Expanding hepatocytes in vitro before cell transplantation: donor age‐dependent proliferative capacity of cultured human hepatocytes

Background: For hepatocyte transplantation as well as experimental purposes, it would be advantageous to be able to expand human hepatocytes in vitro. However, under serum‐free conditions, even with supplements of HGF (hepatic growth factor) and EGF (epidermal growth factor), proliferation of human hepatocytes is hampered. The aim of this study was to identify differences in the proliferative capacity of cultured primary human hepatocytes related to the age of the liver donors. Methods: Proliferation was determined by BrdU‐uptake, ploidy was measured using propidium iodide staining and flow cytometry, and the expression of cell cycle related proteins was determined by Western blotting. Results: During the initial culture, juvenile hepatocytes proliferated better than adult hepatocytes. The proliferation rate declined to barely detectable levels after 8 days in culture in both juvenile and adult hepatocytes. The higher proliferative capacity of juvenile hepatocytes was associated with a larger fraction of diploid cells and a higher viability. The expression of regulatory cell cycle related proteins was higher in juvenile than in adult hepatocytes. Conclusions: The proliferation of human hepatocytes in vitro is critically related to a large fraction of diploid hepatocytes. The expression of regulatory cell cycle proteins reflects the proliferative capacity of cultured human hepatocytes. Juvenile as compared to adult human hepatocytes may be better suited for expansion in culture and could have a stronger repopulation capacity in vivo.