Enzymatic Function of Atginate Immobilized Rat Hepatocytes Ronald G. Tompkins* and Edward A. Carter zyxwvu The Surgical Service of the Massachusetts General Hospital, The Department of Surgery, Harvard Medical School, Boston, Massachusetts 02 1 14 James D. Carlson and Martin L. Yarmush The Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139 Accepted for publication December 30, 1986 zyxwvut The use of immobilized hepatocytes represents a promis- ing approach for the problem of detoxification in acute hepatic failure. Hepatocyte viability and detoxification function of a number of complex enzyme systems were examined before and after immobilization in alginate droplets. Detoxification function was assessed quan- titatively by measuring the kinetics of several specific detoxification systems: the cytochrome P450 system, the urea cycle, and two conjugation systems. Reaction rates for all enzyme systems were similar in immobilized and nonimmobilized cells, and were in good agreement with previously published literature values. These results indi- cate that transport limitations do not occur in these gels and that the intrinsic reaction rate is the limiting step. Feasibility of detoxification replacement by immobilized zyxwvut cells is discussed using measured reaction rates. INTRODUCTION The liver is an indispensable, complex organ that is vital to the functions of metabolism, excretion, detoxification, storage, and phagocytosis. zyxwvutsr At present, the only treatments generally offered for liver failure are supportive care and liver transplants. A number of liver substitutes have been explored including, hernodialysis,'*' hemoperfusion over various ~orbents,~-* exchange transfusions,' and plasma cross perfusions . 'OJ' Of these methods, only coated charcoal hemoperfusion has been studied using a randomized trial in animals and clinical trials in humans with any consistent success. Both a temporary recovery of consciousness and improved survival has been shown with coated charcoal hemoperfusion in acetaminophen-induced fulminant liver failure in humans. 12313 Another promising approach for restoring liver function involves the use of isolated hepatocytes which are entrapped * To whom all correspondence should zyxwvutsr be addressed. Present address: Department of Surgery, ACC 3A, Suite 364, MassachusettsGeneral Hos- pital, Boston, Massachusetts 02114. within a semipermeable membrane as previously suggested by Chang.14 Such a membrane would protect the cells from an immune reaction if implanted, and would similarly pro- tect the cells from shear-related damage if used as an extra- corporeal bioreactor. Alginate, a heteropolysaccaride from brown seaweed, has recently been used to encapsulate a variety of animal and microbial Alginate gels under mild conditions, and gel formation is easily reversed allowing the immobilized cells to be released and their viability and function studied. In addition, gels can be formed in a wide variety of configurations such as spheres, films, or fibers providing versatility in design of a bio- reactor. Alginate would be particularly advantageous as an immobilization substrate for hepatocytes provided the immobilization process did not damage cellular function. The present report documents kinetics of several enzyme systems in immobilized and nonimmobilized hepatocytes. Results indicate that immobilization of the hepatocytes does not alter the viability of hepatocytes nor their enzymatic function. Results of immobilized cells are compared to pre- viously published results for nonimmobilized cells, and dis- cussed in light of eventual feasibility as an artificial liver. METHODS Preparation of Hepatocyte Cultures Hepatocytes were isolated by zyxw in situ collagenase per- fusion of female Sprague-Dawley rats (150-180 g; Charles River Breeding Laboratories) by modification of established methods. 2526 After an intraperitoneal injection of sodium methohexital(O.05 mg/g body wt), the portal vein and infe- nor vena cava above the diaphragm were cannulated and the liver was perfused with calcium-free Krebs-Ringer bicar- bonate buffer (pH 7.4, 37°C) containing 5.5mM glucose at 50-60 mL/min in a temperature-controlled jacketed hood. The perfusate was equilibrated with 95% O2 and 5% C02 using a membrane oxygenator. After perfusion with 500 mL Biotechnology and Bioengineering, Vol. 31, Pp. 11-18 (1988) 01988 John Wiley & Sons, Inc. CCC 0006-3592/88/010011-08$04.00