WO1992007615A1

WO1992007615A1 - A bioartificial liver

Info

Publication number: WO1992007615A1
Application number: PCT/US1991/007952
Authority: WO
Inventors: Russell A. Shatford; Frank B. Cerra; Scott L. Nyberg; Wei-Shou Hu
Original assignee: Regents Of The University Of Minnesota
Priority date: 1990-10-29
Filing date: 1991-10-29
Publication date: 1992-05-14
Also published as: AU9031591A

Abstract

A bioartificial liver (30) to provide liver function to patients with liver insufficiency or failure is disclosed. The bioartificial liver is based on a bioreactor of the type having two fluid paths (42, 46) separated by a permeable medium (38). The bioreactor can be of either hollow fiber or flat-bed configuration. In the configuration using hollow fibers (40), the two fluid paths correspond to the cavity (36) surrounding the hollow fibers (the extracapillary space), and to the lumens of the hollow fibers themselves. Both fluid paths have inlet and outlet ports. Communication between the two fluid paths is across the permeable medium - the hollow fiber material. Hepatocytes (34) are inoculated into the hollow fibers in a solution which quickly forms a highly porous gel. The gel subsequently contracts, leaving an open channel within the hollow fiber adjacent to the gel core entrapped hepatocytes. This channel can be perfused with nutrient media for the hepatocytes.

Description

A BIOARTIFICIAL LIVER Field of the Invention

This invention involves the use of cells cultured in a three-dimensional gel matrix within a bioreactor such as a hollow-fiber or flat-bed system. Specifically, liver cells are maintained in this bioreactor allowing this device to be used as a bioartificial liver for patients with liver failure.

Background of the Invention

* Most patients admitted to an intensive care unit in liver failure do not survive. (Shell an,

R.G.; Fulkerson, W.J.; DeLong, E.; Piantadosi, CA. Prognosis of patients with cirrhosis and chronic liver disease admitted to the medical intensive care unit. Crit Care Med; 1988 Jul; 16(7): 671-8.) Mortality as high as 80-90% has been reported.

(Rueff, B.; Benhamou, J.P. Acute hepatic necrosis and fulminant hepatic failure. GUT; 1983; 14: 805-15.) In 1987, more than twenty-six thousand people died of liver failure. Most of these deaths were not alcohol related. (Blake, J.E.; Compton, K.V.; Schmidt, W. ; Orrego, H. Accuracy of death certificates in the diagnosis of alcoholic liver cirrhosis. Alcoholism (NY); 1988 Feb; 12(1): 168-72.) The patient in hepatic failure, unlike the patient in renal failure, cannot be specifically treated. Renal dialysis, which revolutionized the treatment of renal failure, does not presently have a hepatic equivalent. Currently, the only available treatment for refractory liver failure is hepatic transplantation. Many patients in hepatic failure do not qualify for transplantation due to concomitant infection, or other organ failure. Because of organ shortages and long waiting lists, even those who qualify for liver transplantation often die while awaiting an allograft. UCLA reported that one quarter of their transplant candidates died before a liver could be obtained. Organs suitable for transplant in the pediatric age group are even scarcer. (Busuttil, R. . ; Colonna J. 0 2d; Hiatt, J.R.; Brems, J.J.; el Khoury G.; Goldstein, L.I.; Quinones-Baldrich, W.J.; Abdul-Rasool, I.H.; Ramming, K.P. The first 100 liver transplants at UCLA. Ann Surg; 1987 Oct; 206(4): 387-402.) Multiple Organ Failure Syndrome remains a major cause of death in the surgical intensive care unit. Hepatic failure is believed to be the dominant dysfunction. However, these patients die with histologically normal livers — except for cholestasis. Many investigators believe that outcomes could be improved with short-term hepatic support; the liver, and the patient, would recover given time.

Currently, other organ systems can be externally supported: left ventricular assist devices exist for the injured heart; dialysis units are used for kidney failure; parenteral nutrition are used for the nonfunctioning gastrointestinal tract; ventilators, extracorporeal membrane oxygenators, and veno-venous bypass techniques are employed to support lung function. However, there is currently no substitute for the liver, either to "buy time" for liver recovery or to find a suitable organ for transplantation.

The development of an artificial liver is a complex problem. Many prior attempts, such as plasmapheresis, charcoal and resin hemoperfusion, and xenograft cross circulation, have failed. Unlike the heart, that has one major physiological function, the liver performs many complex tasks necessary for survival. These tasks have been difficult to develop or maintain in mechanical systems.

The liver is the metabolic factory synthesizing glucose, lipids, and proteins — including albumin, enzymes, clotting factors, and carrier molecules for trace elements. The liver maintains appropriate plasma concentrations of amino and fatty acids, as well as detoxifying nitrogenous wastes, drugs, and other chemicals. Waste products, such as bilirubin, are conjugated and excreted via the biliary tree. Hepatic protein synthesis vastly increases the complexity of hepatic support. Culturinα Hepatocytes Systems that employ hepatocytes to provide biochemical function are problematic because hepatocytes can be difficult to maintain in culture. Under standard conditions, non-transformed hepatocytes cultured on plastic lose their gap junctions in about 12 to 24 hours; flatten, become agranular, and lose all their tissue specific functions in 3-5 days; and die within 1-2 weeks. (Reid, L.M.; Jefferson, D.M. Culturing hepatocytes and other differentiated cells. Hepatology; 1984 May-Jun; 4(3): 548-59; Warren, M. ; Fry, Jr. Influence of medium composition on 7-alkoxycoup-arin O-dealkvlase activities of rat hepatocytes in primary maintenance culture. Zenobiotica; 1988 Aug; 18(8): 973-81.)

