Multiporous cellulose microcarrier for the development of a hybrid artificial liver using isolated hepatocytes.

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.

MATERIALS AND 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.