Optimization of hepatocyte spheroid formation for hepatic tissue engineering on three-dimensional biodegradable polymer within a flow bioreactor prior to implantation

We hypothesize that in vitro conditioning of hepatocytes within biodegradab le poly-L-lactic acid (PLLA) polymer matrices prior to implantation may inc rease hepatocyte survival and function after transplantation. The purpose o f this study was to optimize the culture conditions of hepatocytes in a pul satile flow bioreactor. PLLA discs were seeded with rat hepatocytes in a co ncentration of 2.5, 5, 10, 20 and 40 x 10(6) cells/ml. Seeded discs were ex posed to recirculated perpendicular flow of 0, 7, 15, 24, 32, 52 ml/min of supplemented Williams' Medium E and harvested after 6 days in flow culture. Only under flow conditions the hepatocytes formed spheroidal aggregates (S phA) of 50-260 mum in diameter with a liver-like morphology and active meta bolic function. The number of SphA was examined by phase contrast microscop y and the reductive enzyme function of the hepatocytes was tested using MTT . Hematoxylin and eosin histology showed vital hepatocytes within the SphA less than 200 mum in diameter but central necrosis in the SphA exceeding th is size. Immunohistochemical staining confirmed albumin production of hepat ocytes within the SphA. The optimal cell seeding concentration was 10 x 10( 6) cells/ml with a flow speed of 24 ml/min. SphA of hepatocytes cultured wi th this flow bioreactor method may prove useful as a functional unit for ti ssue engineering of an in vivo liver substitute. Copyright (C) 2001 S. Karg er AG, Basel.