A solution to this problem is the use of transformed hepatocytes because they can be grown much more easily. However, transformed hepatocytes are often considered a poor choice because even well-differentiated transformed cells show marked variations in tissue specific function from their parent tissues. (Reid, et al., 1984, supra.) Moreover, many cell lines are transformed by viruses. (Aden, D.P.; Fogel, A.; Plotkin, S.; Damjanov, I.; Knowles, B.B. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature, 1979 Dec 6: 615-6; Knowles, B.B.; Howe, C.C.; Aden, D.P. Human heoatocellular carcinoma cell lines secrete the maior plasma proteins and hepatitis B surface antigen. Science; 1980 Jul 25; 209: 497-9.) These cell lines have the potential to transmit the transforming virus to the patient. As a result, it is doubtful that regulatory agencies would approve the use of transformed cells for humans, even if the risk of transmission were proven minimal.

Many approaches to prolonging the viability and function of cultured hepatocytes and other differentiated cells have been investigated. These approaches have included adding hormones and growth factors to the culture media, adding extracellular matrix constituents, and growing the hepatocytes in the presence of another cell type. Cells routinely used in co-culture work with hepatocytes are endothelial cells, or hepatic nonparenchymal cells such as Kupffer cells.

Effect of Hormones and Growth Factors The addition of corticosteroids to the incubation media has been shown to prolong survival of cultured hepatocytes and to maintain albumin synthesis — particularly in synergy with insulin. (Jefferson, D.M.; Clayton, D.F.; Darnell, J.E. Jr.; Reid, L.M. Post-transcriptional modulation of gene expression in cultured rat hepatocytes. Mol Cell Biol; 1984 Sep; 4(9): 1929-34; Dich, J.; Vind, C; Grunnet, N. Long-term culture of hepatocytes: effect of hormones on enzvme activities and metabolic capacity. Hepatology; 1988 Jan-Feb; 8(1): 39-45.) DMSO (Dimethyl sulfoxide) and phenobarbital also are known to prolong hepatocyte viability and function. (Maher, J.J. Primary hepatocyte culture: is it home away from home? Hepatology; 1988 Sep-Oct; 8(5): 1162-6.) Not all tissue specific functions are equally supported, however. Insulin can promote some functions with an effect that varies with concentration. If only insulin is added to the medium, urea cycle enzyme expression is decreased. This negative effect can be counteracted by the addition of glucagon and dexamethasone. (Dich, et al. , 1988, supra. )

Hormonally defined media can also prolong hepatocyte function and viability. (Jefferson, et al., 1984, supra. ) Using a serum-free hormonally defined medium, good function in baboon hepatocytes has been shown for over 70 days. This medium consisted of epidermal growth factor (100 ng/ml), insulin (lOu/ml), glucagon 4mg/ml), albumin (0.5 mg/ml), linoleic acid (5 mg/ml) , hydrocortisone —6 —7

(10 M) , selenium (10 M) , cholera toxin (2 ng/ml), glycyl-histidyl-lysine (20 ng/ml),

—6 transferrin (5 mg/ml), ethanolamine (10 M) , prolactin (100 ng/ml), somatotropin (1 mg/ml), and thyrotropin releasing factor (10~ M) . (Lanford,

L.E.; Carey, K.D.; Estlack, L.E.; Smith, G.C.; Hay, R.V. Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium. In Vitro Cell Dev Biol; Feb 1989; 25(2): 174-82.)

Effect of Matrices It is now clear that the extracellular matrix has considerable influence on cell function and survival. (Bissell, M.J.; Aggeler, J. Dynamic reciprocity: How do extracellular matrix and hormones direct gene expression. Mechanisms of Signal Transduction bv Hormones and Growth Factors: Alan R. Liss, Inc.; 1987: 251-62.3.) Matrix elements have been shown to reduce or obviate the need for specific growth factors. Using extracted hepatic connective tissue, hepatocytes have been cultured for over 5 months and maintained albumin synthesis for at least 100 days. This extract represented approximately 1% of the liver by weight. One third of the extract was composed of carbohydrates and noncollagenous proteins; the other two thirds were collagens — 43% Type I, 43% Type III, and the remainder an undefined mixture of others including Type IV. (Rojkind, M. ; Gatmaitan, Z.; Mackensen, S.; Giambrone, M. ; Ponce, P.; Reid, L. Connective tissue Biomatrix: Its Isolation and Utilization for Long-term Cultures of Normal Rat Hepatocytes. Journal of Cell Biology; Oct 1980; 87: 255-63.) This mixture may not accurately reflect the local hepatocyte environment — the peri-sinusoidal space or Space of Disse.

The presence of matrix in the Space of Disse has been controversial. Some researchers initially suggested that the peri-sinusoidal space was

"empty." It is now appreciated that all of the major constituents of basement membrane are present in or around the Space of Disse. (Bissell, D.M.; Choun, M.O. The role of extracellular matrix in normal liver. Scand. J. Gastroenterol. ; 1988; 23(suppl 151): 1-7.)

Heparan sulfate proteoglycan binds both cell growth factors and cells. (Saksela, 0.; Moscatelli, D.; Sommer, A.; Rifkin, D.B. Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol; 1988 Aug; 107(2): 743-51; Gordon, M.Y. ; Riley, G.P.; Clarke, D.; AD. Leukemia Research Fund Centre, Institute of Cancer Research, London, U.K.; Heparan sulfate is necessary for adhesive interactions between human earlv hemopoietic progenitor cells and the extracellular matrix of the marrow microenvironment. Leukemia 1988 Dec; 2(12): 804-9.) Heparan sulfate may directly effect the hepatocyte nucleus. (Ishihara, M. ;

Fedarko, N.S.; Conrad, H.E. Transport of heparan sulfate into the nuclei of hepatocytes; J Biol Chem; 1986 Oct 15; 261(29): 13575-80.) Hepatocytes secrete relatively abundant quantities of heparan sulfate in culture. (Arenson, D.M.; Friedman, S.L.; Bissell, D.M. Formation of extracellular matrix in normal rat liver: lipocvtes as a major source of proteoglycan. Gastroenterology; 1988 Aug; 95(2): 441-7.) Immunological studies have identified Type I collagen. Type III collagen, Type IV collagen, fibronectin, and laminin in the Space of Disse. (Geerts, A.; Geuze, H.J.; Slot, J.W. ; Voss, B.; Schuppan, D.; Schellinck, P.; Wisse, E. Immunogold localization of procollaoen III, fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver. Histochemistry; 1986; 84(4-6): 355-62; Martinez-Hernandez, A. The hepatic extracellular matrix. I. Electron immunohistochemical studies in normal rat liver. Lab Invest; 1984 Jul; 51(1): 57-74.) There is normally little Type I collagen in the space of Disse, although hepatocytes in culture show increasing Type I synthesis with de-differentiation. This is at the expense of Type III collagen synthesis. This effect is reversed with culture techniques that support tissue specific hepatocyte activity.

