Long-term culture of primary human hepatocytes with preservation of proliferative capacity and differentiated functions

Background. The aim of this study was to develop a suitable method for the prolonged culture and maintenance of human hepatocytes with preservation of both proliferative capacity and differentiated functions. Materials and methods. Primary human hepatocytes were isolated from small pieces of liver tissue obtained from 15 patients who underwent hepatic resection. Hepatocytes were cultured in keratinocyte-stimulating factor medium supplemented with 10% human serum, 10 mM nicotinamide, 10 ng/ml epidermal growth factor, 0.5 μg/ml insulin, 103⁷ M dexamethasone, and antibiotics. Hepatic differentiation and function were analyzed by immunocytochemistry, Western blot, ELISA, lidocaine metabolism, and urea synthesis. Ultrastructural analysis of cultured hepatocytes was performed by electron microscopy. Results. Many primary hepatocytes were maintained for more than 56 days. Hepatocytes proliferated during the initial 14 days, and bromodeoxyuridine labeling indices were 15.2, 12.2, and 6.2% at days 5, 10, and 15, respectively. Electron micrographs of the hepatocytes at day 28 demonstrated numerous mitochondria, rough endoplasmic reticulum, large peroxisomes, and glycogen granules. Albumin secretion increased for the first 14 days and then gradually decreased thereafter but was maintained at levels greater than 2 μg/ml/h until day 56. α₁-Antitrypsin, α₁-antichymotrypsin, and ceruloplasmin production was also observed at day 56, while lidocaine metabolism and urea synthesis were maintained for a long time. Conclusion. This hepatocyte culture method facilitates the prolonged culture of primary human hepatocytes with preservation of hepatocyte differentiation, function, and proliferative capacity.