Development of a Bioartificial Liver Using Isolated Hepatocytes

Artificial Organs 18(5):371-384, Blackwell Scientific Publications, Inc., Boston 0 1994 International Society for Artificial Organs

Development of a Bioartificial Liver Using Isolated Hepatocytes

Vivek Dixit

Department of Medicine, UCLA School of Medicine, Los Angeles, California, U.S .A.

Abstract: Severe liver disease is very often life-threatening and dramatically diminishes quality of life. Liver support systems based on detoxification alone have been proved ineffective because they cannot correct biochemical disorders. An effective artificial liver support system should be capable of carrying out the liver’s essential processes, such as synthetic and metabolic functions, detoxification, and excretion. It should be capable of sustaining patients with fulminant hepatic failure, preparing patients for liver transplantation when a donor liver is not readily available (i.e., bridge to transplantation), and improving the survival and quality of life for patients for whom transplantation is not a therapeutic option. Recent advances in cell biology, tissue culture techniques, and biotechnology have led the way for the potential use of isolated hepatocytes in treating an array of liver disorders. Isolated hepatocytes may be transplanted to replace

liver-specific deficiencies or as an important element of an auxiliary hybrid, bioartificial extracorporeal liver support device, which are important therapeutic applications for treating severe liver disease. Recently, several hepatocyte-based liver support systems have been proposed. Although there is no current consensus on its eventual design configuration, the hollow fiber hepatocyte bioreactor shows the greatest promise. Furthermore, application of tissue engineering technology, based on cell- surface interaction studies proposed by our group and others, has enhanced interest in the development of highly efficient hybrid, bioartifcial, liver support devices. Key Words: Isolated hepatocytes-Hepatocyte bioreactor-Isolated encapsulated hepatocytes-Bridge to transplantation-Cell (hepatocyte) transplantation-Artificial liver support-Microencapsulation-Bioartificial liver.

ARTIFICIAL LIVER SUPPORT SYSTEMS

The premise for an artificial liver support system is based on the assumption that by supplementing essential functions during the critical phase of severe liver disease or injury, by auxiliary or artificial means, it is possible to improve both quality of life and survival for these patients. Thus, an artificial liver support system should be capable of carrying out the liver’s complex synthetic and metabolic functions, detoxification, and excretion. By providing, or supplementing, essential liver functions, the diseased liver will be able to recover and regener- ate. In the case of bridge-to-transplantation, the

Received October 1993; revised October 1993. Address correspondence and reprint requests to Dr. V . Dixit,

Liver Biosupport and Hepatitis Research Laboratory, UCLA School of Medicine, Department of Medicine, 675 Circle Drive South, MRL Room 1240, Los Angeles, CA 90024-7019, U.S.A.

Presented in part at the 9th World Congress of the Interna- tional Society for Artificial Organs, held July 4-8, 1993, Amster- dam, The Netherlands.

system should provide sufficient support until a do- nor liver can be found and transplanted. For patients with chronic liver disease, an artificial liver support system should be capable of providing essential liver functions to improve both quality of life and its duration.

Over the past 3 decades, a variety of experimental liver support systems has been studied. The his- tory of this effort reflects the complexity of the problem and its inherent frustrations for research- ers. The earliest forms of liver support systems (e.g., total body washout [I]) were essentially he- roic methods to improve the survival of patients with severe fulminant liver failure. Other methods have included animal (baboon or pig) liver cross- circulation (2,3) and various types of detoxification systems. Thus far, only detoxification systems (e.g., adsorbent hemoperfusion, high porosity membrane hemodialysis, plasmapheresis, and he- mofiltration) have demonstrated any significant de- gree of clinical efficacy. These systems are based

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on the hypothesis that liver failure and its compli- cations can be reversed if the associated toxins are removed from the circulation. Indeed, detoxification systems have been reported to promote the re- covery of consciousness in patients in deep hepatic coma (4-6).

Currently, however, intensive critical care man- agement is the only therapy available to patients with severe life-threatening liver disease. Recently, orthotopic liver transplantation has become a successful alternative for such patients (7); however, this procedure is highly costly, complex, and limited by a scarcity of donor livers (8) and by the limited time that donor livers can be preserved (9). A growing population of patients are likely to die waiting for a liver transplant either because of scarcity of organs available for transplantation or because they are too sick to be transplanted. Thus, there is a growing and compelling need for an artificial liver support system for sustaining patients with fulminant hepatic failure; preparing patients for liver transplantation when a donor liver is not readily available (i.e., bridge-to-transplantation), or sustaining patients when a transplanted liver fails; and improving the survival and quality of life for patients for whom liver transplantation is not a therapeutic option.

It is now evident from experimental and clinical data that physical methods alone (i.e., detoxification) cannot sufficiently manage the complex biochemical disorders seen in severe liver disease. Re- cently, advances in biotechnology and tissue engineering have brought back into focus the im- portance of a biological component for a hybrid bioartificial support system. Of the numerous approaches being developed, the biological approach shows the greatest promise of being able to assume the full range of liver functions (10,ll). These experimental endeavors are based on the hypothesis that hepatic assist for a failing or diseased liver can best be achieved with isolated liver cells (i.e., hepatocyte transplantation and the use of isolated hepatocytes in extracorporeal hepatocyte bioreactors).