Hepatocytes also can be cultured on Matrigel, a biomatrix produced by a sarcoma cell line (EHS) . Matrigel contains Type IV collagen, laminin, entactin, and heparan sulfate. On Matrigel, hepatocytes have been shown to maintain normal albumin synthesis for 21 days. (Bissell, et al., 1987, suprg.) Close duplication of the normal environment of the hepatocyte has also been attempted by culturing hepatocytes in a confluent monolayer on collagen. A second layer of Type I collagen is added to recreate the normal matrix "sandwich" formed on the "top" and on the "bottom" of the hepatocyte. This technique has shown significantly improved viability and function with albumin synthesis for more than 42 days. (Dunn, J.C.Y.; Yarmush, M.L.; Koebe, H.G.; Tompkins, R.G. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB; 1989 Feb; 3: 174-7.)

The effect of various proteoglycans and glycosaminoglycans on gap junction protein synthesis and genetic expression has also been carefully examined. The most effective compounds were dermatin sulfate proteoglycan, chondroitin sulfate proteoglycan, and heparan. Heparan extracted from the liver was most effective. Lambda carrageenan, a seaweed extract, was also effective. (Spray, D.C.; Fujita, M. ; Saez, J.C.; Choi, H.; Watanabe, T.; Hertzberg, E.; Rosenberg, L.C.; Reid, L.M. Proteoglvcans and Glycosaminoglycans Induce Gap Junction Synthesis and Function in Primary Liver Cultures. Journal of Cell Biology; 1987 July; 105: 541-55.) Finally, chitosan, a polysaccharide found in crustacean shells and fungal membranes, has been suggested as a factor that can mimic normal matrix and promote function and survival. (Muzzarelli, R.; Baldassarre, V.; Conti, F.; Ferrara, P.; Biagini, G.; Gazzanelli, G.; Vasi, V. Biological activity of chitosan: ultrastructural study. Biomaterials; 1988 May, 9(3): 247-52; Scholz, M.T.; Hu, W-S. A two compartment cell entrapment bioreactor with three different holding times for cells, high and low molecular weight compounds. Cytology, 1990, In Press. )

Cell-Cell Co-Culture Another successful technique for culturing differentiated liver cells involves co-culturing them with nonparenchymal cells. Recently, co-culture of hepatocytes on various endothelial lines was compared. Co-culture showed significantly improved albumin synthesis and maintenance of gap junctions. The cells were g: .wn in the presence of insulin and dexamethasone. The 'addition of serum did not improve the results. The improved survival and function conferred by co-culture occurred only with cells in close proximity, and was not transferred by cell supernatants. (Goulet, F.; Normand, C; Morin, O. Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes. Hepatology; 1988 Sep-Oct; 8(5): 1010-8.)

It is still controversial whether the beneficial effects of co-culture occur through matrix interactions or require cell-cell contact.

There is also evidence that lipocytes play a key role in matrix production. Lipocytes are reported to be as numerous as Kupffer cells, and have been suggested to produce the majority of Type I collagen, Type II collagen, Type IV collagen, laminin, and proteoglycans — particularly dermatin sulfate proteoglycan and chondroitin sulfate proteoglycan. (Friedman, S.L.; Roll, F.J.; Boyles, J.; Bissell, D.M. Hepatic lipocytes: The principle collagen-producing cells of normal rat liver. PNAS; Dec 1985; 82: 8681-5.) It is of particular interest that these specific proteoglycans were those that best support gap junctions. (Spray, et al., 1987, supra. )

Bioartificial Liver — Previous Investigations

Many techniques of artificial support have been utilized over the past three and a half decades. These include simple exchange transfusions (Lee, C; Tink, A. Exchange transfusion in hepatic coma: report of a case. The Med. J. of Austr.; 1958, Jan 11: 40-42; Trey, C, ; Burns, D.G.; Saunders, S.J. Treatment of hepatic coma by exchange blood transfusion. NEJM; 1966; 274(9): 473-81); plasmapheresis with plasma exchange; (Sabin S, Merritt JA. Treatment of hepatic coma in cirrhosis bv Plasmapheresis and plasma infusion Tolasma exchange! . Annals of Internal Medicine; 1968 Jan; 68(1): 1-6); extracorporeal heterologous or homologous liver perfusion (Eisemann, B.; Liem, D.S.; Raffucci, F. Heterologous liver perfusion in treatment of hepatic failure. Annals of Surgery; 1965; 162(3): 329-345; Sen, P.K.; Bhalerao, R.A. ; Parulkar, G.P.; Samsi, A.B., Shah, B.K.; Kinare, S.G. Use of isolated perfused cadaveric liver in the management of hepatic failure. Surgery; 1966, May; 59(5): 774-781); cross-circulation (Burnell, J.M.; Dawlorn, J.K.; Epstein, R.B.; Gutman, R.A. ; Leinbach, G.E.; Thomas, E.D.; Volwiler, W. Acute hepatic coma treated bv cross-circulation or exchange transfusions. NEJM; 1967; 276(17): 953-943); hemodialysis (Opolon, P.; Rapin, J.R. ;

Huguet, C; Granger, A.; Delorme, M.L.; Boschat, M. ; Sausse, A. Hepatic failure coma (HFC) treated bv polγacrylonitrile membrane (PAN) hemodialysis (HP) . Trans. ASAIO; 1976; 22: 701-710); activated charcoal hemoperfusion (Gazzard, B.G.; Weston, M.J.;

Murray-Lyon, I.M.; Flax, H.; Record, CO.; Port ann, B.; Langley, P.G.; Dunlop, E.H.; Mellon, P.J.; Ward, M.B.; Williams, R. Charcoal haemoperfusion in the treatment of fulminant hepatic failure. Lancet, June 29; i: 1301-1307); and, more recently, bioartificial liver systems containing cultured hepatocytes.

