A long-term culture of human hepatocytes which show a high growth potential and express their differentiated phenotypes

The present study succeeded for the first time in cultivating for more than 2 months human normal hepatocytes which showed a high growth potential and expressed their differentiated phenotypes. Constituents of culture medium were critical for this culture, and the medium optimized for their growth c ontained fresh human serum, fetal bovine serum, Swiss 3T3-cell conditioned medium, L-ascorbic acid a-phosphate, epidermal growth factor, nicotinamide, and dimethyl sulfoxide. Hepatocytes steadily replicated and formed colonie s which continued to increase in size up to around 35 days. The number of h epatocytes in the most replicative colonies increased 17-fold during 31 day s. Cells in colonies expressed normal differentiated hepatocytic phenotypes for as long as 35 days. These hepatocytes retained normal liver functions at least for 70 days such as to secrete albumin, and to metabolize lidocain e and D-galactose. (C) 1999 Academic Press.