HEPATOCYTE TRANSPLANTATION

Transplantation of isolated healthy hepatocytes to supplement failing or deficient liver function is an innovative technique that offers a variety of important applications. Treatment of liver disease by transplantation of healthy hepatocytes can also have significant implications for organ replacement therapy (12-14). Compared with whole organ trans-

plantation, it is a technically simple procedure that offers numerous advantages. For example, it may be possible in the future for relatives of patients to donate small samples of liver cells for transplantation, or 1 intact liver could be used to treat many patients. Unlike whole organs, cells could also be cryopreserved for future use (14). During the last decade, various experimental approaches for transplantation of healthy isolated hepatocytes, with previously limited success ( 1 9 , were investigated with very promising results. Hepatocyte transplantation showed considerable promise in being able to assume the full range of liver functions in several animal models of liver disease ranging from fulminant hepatic failure to congenital metabolic liver disease (10,11,1~18) .

Several hepatocyte transplantation sites have been reported in the literature. Among the first sites was the liver itself, which is the intuitively ideal site because it represents an optimal environment for the hepatocytes. However, this procedure led to hepatocyte aggregates in distal portal branches, sinusoids, and central veins, resulting in extensive hepatic necrosis, severe portal hypertension, and pulmonary hypertension (19). Other sites have included subcutaneous tissue, intracapsular dorsal fat pads, muscle, peritoneum, renal capsule, lungs, and spleen (16,20). Among these sites, only the spleen (2 I ,22) and the peritoneal cavity (10,11,21,23-29) have shown any real measure of success. Between these 2 sites, the intraperitoneal route is by far the most advantageous. Intraperitoneal transplantation is a simple technique that causes negligible trauma to the recipient animal (1 1). The intraperitoneal route also allows several orders of magnitude greater quantities of hepatocytes to be transplanted as compared with the intrasplenic route (10,11,22).

Intrasplenic transplantation of hepatocytes Intrasplenic transplantation of hepatocytes is a

simple procedure that has been reported by several groups with varying degrees of success (21,30-33). This technique essentially involves injection of a small suspension of isolated hepatocytes into the splenic pulp. The hepatocytes then attach to and grow in the splenic red pulp to form hepatic cords and sinusoids. Splenic white pulp does not permit proliferation of implanted hepatocytes. Intrasplenic hepatocyte transplantation is not always reproducible because transplanted hepatocytes often embo- lize to the liver or into the circulation, where they may die. Ebata et al. (30). demonstrated the formation of a “hepatized” spleen (Fig. l) following intrasplenic transplantation of syngeneic hepatocytes

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DEVELOPMENT OF A BIOARTIFICIAL LIVER 3 73

FIG. 1. Cut surface of a hepatized spleen 27 months after transplantation. A suspension of freshly isolated syngeneic hepatocytes was injected directly into the splenic pulp of the recipient animal. The transplanted hepatocytes proliferated in the splenic red pulp to occupy nearly two thirds of the splenic mass (Reprinted with permission) (32).

for the treatment of impaired liver function. One year after transplantation, the hepatocytes were functionally active and were reported to correct the inherited glucuronyltransferase deficiency in Gunn rats (3 1 ) . Despite such results, this interesting procedure has many limitations. First, this technique requires long-term immunosuppressive therapy if other than syngeneic hepatocytes are transplanted. Furthermore, the hepatic tissue that can be accom- modated by the spleen (-3% of normal liver weight) is not sufficient to support an animal with severe liver failure. Finally, a very long time ( -1 year) is required to “hepatize” the spleen. Therefore, intrasplenic hepatocyte transplantation does not ap- pear to be feasible.

Intraperitoneal transplantation of hepatocytes Pioneering studies of intraperitoneal transplanta-

tion of hepatocytes produced temporary but encouraging results (15,23). It was not widely recog- nized that the peritoneal milieu did not offer suitable attachment sites for the anchorage-dependent hepatocytes, thus limiting their life span. In-

traperitoneal transplantation of hepatocytes did not advance further until recent developments in microcarrier and microencapsulation technology, which can provide suitable attachment substrates. With microcarrier and microencapsulation techniques, intraperitoneally transplanted isolated hepatocytes have shown tremendous promise in being able to assume the full range of liver functions in several animal models of severe liver disease (10,11,14,17, 18,2 1).

HYBRID BIOARTIFICIAL LIVER SUPPORT SYSTEMS

Hybrid bioartificial forms of liver support couple biological and synthetic components. This process is best exemplified by intraperitoneal transplantation of isolated hepatocytes using microcarrier and microencapsulation technology. Advances in technology allowed a previously limited technique to demonstrate the greatest promise of being able to assume the full range of liver functions in several

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animal models of severe liver disease (10,11,14,17, 18,21). Currently, hybrid liver support systems include microcarrier-attached hepatocytes, microencapsulated hepatocytes, and extrahepatic liver support systems (hepatocyte bioreactors).