Examples of bioartificial liver systems currently being investigated for support of liver failure include extracorporeal bioreactors (Arnaout, W.S.; Moscioni, A.D.; Barbour, R.L.; Demetriou, A.A. Development of bioartificial liver: bilirubin conjugation in Gunn rats. Journal of Surgical Research; 1990; 48: 379-382; Margulis MS, Eruckhimov EA, Ahdieimann LA, Viksna LM. Temporary organ substitution bv hemoperfusion through suspension of active donor hepatocytes in a total complex of intensive therapy in patients with acute hepatic insufficiency. Resuscitation; 1989; 18: 85-94); and implantable hepatocyte cultures, such as microencapsulated gel droplets (Cai, Z.; Shi, Z.; O'Shea, G.M.; Sun, A.M. Microencapsulated hepatocytes for bioartificial liver support. Artificial Organs; 1988 May; 12(5): 388-393) and spheroid aggregates (Saito, S.; Sakagami, K. ; Koide, N.; Morisaki, F.; Takasu S, Oiwa T, Orita K. Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen. Transplantation Proceedings; 1989 Feb; 21(1): 2374-77.). These bioartificial liver systems have the advantage of performing detoxification, synthesis and bioprocessing functions of the normal liver. Only a few extracorporeal bioreactors have been used in the clinical setting (Matsumura, K.N.; Guevara, G.R.; Huston, H. ; Hamilto, W.L.; Rikimaru, M. ; Yamasaki, G.; Matsumura, M.S. Hybrid bioartificial liver in hepatic failure: preliminary clinical report. Surgery; 1987 Jan; 101(1): 99-103; Margulis, et al.; 1989, supra) . Implantable hepatocyte cultures remain clinically untested.

The technique for hepatocyte entrapment within microencapsulated gel droplets (hepatocyte microencapsulation) is similar to the technique successfully used for pancreatic islet encapsulation (O'Shea, G.M.; Sun, A.M. Encapsulation of rat islets of Lanoerhans prolongs xenograft survival in diabetic mice. Diabetes; 1986 August; 35: 943-46; Cai, et al., 1988, supra) . Microencapsulation allows nutrient diffusion to the hepatocytes, and metabolite and synthetic production diffusion from the hepatocytes. Microencapsulation also provides intraperitoneal hepatocytes with "immuno-isolation" from the host defenses (Wong, H.; Chang, T.M.S. The viability and regeneration of artificial cell microencapsulated rat hepatocvte xenograft transplants in mice. Biomat. Art. Cells, Art. Org.; 1988; 16(4): 731-739.) Plasma protein and albumin synthesis (Sun, A.M.; Cai, Z.; Shi, Z.; Fengzhu, M. ; O'Shea, G.M.; Gharopetian, H. Microencapsulated hepatocytes as a bioartificial liver. Trans. ASAIO; 1986; 32: 39-41; Cai, et al., 1988, supra) ; cytochro e P450 activity and conjugation activity (Tompkins, R.G.; Carter, E.A. ; Carlson, J.D.; Yarmush, M.L. Enzvmatic function of alginate immobilized rate hepatocytes. Biotechnol. Bioeng.; 1988; 31: 11-18); gluconeogenesis (Miura, Y. ; Akimoto, T.; Yagi, K. Liver functions in hepatocytes entrapped within calcium aloinate. Ann. N.Y. Acad. Sci.; 1988; 542: 531-32); ureagenesis

(Sun, A.M.; Cai, Z.; Shi, Z.; Ma, F.; O'Shea, CM. Microencapsulated hepatocytes: an in vitro and in vivo study. Biomat. Art. Cells, Art. Org.; 1987; 15: 483-486); and hepatic stimulating substance production (Kashani, S.A.; Chang, T.M.S. Release of hepatic stimulatory substance from cultures of free and microencapsulated hepatocytes: preliminary report. Biomat., Art Cells, Art. Org.; 1988; 16(4): 741-746) have all been reported by calcium alginate entrapped hepatocytes.

Spheroid aggregate cultured hepatocytes have also been proposed for the treatment of fulminant hepatic failure. Multiple techniques exist for hepatocyte aggregation into spheroids (Saito, S.; Sakagami, K. ; Koide, N.; Morisaki, F.; Takasu, S.; Oiwa, T.; Orita, K. Transplantation of spheroidal aggregate cultured hepatocytes into rat spleen. Transplanatation Proceedings; 1989 Feb; 21(1): 2374-77; Koide, N.; Shinji, T. ; Tanube, T.; Asano, K.; Kawaguchi, M. ; Sakaguchi, K. ; Koide, Y. ; Mori, M.; Tsuji, T. Continued high albumin production bv milticellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans. Biochem. Biophys. Res. Co m. ; 1989; 161(1): 385-91.) It is hypothesized that hepatocyte aggregation would improve the beneficial results of intraperitoneal hepatocyte injection therapy. Such therapy has been used experimentally in the treatment of enzyme deficiency diseases, acute liver failure and hepatic cirrhosis with varying degress of success (Saito, et al., 1989, supra) .

Extracorporeal bioreactor designs for the purpose of artificial liver support have included perfusion of small liver cubes (Lie TS, Jung V,

Kachel F, Hohnke C, Lee KS. Successful treatment of hepatic coma bv a new artificial liver device in the pig. Res. Exp. Med.; 1985; 185: 483-494); dialysis against a hepatocyte suspension (Matsumura, et al., 1987, supra: Margulis, et al., 1989, supra) ; perfusion of multiple parallel plates (Uchino, J.; Tsuburaya, T.; Ku agai, F.; Hase, T.; Ha oda, T.; Kornai, T.; Funatsu, A.; Hashimura, E.; Nakamura, K. ; Kon, T. A hybrid bioartificial liver composed of multiplated hepatocyte monolayers. Trans. ASAIO; 1988; 34: 972-977); an hollow fiber perfusion. Human studies using extracorporeal hepatocyte suspensions have been reported. The first clinical report of artificial liver support by dialysis against a hepatocyte suspension was released in 1987 (Matsumura, et al., 1987, supra) . The device consisted of a rabbit hepatocyte liquid suspension (1-2 liters) separated from the patient's blood by a cellulose acetate dialysis membrane. Each treatment used fresh hepatocytes during a single four to six hour dialysis (run). Multiple runs successfully reduced serum bilirubin and reversed metabolic encephalopathy in a single case.

