Large-scale isolation and production of microcarrier-attached porcine hepatocytes for bioartificial livers

Abstract

The use of hepatocytes in the construction of bioartificial livers (BALs) is currently being investigated. The loading of microcarrier-attached hepatocytes into hollow fibres is one BAL system that has been successful in clinical trials. A reliable method for the isolation and production of large numbers of metabolically-active microcarrier-attached hepatocytes is necessary for this system. In this study, a large volume of pre-perfusion medium and collagenase P was used for harvesting hepatocytes from porcine liver; a cell yield of 1.16 x 10 10 ± 0.36 x 10 10 was obtained and the viability of hepatocytes was more than 92%. By incubating the hepatocytes in DMEM/F12 medium supplemented with 10% foetal calf serum, insulin (0.5 U/ml) and dexamethasone (7.5 μg/ml) in a microcarrier spinner vessel, 80% hepatocyte attachment to microcarrier was achieved at a cell concentration of 2 x 10 9, with cell viability of more than 85% after 24 hours' incubation. These cells were shown to be metabolically active. The method described here could provide sufficient microcarrier-attached hepatocytes for the construction of a BAL.