Microcarrier-attached hepatocytes Microcarriers have been available for use in tis-

sue culture and biotechnology for more than 1 decade; however, their application in cell transplantation studies is recent (10,21,24). Microcarriers provide essential attachment substratum for the hepatocytes. Their enormous surface area-to-volume relationship facilitates transplantation of huge quantities of hepatocytes in very small volumes of microcarrier suspensions (Fig. 2). The first cell transplantation studies with microcarrier-attached hepatocytes were reported by Demetriou et al. (lo), who demonstrated the feasibility for restoration of deficient liver function in 2 animal models of congenital metabolic liver defects: hyperbilirubinemia and analbuminemia. In these studies, microcarrier- attached hepatocytes were intraperitoneally transplanted into hyperbilirubinemic Gunn rats and analbuminemic Nagase rats, resulting in a significant decrease in hyperbilirubinemia and elevation in albumin levels in the respective animal models (Fig. 3). In cross-species experiments with isolated human hepatocytes, this group demonstrated similar results in these animal models (25). They also showed that intraperitoneally transplanted microcarrier-attached hepatocytes could significantly improve the survival of rats undergoing 90% partial hepatectomy (17), demonstrating that the transplanted hepatocytes provided significant metabolic support while the hepatectomized animals’ livers regenerated (17).

Subsequently, other investigators demonstrated significant reduction of serum LDL levels in

Microcarriers / I

FIG. 2. Microcarrier-attached hepatocytes. The enormous surface area-to-volume relationship of the microcarriers can facilitate transplantation of large quantities of hepatocytes in very small volumes of microcarrier suspensions.

congenitally hyperlipidemic Watanabe rabbits following intraperitoneal transplantation of microcarrier-attached hepatocytes (21). Intraperitoneal transplantation of microcarrier-attached hepatocytes has also been shown to significantly improve the long-term survival of rats with galactosamine- induced fulminant hepatic failure (27). Recently, we developed biologically modified polyhydroxymethylmethacrylate (Poly HEMA) microcarriers (Fig. 4) for high hepatocyte immobilization (>75% coverage of the microcarriers as compared with approx- imately 25% coverage with collagen-coated dextran microcarriers) and function to provide optimal liver-specific metabolic function in experimental studies (34). Thus, the data from several centers strongly suggest that intraperitoneally transplanted, microcarrier-attached hepatocytes can provide significant metabolic support in animal models of both acute and chronic (congenital) metabolic liver disease. However, without adequate immunosuppression, hepatocyte rejection remained a significant problem in these trials (10,17,24).

Microencapsulated hepatocytes Microencapsulation techniques for hepatocyte

transplantation evolved after the successful experiments with microcarrier-attached hepatocytes described. Unlike microcarrier-attached hepatocytes, microencapsulating hepatocytes circumvent the need for immunosuppression. The technology for microencapsulation was derived from Chang’s pioneering work (35) and the more recent application of this technique by Lim (36) and Sun et al. (37) for encapsulating islets of Langerhans. Microcapsules are synthetic, spherical, polymeric structures that are composed of ultrathin, semipermeable membranes of cellular dimensions (35). The microcapsule membrane can be prepared from various poly- mers; its contents can consist of tissue or cells, enzymes, or other biologically active materials (38). The membrane prevents the contents of the microcapsule from leaking out and causing immunological reactions, but still allows those contents to interact freely in biological reactions. In addition, this membrane keeps unwanted substances, such as cells and antibodies, from entering the microcapsule (Fig. 5) .

In contrast to the microcarrier studies described, in which hepatocytes are cultured on the exterior surface of collagen-coated microcarriers (see Fig. 2), hepatocytes are microencapsulated within a three-dimensional collagen matrix separated from the external environment by a semipermeable membrane (see Fig. 5) . This three-dimensional orienta-

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FIG. 3. (A) Serum bilirubin concentrations following intraperitoneal transplantation of normal microcarrier-attached hepatocytes into allogeneic (open squares) or congeneic (solid circles) Gunn rat recipients. Data represent mean ? SEM; n = 5 rats for each time point. (B) Serum albumin concentrations following intraperitoneal transplantation of normal microcarrier-attached hepatocytes into allogeneic Nagase analbuminemic rat recipients with (open squares) or without (solid circles) cyclosporin immunosuppression. Data represent mean ? SEM; n = 5 rats for each time point (Reprinted with permission) (58).

tion has been suggested to have an important role in the maintenance of hepatocyte function through improved cell-to-cell interaction (12). Thus microencapsulation provides a unique and innovative technique for transplantation of foreign tissue and cells that eliminates the need for immunosuppression. Indeed, microencapsulation techniques have been successfully used for transplanting a variety of cells, such as islets of Langerhans (39,40) for the treatment of diabetes in rats, parathyroid cells for a

bioartificial parathyroid (4 l ) , and adrenal cortex cells for neurological disorders (42).