A controlled study from the USSR comparing dialysis against a hepatocyte suspension with standard medical therapy for support of acute liver failure was recently reported (Margulis, et al.; 1989, supra) . The bioartificial device consisted of a small 20 ml cartridge filled with pig hepatocytes in liquid suspension, along with activated charcoal granules. The cartridge was perfused through a Scribner arteriovenous shunt access. Patients were treated daily for six hours. The hepatocyte suspension was changed hourly over each six hour treatment period. Improved survival was demonstrated in the treated group (63%) when compared with the standard medical therapy control group (41%). Culturing hepatocytes with a hollow fiber cartridge is another example of bioartificial liver support. Traditionally, hepatocytes are loaded in the extracapillary space of the hollow fiber cartridge, while medium, blood or plasma is perfused through the lumen of the hollow fibers. Cells may be free in suspension (Wolf, C.F.W.; Munkelt, B.E. Bilirubin conjugation bv an artificial liver composed of cultured cells and synthetic capillaries. Trans. ASAIO; 1975; 21: 16-27); attached to walls (Hager, J.C; Carman, R. ; Stoller, R.; Panol, C; Leduc, E.H.; Thayer, W.R.; Porter, L.E.; Galletti, P.M.; Calabresi, P. Trans. ASAIO; 1978; 24: 250-253); or attached to microcarriers which significantly increase the surface area within the extracapillary space (Arnaout, et al., 1990, supra) .

Bilirubin uptake, conjugation and excretion by Reuber hepatoma cells within a hollow fiber cartridge was reported in 1975. (Wolf, et al., 1975, supra) . Tumor cell suspensions were injected by syringe into the shell side of the compartment while bilirubin containing medium was perfused through the hollow fiber intraluminal space. This technique has not been reported clinically, possibly due to the risk of tumor seeding by hepatoma cells.

Another hollow fiber device developed for liver support uses hepatocytes attached to microcarriers loaded into the extracapillary cavity of a hollow fiber cartridge. In this device, blood flows through semi-permeable hollow fibers allowing the exchange of small molecules. Using this system, increased conjugated bilirubin levels have been measured in the bile of glucuronosyl transferase deficient (Gunn) rats. (Arnaout, W.S.; Mosicioni,

A.D.; Barbour, R.L.; Demetriou, A.A. Development of Bioartificial Liver: Bilirubin Conjugation in Gunn Rats. J. Surg. Research; 1990; 48: 379-82. Since the outer shell is not perfused, all oxygen and

SUBSTITUTESHEET nutrients are provided by the patient's blood stream. In addition, this system may require an intact in vivo biliary tree for the excretion of biliary and toxic wastes. Summary of the Invention

A hollow fiber bioreactor, in its "conventional" configuration, may not be optimal for a bioartificial liver. In a "conventional" hollow fiber configuration, such as the two described above, cells are loaded in the extracapillary cavity (shell) while media flows through the lumen of the fibers. Potential problems exist in the extracapillary space such as uncontrolled fluid flow, fluid channelling, and location dependent cell concentration and viability. The present invention thus proposes a new hollow fiber bioreactor configuration, as well as a new flat-bed configuration.

Accordingly, the present invention presents a novel bioreactor configuration for cell culture, which is particularly suitable for supporting viable hepatocytes in vitro. In one embodiment, this novel bioreactor is a hollow fiber cell culture bioreactor employing cells entrapped within a fibrous and highly porous collagenous gel matrix within the hollow fiber lumen. In another embodiment, this novel bioreactor is a flat-bed bioreactor with cells entrapped within a matrix but separated from a media stream by a porous membrane.

This invention also relates to a cell gel matrix and a method of preparing such a cell gel matrix for cell cultivation. A bioartificial liver employing this novel bioreactor for supporting hepatocyte function in a patient suffering from hepatic failure is also provided by this invention. Tissue-specific function of other mammalian cells can also be supported us ng the cell gel matrix and the novel bioreactor provided by this invention, while also withdrawing desirable products or by-products therefrom. These and other advantages of the present invention will be further described herein. Brief Description of the Drawings Figure 1: Schematic of novel hollow fiber bioreactor. Figure 2: Schematic detail of single fiber showing a contracted core of gel which contains hepatocytes.

Figure 3: Contraction in hepatocyte gel discs. Figure 4: Bilirubin conjugation rate in spinner flasks containing hepatocyte-gel cores.

Figure 5: Oxygen consumption rate in the hollow fiber bioreactor over 120 hours.

Figure 6: HPLC analysis of bilirubin. Figure 7: Bilirubin conjugation (HPLC) data.

Figure 8: Conjugated and unconjugated bilirubin levels (Ektachem 700XR) .

Detailed Description of the Invention

We have now developed a novel bioreactor 30 for cell culture, diagrammed in Figures 1 and 2.

(Schultz, et al., 1990, supra. ) The stream (blood or plasma) to be detoxified flows through the shell side. Rather than residing in the extraluminal shell space 32, cells 34, such as hepatocytes, are within the hollow fiber lumen 36, entrapped in a gel matrix 38. This configuration is accomplished by first suspending hepatocytes 34 in a solution of collagen or a mixture of collagen and extracellular matrix components. The pH is then adjusted to 7.4 and the cell suspension inoculated into the lumen 36 of the hollow fiber 40. A 'temperature change from 4 °C to 37°C induces collagen fiber formation. This results in cell entrapment in an insoluble fibrous and highly porous cylindrical gel 38.

