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It has been reported that small hepatocytes need to be cocultivated with nonparenchymal cells for proliferation in vitro. The purpose of this study was to investigate whether purified small hepatocytes would proliferate in the absence of nonparenchymal cells and to determine the their metabolic activities in culture. Purified small hepatocytes were obtained by centrifugation in Percoll density gradients. Small hepatocytes were seeded on collagen-coated dishes at a cell density of 4 × 104 cells/cm2 and were cultured in a hormonally modified Williams' medium E with 10 mM nicotinamide (WE-HN: group I). Comparision of 1 mM dimethyl sulfoxide (DMSO) (group II) or 5 mM retinoic acid (group III) in WE-HN was performed. Cell proliferation, as indicated by DNA content, and metabolic function, as assessed by urea synthesis, gluconeogenesis, and albumin production, were measured for 14 days. In each group, DNA content increased significantly during 14 days in culture. An increase of approximately 400% was observed in group II. The cell proliferation of group II was confirmed by microscopic observations. Urea synthesis and gluconeogenesis in each group declined gradually from days 1 to 14. Albumin production also fell, but to a less extent. We found that the purified small hepatocytes could proliferate without cocultivation of nonparenchymal cells. Further, they expressed metabolic activities of mature hepatocytes. |
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