The feasibility of microencapsulated hepatocyte transplantation for the replacement of liver function was also demonstrated by several groups in various animal models of fulminant hepatic failure and congenital metabolic liver disease (1 1,14,18,28,29,43). In controlled experiments using the well-characterized galactosamine-induced fulminant hepatic failure rat model (44), Wong and Chang (45) ob-

FIG. 4. Biologically modified polyhydroxymethylmethacrylate (Poly HEMA) microcarriers were developed for high hepatocyte immobilization and function. More than 75% coverage of the biologically modified Poly HEMA microcarriers was seen compared with 0% coverage on untreated Poly HEMA microcarriers. (A) Untreated Poly HEMA microcarriers. (B) Biologically modified Poly HEMA microcarriers. Bar represents 20 pm.

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100 - 75 - 50 -

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V . DIXIT

IMPERMEABLE SUBSTANCES eg. Hepdtocytes, Lymphocytes, Antibodies, Bacteria, etc.

PERMEABLE SUBSTRATES ( S ) , PRODUCTS (P) eg. Ammonia, Phenols, Mercaptans, Fatty Acids, Bile Salts.

Albumin, Clotting Factors, etc. - FIG. 5. Microencapsulated hepatocytes. Microcapsules are synthetic, spherical, polymeric structures that are composed of ultrathin, semipermeable membranes of cellular dimensions. The membrane prevents the contents of the microcapsule from leaking out and causing immunological reactions, but still allows those contents to interact freely in biological reactions. In addition, this membrane keeps unwanted substances (e.g., cells and antibodies) from entering the microcapsule.

served that the survival time of rats with liver failure could be significantly increased if microencapsulated hepatocytes were transplanted in late stages of hepatic coma. Furthermore, we reported (18) in the same animal model that if microencapsulated hepatocytes were transplanted in earlier stages of liver failure, a highly significant increase in the

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overall survival rate of rats was achieved when compared with untreated control animals or control animals treated with empty microcapsules (Fig. 6). The animals in these studies received no immunosuppression. Because fulminant hepatic failure essentially requires support only during the critical phase of liver injury, these studies demonstrated the effectiveness of microencapsulated hepatocyte transplantation for short-term liver support.

A major requirement of an acceptable artificial liver support system is that it must be effective for long-term supplementation of liver function. Sev- eral studies evaluated long-term effectiveness using the Gunn rat model. Gunn rats are congenitally deficient in the liver enzyme bilirubin-uridine diphos- phate glucuronyltransferase and are therefore un- able to conjugate bilirubin. This enzyme deficiency results in lifelong, nonhemolytic, unconjugated hyperbilirubinemia in these animals (46). In long-term studies, the effectiveness of microencapsulated hepatocytes was evaluated for the replacement of a specific liver function in Gunn rats (1 1,14,29). These studies showed that transplanted microencapsulated hepatocytes significantly lowered the level of hyperbilirubinemia (Fig. 7) in Gunn rats following a single transplantation of microencapsulated hepatocytes (1 1,14). Also, concurrent with the decrease in serum bilirubin levels a significant increase in conjugated bilirubin in bile (Fig. 8) was also detected (12,14). These studies effectively demonstrated that transplanted microencapsulated hepatocytes were as competent as transplanted un- encapsulated, microcarrier-attached hepatocytes in providing liver function in Gunn rats (10,11,14).

B Grade I Coma

I U

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FIG. 6. Survival time and survival rate data following intraperitoneal transplantation of isolated encapsulated hepatocytes (IEH) in galactosamine-induced fulminant hepatic failure rats. A highly significant (p < 0.01) increase in survival time and rate was observed following IEH transplantation. (A) Animals treated in Grade II coma (mean * SEM, n = 14) (Reprinted with permission) (18). (B) Animals treated in Grade I coma (mean 2 SEM, n = 16) (Reprinted with permission) (18).

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3 -

2 -

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377

+ MICROENCAPSULATED HEPATOCYTES --0 -- CONTROL MICROCAPSULES

4- FREE HEPATOCYTES ---).- NON-TRANSPLANTED CONTROLS

FIG. 7. Serum bilirubin levels in Gunn rats following transplantation of isolated encapsulated hepatocytes (solid circles), free hepatocytes (open squares), control (empty) microcapsules (open circles), or no transplant (solid triangles). Gunn rats transplanted with microencapsulated hepatocytes show a highly significant (p < 0.001) decrease in serum bilirubin levels. No significant (p < 0.70) change in serum bilirubin levels was observed in the other groups of animals. Data represent (mean 2 SEM; n = 12) rats for each time point.

However, unlike the latter, microencapsulated hepatocytes offered the advantage of circumventing the need for immunosuppression (10,18,14).

One of the main drawbacks observed in hepatocyte transplantation studies (with both microcarrier-attached hepatocytes and microencapsulated hepatocytes) is that the transplanted cells had a limited (4-6 wk) functional viability in the peritoneal cavity of Gunn rats (10,26). Recently, the decline in the functional viability of microencapsulated hepatocytes was related to the specific attachment re- quirements of the hepatocytes (12). Indeed, by using superior attachment substrates such as Matrigel (a mouse sarcoma-derived complex of proteins that closely resembles liver basement membrane proteins), a significant increase in the functional viability of transplanted microencapsulated hepatocytes was reported (12). Other methods to improve long- term functional viability may be achieved through co-culture techniques (47).