After inoculation, the cross-sectional area of the gel-matrix cylinder can contract as much as 75%. This permits perfusion of hollow fiber lumen 36 even after it had been initially filled with gel matrix 38. Figure 2 illustrates that media or blood or plasma with low molecular weight nutrients flows around hollow fibers 40 in the extraluminal shell space 32 from extraluminal inlet 42a to extraluminal outlet 42b. Molecular exchange occurs through the pores in the hollow fiber 40. Media with high molecular weight constituents flows through the hollow fiber 40 containing a contracted core of hepatocytes 34 embedded in biomatrix 38 through hollow fiber inlet 46a to hollow fiber outlet 46b. This technique is useful with multiple cell lines including Chinese Hamster ovary cells, Hep2, HepG2, Vero, 293 cells, and normal diploid human cells. Study of a hematoxylin and eosin (H & E) stained thin section of human hepatoblastoma (HepG2) cells within a contracted gel matrix after 7 days showed that tissue density and cytoarchitecture closely resemble in. vivo histology.

This bioreactor offers distinct advantages over other configurations. Cells can be cultured at density close to that of tissue. At high density, cells occupy much less space, thus reducing the size of the bioreactor. Cells also obtain the benefits of close contact with minimal oxygen and nutrient limitations. Mammalian cells, at high density, may better preserve tissue specific function. This has been shown in hepatoma lines. (Kelly, J.H.;

Darlington, G.J. Modulation of the liver specific phenotype in the human hepatoblastoma line Hep G2. In Vitro Cell Dev Biol; Feb 1989; 25(2): 217-22.)

This bioreactor configuration also allows manipulation of the hepatocytes' local environment.

Matrix constituents that support differentiated hepatocyte function can be incorporated into the gel. The ability to perfuse the inner lumen provides high molecular weight growth factors at high concentrations.

Another advantage of such a system is that different cell types can be co-entrapped in the gel to provide possible synergistic effects which may improve tissue specific function.

This invention is thus capable of incorporating many factors (medium, gel matrix, co-culture, high cell density) necessary or beneficial to sustain liver specific functions. It can be used as a bioartificial liver to support patients in liver failure.

Example I: Hybrid Bioreactor

The new hollow fiber bioreactor 30 is illustrated in Figures 1 and 2. The hollow fiber 40 cartridge allows a large surface area for oxygen and

7 nutrient exchange; cell density exceeding 10 cells/ml is possible with gel entrapment.

Figure 1 and Figure 2 show that blood or plasma from the patient flows continuously through the extraluminal shell space 32 and semi-permeable hollow fibers 40 which separate this fluid from the hepatocytes 34. Intraluminal stream 46 containing high molecular weight constituents flows through hollow fibers 40 containing hepatocytes 34 in biomatrix 38. The extraluminal stream 42 containing the patient's blood or plasma flows in either a counter-current, cross-current, or co-current direction to the intraluminal stream 46. Molecular exchange occurs through the pores in the hollow fiber 40. It is probable that blood—particularly from a patient in liver failure—does not provide the optimal chemical environment to sustain hepatocyte function and viability. Intraluminal stream 46 containing growth factors and nutrients is passed through the hollow fiber lumen. Intraluminal stream 46 can also provide toxin or metabolic product removal. Our two channel hollow fiber design supplies both a "life support system" for the hepatocytes 34, and a stream of waste products. The microporous hollow fibers 40 can allow diffusion of waste products, such as ammonia and bilirubin from the blood, for detoxification by the hepatocytes. Waste products are then cleared in the hollow fiber intraluminal stream 46. These conditions can result in improved hepatocyte survival and continuous function. Several fundamental aspects of hepatocyte cultivation have been addressed. Prolonged hepatocyte viability and function have been demonstrated in monolayer cultures. The contraction of three-dimensional collagen gels by rat hepatocytes has also been demonstrated. Energy metabolism and bilirubin conjugation by hepatocytes in these contracted gels have been shown. Finally, viable and functional hepatocytes within the bioreactor have been demonstrated through vital dyes, oxygen consumption, glucose consumption, and bilirubin conjugation.

Example II: Three-Dimensiσnal Collagen Gels

In order to achieve a high cell density and simulate a natural environment, hepatocytes were cultured in three dimensional collagen gels. Dime-sized collagen "discs" and thin diameter cylindrical collagen "cores" of 0.5 or 1.1 mm in diameter were studied. Gels contained 2 gm/1 of Type I collagen in isotonic DMEM. Collagen gel discs were made by adding a mixture of collagen/DMEM and hepatocytes to empty tissue culture plates. Collagen gels have been made with other isotonic media, such as William's E medium. Media was added following gel formation. Silicone tubing was used to form thin diameter collagen gel cores. After 10 minutes of incubation at 37°, the cylindrical gel cores were extruded into media containing wells. All collagen gel experiments including bioreactor trials were done using William's E medium supplemented with 10% calf serum, insulin, L-glutamine (Modified William's E medium) or a serum-free hormonally defined media. (Lanford, supra) .

Gel Contraction Measurements Collagen gel discs were used to study gel contraction. Many combinations of gel thickness and cell density were compared. Gel diameters were measured daily for 10 days and the average of greatest width and its perpendicular width was recorded. Figure 3 summarizes the average daily gel contraction resulting from several hepatocytes cultures. Gel discs containing dead cells or no cells were used as controls. Error bars show standard error. Control gels without cells or with dead cells did not contract. Thus, gel contraction becomes a criterion for viability. The cell

7 concentrations tested ranged from 0.2 to 2.0 x 10 cells/ml of gel. Both cell density and gel thickness effected the rates of contraction. The examples shown in Figure 3 had an average decrease in diameter of 40% at ten days, which corresponds to a 64% reduction in cross-sectional area. Further studies were carried out in hollow fibers. After contraction, the collagen matrix leaves a residual lumen of sufficient size to allow growth factor, media, or waste stream perfusion.

Metabolic Results in Gel

Collagen gel cores were used to measure metabolic activity. After formation in the silicone tubing, the gel cores were placed in spinner flasks and incubated for 30 hours. Media samples were taken for analysis at six hour intervals.

