane oxygenator equilibrating with 95% oxygen into the bioreactor. Alternatively, one could incorporate a separate oxygen supply into the bioreactor in a crossflow configuration. The oxygen content could also be increased with the use of blood as the perfusate, although immunological protection may be more problematic. For implantation, the rate of oxygen transfer is limited by the available surface area for exchange. The peritoneum has an estimated surface area of 1.5 m 2 • A monolayer of 25 billion hepatocytes occupies roughly 10 m 2 • Experimental evidence suggests that passive diffusion can


support at most two to three cell-layer thick of hepatocytes. Methods to increase the available oxygenated surface area such as vascularization of porous matrices will be critical to the development of implantable systems. CELL SOURCE AND STORAGE

The large number of cells needed for bioartificial liver raises questions concerning the source of these cells. Human hepatocytes can be obtained from surgical biopsies (78). In theory, these cells might be expanded in vitro to provide the quantities needed. In practice, however, the problem of liver regeneration in vitro has yet to be solved (53). While many factors such as insulin and epidermal growth factor are important for regeneration, it is unknown what the necessary and sufficient conditions are for the in vitro expansion of hepatocytes. Recently the molecular cloning of a hepatotrophic factor that stimulates DNA replication in hepatocyte culture was reported (61). However, it must be noted that demonstrating DNA replication is far from demonstrating successful cell mass amplification. A potential large source of human hepatocytes may come from pretransplant livers through the use of adult donor livers for pediatric recipients. Due to physical size limitation, usually only the left lateral lobe of the liver is transplanted. The remaining portion is potentially available for cell procurement. Furthermore, it was shown that a small liver transplanted into a large adult grew to match the expected size for the recipient. Since the left lateral lobe usually represents more than 10070 of the normal liver mass, one may potentially transplant only the left lateral lobes routinely for every recipient, thereby increasing the availability of cells for bioartificial liver support tremendously. The clinical applicability of these ideas remains to be tested. An alternative source of hepatocytes is from other animals. Although they represent an essentially limitless supply, the xenogeneic barrier is immunologically prohibitive. Whether the foreign components can carry out the same functions as their human counterpart is unclear. Finally, the use of hepatoma cells has also been proposed. Although tightly encapsulated tumor cells might serve well in certain cases (e.g., replacement of dopamine-producing tissue in Parkinson's disease), this would not be the case for liver failure (where release of oncogenic substances through larger pore membranes is possible). The storage of the hepatocyte systems will also need to be addressed. When a large number of cells become available at once, multiple recipients can potentially benefit from one donor if the excess can be stored. Alternatively if small portions of cells need to be pooled,

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then there is also a need for storage. Keeping hepatocytes in culture is not only labor intensive and expensive, but is also finite. Cryopreservation will most likely be necessary for storage. There is some controversy over the efficacy of cryopreserved hepatocytes. While some investigators reported successful cryopreservation using standard cryoprotectant and freezing protocol, others reported essentially no survival of hepatocytes after standard cryopreservation (24,27,62). Some of the confusion is attributable to different assays used to evaluate viability and function. Trypan blue dye exclusion is the standard test of viability in freshly isolated cells. Its applicability in cryopreserved cells is questionable. Cryopreserved hepatocytes that exclude trypan blue may not attach to the culture dish even if they are temporarily enzymatically active. A more rigorous test is to demonstrate functional capability of cryopreserved hepatocytes long term in vitro. Very few studies have shown such conclusive evidence. Recently it was demonstrated that the use of hepatocytes cultured for I wk in a collagen sandwich system prior to cryopreservation appears to be a promising new approach to recover functional hepatocytes (41). CONCLUDING REMARKS

It is generally agreed that methods of liver support without hepatocytes have provided, at best, minimal benefit in overall survival and clinical improvement in patients with liver failure. Because these devices are poor substitutes for normal liver function, investigators have focused on methodologies that include hepatocytes to take advantage of the cells' ability to perform a wide variety of functions. These techniques have often been based on systems that maintained hepatocyte function for only a short while because it was believed that only temporary support was necessary for the treatment of acute liver failure. A number of studies that used hepatocyte systems showed improved survival rate in animal models of liver failure. However, it is unclear whether the observed benefit is the result of support provided by the hepatocytes or by some other nonspecific mechanism. This raises questions regarding the applicability of the animal models of acute liver failure to the clinical situation. Clinical experiences with charcoal hemoperfusion that failed to demonstrate any survival benefit despite the experimental success in animals with hepatic failure confirm the need for better models. Today, more than 30 yr after the initial efforts to develop an artificial liver, most strategies focus on the use of hepatocytes as the basis for liver support. While the emerging hepatocyte-based devices face many challenges ahead they, indeed, represent the most promis-


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ing approach for the replacement of normal liver function. For a clinically useful device to be realized, one needs to have sufficient cell mass in a stable environment that can be stored for a long period of time. While reasonable methods to procure hepatocytes and to maintain them in long-term cultures exist, technologies to expand the cell mass and to preserve them are lagging behind. Issues dealing with device scaleup such as the distribution of oxygen and miniaturization of the supporting structure need to be thoroughly addressed. Finally, before serious clinical trials are undertaken, the full-scale device should be tested in an adequate large animal model of liver failure. In summary, hepatocyte-based systems appear to be the most promising approach to artificial liver support. The bioartificialliver needs to be tested in an adequate model to demonstrate that the functional support provided by the exogenous hepatocytes is necessary for the survival of the organism. Further research on:

8.

9.

10.

11. 12.

13.

14.

1. methods of functional maintenance of hepatocytes; 2. technologies for in vitro cell growth, cell preservation, and nutrient supply; and 3. identification of reliable sources of hepatocytes will help develop the artificial liver support dream into clinical reality. Acknowledgments- This work was supported in part by grants from the NIH (DK-43371 and DK-41709) and from the Shriners Burns Institute (15877). M. L. Yarmush is a Lucille P. Markey Fellow in Biomedical Science. J. C. Y. Dunn was supported-by the Medical Scientist Training Program.

15.

16.

17.

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