LONG-TERM CULTIVATION OF ADULT-RAT HEPATOCYTES THAT UNDERGO MULTIPLE CELL DIVISIONS AND EXPRESS NORMAL PARENCHYMAL PHENOTYPES

The present study succeeded in cultivating normal adult rat hepatocyte s for at least 85 days without losing their replicative potential and differentiation capacity. Small pieces of hepatocyte aggregates (clust ers) were prepared from the primary culture hepatocytes and used as st arting material for the growth experiment. Some of the hepatocytes sta rted to proliferate at 3 days when the clusters were cultured in Dulbe cco's modified Eagle's medium containing 10% fetal bovine serum, 10 ng /ml epidermal growth factor, 10 mmol/L nicotinamide, 0.2 mmol/L L-asco rbic acid 2-phosphate, and 1% dimethylsulfoxide. Clusters continued to grow and formed colonies. All the cells covering colonies expressed n ormal hepatocyte-specific proteins. The number of albumin-expressing c ells in the most replicative colonies increased sixfold during 32 days . Most of the cells were mononucleate and small in size and some of th em expressed immature hepatocyte markers such as alpha-fetoprotein. El ectron microscopy of cells in colonies revealed the presence of peroxi somes in the cytoplasm and desosomes, tight junctions, and bile canali culus-like structures between the cells. Depletion of one of the addit ives inhibited the growth of hepatocytes. The culture medium used also supported the growth of stellate cells (Ito cells) that had contamina ted the original preparation in small numbers and seems to cooperative ly stimulate a proliferative population of hepatocytes.