A glucose consumption rate of 1.1 mg/hour

7 was calculated for gels containing 2.3 x 10 hepatocytes. Glucose consumption was negligible in the control spinner flasks that contained media and gels without cells. Bilirubin conjugation, a function unique to hepatocytes and catalyzed by UDP glucuronosyl transferase, was measured by high performance liquid chromatography (HPLC) . (Figure

4.) A conjugation rate of 1.8 mg/hour was measured by linear regression analysis. The level of conjugated bilirubin remained negligible in the control gels without cells.

EXAMPLE III: Hollow Fiber Reactor

Apparatus

A hollow-fiber system assembly consisted of an Amicon HI hollow-fiber cartridge with Delrin end caps. The hollow fibers are made of porous polysulfone with a 30,000 molecular weight cut-off. The extracapillary space (outer shell) was perfused with Modified William's E medium. The inner channel was not perfused. The hollow fiber reactor was kept in a 37°C warm room following inoculation.

Metabolic Results The following results relate to the o hepatocyte hollow-fiber reactor. 1.20 x 10 rat

₇ hepatocytes at a final concentration of 0.9 x 10 cells/ml of gel were cultured for 120 hours. Partial pressure of oxygen was measured in the inflow and the outflow streams, and oxygen uptake rate (OUR) was calculated from the following equation:

OUR = tC_in - C_out] • F

C. is the inlet oxygen concentration; C . is the outlet oxygen concentration; F is the media flow rate.

The oxygen uptake rate increases with increasing flow rate at low flow rates, and becomes flow independent at high flow rates. A flow rate of 30 ml/min was sufficient to maintain maximum oxygen uptake without inducing the larger pressure drop seen at higher flow rates, and was used in this example.

The oxygen consumption rose during the first 20 hours and then declined gradually until termination at 120 hours. (Figure 5.) Glucose concentration in the perfused media was measured by a spectrophotographic assay. Glucose consumption rate as determined by linear regression was 1.0 mg/hour. Judging from the consumption of oxygen and glucose, hepatocytes cultivated in this bioreactor were metabolically active.

Bilirubin Clearance

Perfusion of this same bioreactor with fresh media containing unconjugated bilirubin at 2.1 mg/dl was begun at 40 hours (t=0) and continued for 42 hours. Samples were taken from the media circulating on the shell side. Bilirubin conjugation was measured by both HPLC and Kodak Ektache absorbance. A significant conjugation rate was detected by both techniques. This accumulation of conjugated bilirubin can be visualized from the raw HPLC data; onoconjugate and diconjugate peaks are shaded (Figure 6). Bilirubin conjugation rates of 1.7 mg/hour and 8.2 mg/hour were obtained by linear regression analysis of HPLC (Figure 7) and Ektachem data (Figure 8), respectively. Unconjugated bilirubin levels are also included on Figure 8. Judging from the appearance of conjugated bilirubin in the medium, hepatocytes cultivated in the hollow fiber bioreactor are capable of liver specific function - namely, bilirubin conjugation.

CONCLUSION We have created a novel bioreactor system for short term support in cases of liver failure. A system using the gel matrix concepts described herein provides constant optimal media perfusion to detoxify blood and facilitates liver cell metabolic function. A device using this concept is designed such that the blood flow and media flow allow proper oxygenation, toxin transfer, and toxin-metabolite removal. Likewise, membrane pore size must allow proper diffusion rates for toxin removal and liver cell metabolic function.

While many specific embodiments have been shown and described in detail to illustrate the application of the principles of this invention, it will be understood by those skilled in the art that the invention may be embodied otherwise without departing from such principles. For example, while a hollow fiber system is described herein using the gel matrix/biomatrix concept, a flat-bed bioreactor could be used. A suitable flat-bed reactor is shown in U.S. patent application Serial No. 7-355,115, filed May 18, 1989 entitled Bioreactor Device. The disclosure contained therein is incorporated herein by reference. In such a system, the entrapped cells would be hepatocytes. Moreover, cell gel matrix other than collagen may be employed such as Type III collagen, chitosan or fibronectin. The selected material need only be a biocompatible and capable of forming a cell gel matrix.

Claims

What Is Claimed Is:

1. In a hollow fiber cell culture bioreactor of the type containing a housing having first inlet and outlet ports defining a fluid flow cavity therebetween, said cavity also enclosing a plurality of microporous hollow fibers in fluid flow communication with second inlet and outlet ports in said housing, such that communication between the cavity and the lumen of the hollow fibers is exclusively through the microporous hollow fiber walls, the improvement comprising providing a highly porous fiber containing cell gel matrix within the hollow fiber lumen.

2. A hollow fiber bioreactor according to claim 1, wherein the cell gel matrix is contracted within the hollow fiber lumen to facilitate perfusion thereof .

3. A hollow fiber bioreactor according to claim 1, wherein fluid such as plasma, serum, or blood flows between the first inlet and outlet ports around the hollow fibers in the cavity, and media containing nutrients flows between the second inlet and outlet ports through the hollow fiber lumen.

4. A hollow fiber bioreactor according to claim 1, wherein the cell gel matrix is comprised of a collagen mixture of cells in a solution of collagen, collagen and heparan or other matrix additives or cell components.

5. A hollow fiber bioreactor according to claim 4 wherein the collagen is Type I collagen or some other matrix such as Type III collagen, chitosan, or fibronectin.

6. A hollow fiber bioreactor according to claim 1 wherein the cells contained within said matrix are mammalian cells.

7. A hollow fiber bioreactor according to claim 6 wherein the cells are selected from liver tumor lines or normal liver cells.

8. A method of forming an insoluble fibrous and highly porous fiber containing cell gel matrix comprising:

admixing cells with a solution of an appropriate matrix forming material; and inducing fiber formation therein.

9. The method according to claim 8 wherein said cell gel matrix provides entrapment of cells within a cell culture environment.

10. The method according to claim 8 wherein matrix formation is facilitated by adjusting pH and temperature.

11. The method according to claim 10 wherein pH is adjusted to 7.4 and temperature is adjusted from 4° to 37° C

12. The method according to claim 8 wherein the matrix forming material is Type I collagen, Type III collagen, chitosan, or fibronectin.

