Derivation of phenobarbital-responsive immortal rat hepatocytes

Two lines of rat hepatocytes, designated 6/15 and 6/27, were obtained from carcinogen-treated livers by cultivation in medium containing the liver tumor promoter, phenobarbital (PB). Both lines appeared to be PB-responsive and to have an unlimited in vitro proliferative lifespan, i.e., immortality. The ability of pure 6/27 hepatocytes to form colonies from single cells was strictly dependent upon PB; it was reduced by 97 to 99% in the absence of PB. These hepatocytes were not tumorigenic. For 6/27 hepatocytes in early passages where cultures contained fibroblast contaminants and later when they were a pure culture, PB was able to enhance colony growth from single cells and facilitated population expansion by sustaining DNA synthesis and by inhibiting cell lysis. The 6/15 line displayed PB-dependent colony formation and was not tumorigenic at early passages. At later passages 6/15 hepatocytes were less dependent on PB for colony formation, and they formed hepatocellular carcinoma when transplanted into livers of syngeneic rats. The demonstration that PB sustained the proliferation and viability of hepatocytes with enhanced growth capacity and indefinite proliferative lifespan suggests that PB may be necessary for progression of these chemically initiated hepatocytes to immortal and tumorigenic lines in vitro.