If microencapsulated hepatocyte transplantation is to be considered a therapeutic option for acute and chronic liver disease, the long-term fate of the hepatocytes following transplantation must be considered. Recently, the fate of microencapsulated hepatocytes 6 months after intraperitoneal trans-

plantation was investigated (26). The microencapsulated hepatocytes were observed to function adequately for up to 6 to 8 weeks posttransplantation (Fig. 9). Thereafter, a process of degeneration began that destroyed the hepatocytes and resulted in physical breakdown of the microcapsule (26). Re- searchers speculate that degeneration of the microencapsulated hepatocytes (autolysis) leads to ionic and pH changes within the microcapsule mi- croenvironment (26). These pH changes may weaken the alginate-polylysine co-polymer membrane of the microcapsule by altering the charges on the molecules that comprise the membrane (26). Thus, degenerative changes observed in the microcapsules beginning 2 months posttransplantation suggest that repeated transplantation may be necessary for long-term effectiveness of microencapsulated hepatocytes (13). In a 6-month study, repeated monthly transplantation of microencapsulated hepatocytes in Gunn rats demonstrated a prolonged and sustained decrease in serum bilirubin levels (13). Repeated monthly transplantation of microencapsulated hepatocytes, performed before the previous month's microencapsulated hepatocyte transplant began to deteriorate, provided sustained reduction in hyperbilirubinemia (Fig. 10). The pro-

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Transplantation of Microencapsulated Hepatocytes or Control Microcapsules in Gunn Rats

Normal Wistar Rats Microencapsulated Hepatocytes

Normal Gunn Rats Control Microcapsules

0 7 14 21 28

DAYS POST TRANSPLANTATION FIG. 8. Data of bilirubin conjugation in the bile of Gunn rats transplanted with isolated encapsulated hepatocytes (IEH). Com- pared with the donor Wistar rats, the recipient Gunn rats had more than 5 times less conjugated bilirubin in their bile. After IEH transplantation, a significant (p < 0.01) increase in the level of conjugated bilirubin in the bile over pretransplantation levels was observed. Animals transplanted with control microcapsules (containing no hepatocytes) showed no such increase in the level of conjugated bilirubin in bile at day 7 posttransplantation. Data represent mean & SEM; n = 12 rats for each time point.

cess can be compared with a relay race: the newly transplanted microencapsulated hepatocytes take the baton from the previously transplanted microencapsulated hepatocytes, thereby sustaining

VIABILITY OF TRANSPLANTED MICROENCAPSLLATED HEPATOCYTES

- 3 MONTHS FOSTTRANSPLANWION ' 2" 1 I = WTACT MICROCAPSULES 0 FREE HEPALYTES

= BROKEN MIcRoc~psuLEs 0 MICROENCAPSULATED HEPATOCYTES A CONTROL MICROCAPSULES

il 1 ; 3 i ; 4

MONTHS POST-TRANSPLANTAHON

FIG. 9. Composite graph summarizing 6 months' data showing the total serum bilirubin measurements, the viability of transplanted hepatocytes, and the physical fate of transplanted isolated encapsulated hepatocytes (IEH) from 72 Gunn rats following transplantation of IEH, free unencapsu- lated hepatocytes, and control microcapsules containing no hepatocytes. The long-term physical fate of microencapsulated hepatocytes following 6 months' intraperitoneal transplantation showed that the microencapsulated hepatocytes function adequately for up to 6 to 8 weeks posttransplantation.

hepatic function. In this study, investigators were successful in keeping the race going for 6 months in the Gunn Rat model. In contrast, the control rats that received empty microcapsules experienced no

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FIG. 10. Data obtained during a 6-month study period during which Gunn rats received repeated intraperitoneal transplantation (arrows) of either microencapsulated hepatocytes (IEH) or control (empty) microcapsules at 4-week intervals. A significant (p < 0.01) and sustained decrease in total serum bilirubin levels was observed following monthly IEH trans- plantations. No such decrease was seen in the control Gunn rats. Data represent mean k SD; n = 8 (Reprinted with permission).

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DEVELOPMENT OF A BIOARTIFICIAL LIVER 3 79

reduction in hyperbilirubinemia (see Fig. 10). The experimental animals tolerated the transplanted microcapsules well, and there were no adverse side effects (e.g., inflammation, sepsis, irritation).