13. The method according to claim 8 additionally comprising inoculating the cell admixture into a hollow fiber lumen prior to inducing fiber formation.

14. The method according to claim 8 wherein the cells are mammalian cells.

15. The method according to claim 8 wherein the cells are selected from liver tumor lines and normal liver cells.

16. A bioartificial liver comprising:

a hollow fiber cell culture bioreactor containing a housing having first inlet and outlet ports defining a fluid flow cavity therebetween for flowing therethrough blood or plasma in vivo from a living patient;

said cavity also enclosing a plurality of hollow fibers in fluid flow communication with second inlet and outlet ports in said housing for flowing therethrough media to support hepatocyte function and viability, such that communication between the cavity and the lumen of the hollow fibers is exclusively through the hollow fiber walls, said hollow fibers initially containing a collagen fiber forming solution of collagen or a mixture of collagen and extracellular matrix components or other supporting cells such as the macrophage, lipocyte, or endothelial cells;

such that, hepatocytes are separated into the hollow fiber lumen wherein said cells form with the sol_tion therein a highly porous collagen fiber containing hepatocyte gel matrix, and said flowing of hepatocyte media effects hepatocyte waste removal, while supporting hepatocyte function and viability.

17. The bioartificial liver of claim 16 wherein said blood flow and said hepatocyte support medium flow are continuous or intermittent and co-current or cross-current or counter-current to each other.

18. A method of supporting hepatocyte function and viability in a patient suffering from hepatic failure comprising:

providing first fluid flow means for flowing therethrough blood or plasma in vivo from said patient in communication with a gel fiber containing hepatocytes, such that communication between said blood or plasma and said fiber is exclusively through a microporous means which permits separation of hepatocytes from said blood into said fiber, thereby forming a highly porous fiber gel matrix containing hepatocytes separated from said blood by said microporous means; providing second fluid flow means for flowing therethrough, in contact with said hepatocyte gel matrix and separated from said blood by said microporous means, medium to support hepatocyte function and viability;

such that, as blood or plasma flows through the first fluid flow means and hepatocyte support media flows through the second fluid flow means, said flowing of medium effects hepatocyte waste removal, while supporting hepatocyte function and viability.

19. The method of claim 18 wherein said blood flow and said hepatocyte support media flow are continuous and counter-current to each other.

20. The method of claim 18 wherein said microporous medium comprises hollow fibers and the hepatocyte gel matrix is within the hollow fiber lumen.

21. The method of claim 18 wherein said microporous medium comprises a membrane forming a cell chamber, and said cell chamber contains the hepatocyte gel matrix.

22. A method of preserving tissue-specific function of mammalian cells comprising:

combining tissue-specific mammalian cells with a fiber forming solution thereby forming an insoluble fibrou., and highly porous fiber containing tissue-specific mammalian cell gel matrix; and

perfusing said cell gel matrix with medium to support the function and viability of said tissue-specific mammalian cells.

23. The method of claim 22 wherein said cell gel matrix and said perfusing media are separated by a microporous medium, such that communication between said cell gel matrix and said perfusing media is exclusively through said medium.

24. The method of claim 22 wherein said cell gel matrix is maintained within the lumen of hollow fibers and communication between said cell gel matrix and said perfusing medium is through said hollow fiber walls.

25. The method of claim 22 wherein said cell gel matrix is maintained within a cell chamber created by a membrane and communication between said cell gel matrix and said perfusing medium is through said membrane.

26. The method of claim 22 wherein the tissue-specific cells are pancreatic islet cells, bone marrow stem cells, liver cells or liver tumor cells.

27. A hollow fiber cell culture bioreactor of the type containing a housing having first inlet and outlet ports defining a fluid flow cavity therebetween, said cavity also enclosing a plurality of semi-permeable hollow fibers in fluid flow communication with second inlet and outlet ports in said housing, such that communication between the cavity and the lumen of the hollow fibers is exclusively through the microporous hollow fiber walls, said bioreactor having three separate cell culture zones: a first zone comprising an insoluble fibrous and highly porous fiber containing cell gel matrix contracted within the hollow fiber lumen; a second zone within the hollow fiber lumen around the contracted cell gel matrix; and a third zone within the fluid flow cavity around the hollow fibers.

28. A bioartificial liver comprising:

(a) a housing having a first and a second base plate, the first base plate having first and second fluid inlet means, and the second base plate having first and second fluid outlet means;

(b) at least one cell growth plate having windows extending substantially the distance from the second fluid inlet means to the second fluid outlet means;

(c) at least one nutrient medium plate having windows extending substantially the distance from the first fluid inlet means to the first fluid outlet means; (d) at least one microporous membrane, the base plates, growth plate, medium plate and membrane assembled together such that each growth plate alternates with each medium plate with said membrane separating each medium plate from each growth plate;

thereby each growth plate forms, with the adjacent membrane a cell chamber in flow communication between the second fluid inlet means and the second fluid outlet means, and thereby each medium plate forms with the adjacent membrane a nutrient chamber in flow communication between said first fluid inlet means and the first fluid outlet means.

29. The bioartificial liver of claim 27 wherein the cells in each cell chamber are entrapped by a substantially insoluble, biocompatible matrix means formed in situ in said cell chamber.

30. The bioartificial liver of claim 27 further comprising first and second fluid flow inlet manifolds on the first base plate, and first and second fluid flow outlet manifolds on said second base plate.

31. The bioartificial liver according to claim 28 wherein the base plates, media plates and membranes are secured together by fittings to provide a fluid tight device capable of asceptic operation.

32. The bioartificial liver according to claim 28 wherein said cell gel matrix is comprised of a collagen fiber forming mixture of cells in a solution of collagen or collagen and heparan.

33. The bioartificial liver of claim 29 wherein the matrix means is Type I collagen.

34. The bioartificial liver of claim 29 wherein the cells contained within said matrix is liver tumor cells or normal liver cells.

35. The bioartificial liver of claim 16 which is sterilizable and can be loaded with said solution of cells and collagen by injection into the hollow fiber intraluminal space.