Immortalization of differentiated human hepatocytes by a combination of a viral vector and collagen gel culture.

Adult human and chimpanzee hepatocytes, which have no proliferative potential in vitro, were immortalized by introducing an oncogene encoding simian virus 40 large tumor(T) antigen by means of infection with a recombinant adenovirus vector. The frequency of immortalization was enhanced by a suspension method for virus adsorption and especially by using a collagen gel culture. The replicating hepatocyte colonies were isolated and selected by albumin secretion which is specific for parenchymal hepatocytes, then continuously subcultured. The immortalized hepatocyte lines also secreted other hepatocyte-specific proteins (complement C3, transferrin, fibrinogen). However, the amount of protein secretion declined with subculture. The subline RY5, cloned from a human immortalized line, showed relatively stable albumin secretion. Therefore, chromosomal studies and anchorage-independent growth properties were discussed and compared with its original line.