For microencapsulated hepatocyte transplantation to provide a viable form of artificial liver support (e.g., as a bridge-to-transplantation), or as an alternative to orthotopic liver transplantation, it must not only offer substantial therapeutic benefit to the recipient but also must be simple and convenient to use. Large quantities of healthy microencapsulated hepatocytes must be readily available for transplantation when required. In concept, a fa- cility akin to a blood bank, in which microencapsulated hepatocytes could be stored and used as needed, would be ideal. Unfortunately, free hepatocytes in suspension are far more sensitive than other types of cells and are easily damaged by ca- sual handling. Furthermore, primary cultures of isolated hepatocytes do not store well because hepatocytes start to dedifferentiate within a few days (48). Although cryopreservation is a standard procedure for preserving bone marrow as well as various cell lines, it can inflict considerable injury to free suspensions of hepatocytes (48). Recently, we cryopreserved microencapsulated hepatocytes and showed that microencapsulation protects the hepatocytes from damage and allows them to be stored simply, conveniently, and inexpensively (14). Fur- thermore, cryopreserved, microencapsulated hepatocytes functioned as well as freshly prepared, non- frozen microencapsulated hepatocytes both in vitro and in vivo (14). Thus, transplantation of cryopreserved microencapsulated hepatocytes may provide a convenient therapeutic option for the manage- ment of fulminant hepatic failure, as well as of in- born errors of metabolism, without need for immunosuppression.

Isolated hepatocyte and extracorporeal hepatocyte bioreactors

Hepatocyte bioreactors are extracorporeal devices for auxiliary supplementation of liver function during disease. Such bioreactors usually consist of hepatic tissue cultivated as primary cultures of isolated hepatocytes or as a liver cell line (4%51). In the past, pieces of liver tissue and liver slices were also used with limited success (52,53). Although suspension cultures are the easiest to cultivate, they also are the most short-lived because hepatocytes are anchorage-dependent (48,54). Thus, hepatocyte cultures on membranes, microcarriers, and microcapsules, which have a longer life span, are usually used. In conjunction with cryopreserved, microcar-

rier-attached hepatocytes and cryopreserved microencapsulated hepatocytes, bioreactors have the distinct advantages of long-term storage; they can be prepared in advance and can be used off-the- shelf as needed (50,SS). The design characteristics of hepatocyte bioreactors have been based on the conventional renal hemodialysis system because of its physiological approach (i.e., counter-current blood flow design) to blood detoxification and its mass transfer characteristics (56). These designs are also advantageous because they are engineered for compactness and adaptability to high pressure op- eration in a form convenient for perfusion by blood. Thus far, 3 basic bioreactor design configurations have been proposed: capillary hollow fiber systems, plate dialyzer systems, and hemoperfusion systems (Fig. 11).

Capillary hollow fiber hepatocyte bioreactors The capillary hollow fiber hepatocyte bioreactor

design was first proposed by Wolf and Munkelt (49). This design involves cultivation of hepatoma cells (or primary hepatocytes) on the exterior surfaces of semipermeable capillary hollow fiber membranes that are bundled together within an enclosing plastic shell (Figs. 1 IA, 12). Nutrient medium is circulated within the capillaries until the hepatocytes attach and form a mass of liver tissue. The capillary membranes are then perfused with the host animal’s blood. In theory, the cells resting on the capillary membranes’ 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 pro- tected from the body’s immune system by the semipermeable capillary membrane.

Recently, 4 prototype versions of capillary hollow fiber membrane-based bioreactors were reported in the literature. Sussman et al. (57) described a system that is essentially the same as the pioneering design of Wolf and Munkelt (49) described (see Figs. 11A, 12); however, instead of the Reuber hepatoma cell line, this group used a new cell line known as C3A (57). The C3A cell line was described as a stable, well differentiated, clonaly- derived, hepatoma cell of human origin that grows to very high densities and is strongly contact- inhibited (57). The rationale for a hepatoma cell line is that it can provide a steady supply of large quantities of hepatocyte-like cells: It is not known whether hepatoma cells will cause any detrimental effects to the recipient. Limited experimental stud-

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Capillary Hollow Fiber

Wolf I975 Arnaour er al. I990 Sussman et al. I992 Dixit et al. 1992

A

Hepatoma cells +J=P or rnrcrocamer-attached < hepatocytes in exaacaplllary space

Nyberg et al. I992 B

- Hepatocytes in capillary fiber lumen

Dixit er al. 1993 Seedina Pon

C

Hepatocytes on tissue engineered capillary fibers in exaacapillary space

Plate (Kiil) Dialyzer

Matsumura et al. 1987 D

Hepatocytes - in dialysate

compartment

Hemoper fusion

Dixit 1991 E

Microencapsulated hepatocytes in perfusion chamber

Blood Flow -3 Nutrient Medium Flow

FIG. 11. Current hepatocyte bioreactor designs: (A-C) Capillary hollow fiber design, (D) plate (Kiil) dialyter design, (E) and hemoperfusion design.

ies have demonstrated encouraging results in pro- longing the long-term survival of acetaminophen- induced fulminant hepatic failure in dogs (57). Recent, uncontrolled clinical trials with this system have been inconclusive at best.

