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BACKGROUND This study was aimed at developing an optimal method for immobilizing isolated hepatocytes in cellulose multiporous microcarriers (MCs) and evaluating the metabolic activity of MC-immobilized hepatocytes. METHODS Hepatocytes isolated from the livers of male Wistar rats were immobilized in collagen-coated MCs by intermittent stirring (30 rpm for 2 min per 15 min) for 180 min or accumulation methods. The accumulation method was performed by pouring aliquots of hepatocyte suspension (8 x 10(5)) and MC suspension (1 mg) in turn onto a nylon mesh (pore size: 100 micron). The metabolic activity of MC-immobilized hepatocytes in floating culture and in a newly developed bioreactor was evaluated. The metabolic activity of MC-immobilized hepatocytes in the bioreactor was also evaluated in in vitro perfusion of a hollow-fiber-based hybrid artificial liver support system. RESULTS The accumulation method immobilized 20 times more hepatocytes in collagen-coated MCs than the intermittent stirring method (P < 0.01). Morphological observation of hepatocyte-immobilized MCs revealed that many hepatocytes were immobilized deep within the MCs maintaining a spherical shape and normal microvilli on their surface. MC-immobilized hepatocytes in floating culture revealed similar NH3 metabolism and glucose synthesis to monolayer-cultured hepatocytes, and this metabolic activity was maintained during 9h of floating culture. MC-immobilized hepatocytes in a bioreactor also showed similar NH3 metabolism to monolayer-cultured hepatocytes. The NH3 metabolism of MC-immobilized hepatocytes in in vitro perfusion of a hybrid artificial liver support system was 241.5 microg/h/mg protein/m2 membrane surface. CONCLUSIONS The results of this study indicate that the accumulation method was optimal for immobilizing isolated rat hepatocytes in MCs and that MC-immobilized hepatocytes maintained their metabolic activity for a long period.
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