Arnaout et al. (50) described another modifica- tion of the capillary hollow fiber bioreactor that in-

Capillary Hollow

Blood Flow

FIG. 12. Capillary hollow fiber hepatocyte bioreactor with cultured hepatoma cells. Hepatoma cells are cultivated on the exterior surfaces of semipermeable capillary hollow fiber membranes that are bundled together within an enclosing plastic shell. After the cells have attached and formed a mass of liver tissue, the capillary membranes are perfused with the host animal's blood (57).

volves (instead of a hepatoma cell line) primary cultures of microcarrier-attached hepatocytes placed in the extracapillary space on the exterior surface of the capillary hollow fiber membranes (Figs. 11A, 13). Intraperitoneal transplantation of microcarrier- attached hepatocytes has previously been shown to be highly successful for supplementing liver function in several animal models of liver disease (10,58). Recent experimental studies with the device have demonstrated its efficacy in animal models of both acute liver failure and congenital metabolic liver disease (5039). By using cryopreserved

Extracapillary Space

Fibers Hepatocytes

FIG. 13. Hepatocyte bioreactor. with microcarrier-attached hepatocytes. The extracapillary space is filled with primary cultures of microcarrier-attached hepatocytes. The capillary membranes are then perfused with the host animal's blood.

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DEVELOPMENT OF A BIOARTIFICIAL LIVER 381

Blood Flow

Hepatocytes Entrapped in Collagen Gel

Extracapillary Space

FIG. 14. A hollow fiber bioreactor showing gel-entrapped hepatocytes located in the intraluminal capillary space of the device. Metabolic exchange between blood and hepatocytes occurs through pores in the wall of the capillary hollow fibers. Culture (nutrient) medium is perfused through and over the gel-entrapped hepatocytes. The host animal’s blood is circulated in the extracapillary compartment between the capillary hollow fibers and the plastic shell of the device (62).

microcarrier-attached hepatocytes, this system also offers the convenience of ready availability. In unpublished and limited clinical trials, this system was reported to prolong the clinical course of acute liver failure and also served as a bridge-to-transplantation in at least 10 patients. This device is currently undergoing controlled clinical trials. Recently, Ya- mamoto et al. (60) also reported encouraging results with a similar system that used microencapsulated hepatocytes instead of microcarrier-attached hepatocytes in a lipophilic capillary hollow fiber device.

Recently, Nybert et al. (61) have proposed a novel capillary hollow fiber-based bioartificial liver support system. Unlike the 2 systems described, this bioreactor design entraps primary hepatocytes in cylindrical collagen gels (Figs. 1 lB, 14) inside the lumen of the hollow fibers (62). The gel entrapment technique facilitated the use of large numbers of hepatocytes in this design of a bioartificial liver. In addition, this technique also provides a three- dimensional framework for cultured hepatocytes, allowing close contact between adjacent hepatocytes, which may be important in maintaining normal hepatocyte function within the bioartificial liver (12,62). In this system, culture medium is circulated through the capillaries and over the gel-entrapped hepatocytes. Blood is circulated in the extracapillary compartment between the capillary hollow fibers and the plastic shell of the device. Recent in vitro studies with this system have reported significant albumin production and cytochrome P-450 ac- tivity by the hepatocytes (63). Limited in vivo stud-

ies have demonstrated improved biochemical function in anhepatic rats with this system (63).

We recently described an interesting bioreactor design still in its preliminary stages of development (64). This design involves 2 different sets of hollow fiber bundles placed parallel to each other in a poly- carbonate shell (Figs. 11C, 15). One set of fibers is made of polysulfone, with a pore size of 2 pm. The other set of fibers is made of cellulose acetate, with a permeability cutoff of 150 to 200 kd. The polysulfone fibers are chemically modified by glow- discharge treatment and biologically modified with fibronectin. These modifications optimize the surface for maximum hepatocyte attachment and facilitate the formation of a mass of liver tissue on the exterior surface of the capillary fibers (34). Nutrient medium is circulated through the fibers to nourish and maintain the hepatocytes in culture. The second set of fibers (cellulose acetate) are chemically modified by glow-discharge treatment to resist hepatocyte attachment (34).

The host animal’s blood is perfused (counter- current to the nutrient medium) through the cellulose acetate hollow fibers that are juxtaposed parallel to the hepatocyte-cultured polysulfone capillary fibers. In theory, toxins and metabolites produced during liver disease would diffuse out of the highly permeable cellulose acetate capillary hollow fibers along a concentration gradient and be acted on by the hepatocytes resting on the adjacent polysulfone hollow fibers. The metabolic end products would then be “excreted” into the lumen of

Nutrient Medium

Hepatocytes FIG. 15. A bioreactor design showing 2 different sets of hollow fiber bundles placed parallel to each other in a polycar- bonate shell. One set of fibers (polysulfone) is biologically modified for maximum hepatocyte attachment and function. Nutrient medium is circulated through these fibers to nourish and maintain the hepatocytes in culture. The second set of fibers (cellulose acetate) is chemically modified to resist hepatocyte attachment. The host animal’s blood is perfused (counter-current to the nutrient medium) through the cellulose acetate fibers that are juxtapositioned parallel to the hepatocyte-cultured polysulfone capillary fibers. Metabolic exchanges between the host animal’s blood and cultured hepatocytes would be facilitated by concentration gradients between the 2 sets of capillary hollow fibers, analogous to the physiological condition.

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the polysulfone capillary fibers along a concentration gradient and be removed by the circulating nutrient medium. Concurrently, essential proteins and factors produced by the hepatocytes would diffuse along a concentration gradient and be taken up by the cellulose acetate capillary fibers lying alongside the hepatocytes. Thus, adequate liver function could be simulated, as in the physiological condition. The semipermeable capillary hollow fiber membranes would protect the cultured hepatocytes from the body's immune system. Preliminary studies show that tissue engineering techniques are effective in creating highly specific hepatocyte attachment surfaces for optimal hepatocyte function in this bioreactor design. Current problems with this design relate mainly to fabrication of the bioreactor.

PLATE DIAL.YZERS The plate, or Kiil, hemodialyzer hepatocyte

bioreactor system was first proposed by Matsumura et al. (65). A suspension, monolayers, or primary hepatocytes are cultured on multiple membranous plates in a configuration that allows blood to flow in a counter-current direction over the opposite side of the semipermeable membrane (Fig. 11D). Thus far, except for one case report on the dramatic improve- ment of a single fulminant hepatic failure patient, no experimental data with animal models have been published (65). Another Japanese group reported inconclusive in vitro data with this system (66). Basic in vitro and in vivo studies are needed to evaluate the efficacy of this system.

HEPATOCYTE HEMOPERFUSION The hepatocyte hemoperfusion bioreactor design

essentially allows for the direct perfusion of blood over microencapsulated hepatocytes placed in an extracorporeal chamber. Because free hepatocytes can cause immunological reactions, we proposed that microencapsulated hepatocytes could be placed directly in a chamber through which the patient's blood would be perfused (55). Because a spherical microcapsule membrane has the greatest surface area-to-volume relationship of any shape, maximum nutrient/product exchange across the microcapsule membrane may be achieved by using microencapsulated hepatocytes in a liver bioreactor (55) . Our group previously demonstrated the efficacy of transplanted microencapsulated hepatocytes in providing significant metabolic support in animal models of severe liver disease. However, recent unpublished results showed that current microencapsulation techniques do not consistently produce microcapsules that can withstand the high

shear stresses that would be expected in an extracorporeal device with high blood flow velocities. Thus, until a more durable microcapsule membrane is developed, this system may not be effective in providing adequate hepatic function. Recently, groups in Japan have reported a similar system in which hepatocytes entrapped in calcium alginate hydrogels exhibited significant ammonia metabolism and urea synthesis (67). Also, recent in vivo perfusion experiments in cats with acute liver failure indicated that such a system is able to provide liver function for up to 4 h (67).

FUTURE PERSPECTIVES The development of artificial liver support sys-

tems is still in its infancy. Several types of artificial liver support systems have been described. No one system has been proven as the ideal form of artificial liver support, which is not surprising because the liver is a highly complex metabolic organ. It is increasingly evident that a multifactorial approach to liver support is necessary. For example, depend- ing on the type and severity of liver disease, detoxification and biologically based liver support systems may need to be combined to provide adequate liver support.

Recent cell culture and biotechnological advances have brought bioartificial liver support systems into a very exciting period of development. Many groups are actively developing bioreactors with an enthusiasm that has prompied some to limit animal testing and to forge ahead with clinical trials. Unfortunately, such trials do not permit the careful, controlled testing of devices that is warranted. Thus, to avoid the pitfalls of previous generations of artificial liver support research and to facilitate ob- jective comparison of the efficacy of various bioartificial liver support systems, we propose the following guidelines. All liver biosupport systems should be tested in controlled animal experiments with well-characterized and reproducible animal models of liver diseases. The number of hepatocytes, or mass of liver tissue, to be used in bioreactor devices should be standardized. Currently, different systems utilize widely divergent quantities of cells or liver tissue. Experimental studies should clearly define treatment end-points. Hepatocyte bioreactor treatment protocols should be standardized with regard to duration and frequency of treatment. Current protocols suggest treatment times ranging from 2 to 48 hours. Also, some protocols disagree as to whether treatment should be contin- uous or intermittent. Reliable assessment criteria should be established for evaluating hepatocyte

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bioreactors using transformed cell lines and primary hepatocyte cell cultures. Currently, there are some conflicting reports on whether transformed cell lines exhibit essential liver metabolic functions such as those involving P450 enzyme systems.

Other areas of future interest include the use of fetal liver tissue for both hepatocyte transplantation and extrahepatic liver assist systems. Fetal tissue offers significant advantages in that it may be transplanted across the histocompatibility barrier, poten- tially eliminating the need for immunosuppression following transplantation. However, important eth- ical questions must be resolved before fetal tissue can be freely used. Finally, recent tissue engineering studies suggest it may be possible, given the right polymer supports (scaffoldings) and hepatocyte attachment substrates, to produce liver organoids. These organoids could be transplanted or used in extracorporeal hepatocyte bioreactor systems (34,68-70). In the future, gene therapy techniques may enable a functional gene to be inserted into defective liver cells of patients. Autologous transplantation of such corrected cells would re- store a previously deficient liver function. The future of hepatocyte transplantation and its use in extracorporeal bioreactor devices is promising. Carefully controlled animal trials are now necessary to bring this technology from the bench to the bedside.

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