Quantitative assay for albumin-producing liver cells after simian virus 40 transformation of rat hepatocytes maintained in chemically defined medium

Abstract

Transformation of rat hepatocytes by simian virus 40 in chemically defined medium was examined. When hepatocytes plated on collagen-coated plates were infected with simian virus 40, colonies of replicating cells appeared as early as 40 days after infection, whereas no colonies appeared in control cultures. Medium from 85% of the transformed cultures contained albumin. When collagen was eliminated and hepatocytes were plated on Primaria plastic cell culture dishes, transformation occurred; medium from 86% of the transformed cultures contained albumin but the maximum albumin level secreted per culture was only 62% of that produced by cultures on collagen-coated plates. Quantitative assays for transformation were established. Transformation was linear after infection with 2-50 plaque-forming units of virus per hepatocyte, and the transformation frequency was the same on the two plating surfaces. An immuno-overlay technique made it possible to identify, purify, and determine the morphology of the albumin-producing cells. When ornithine was substituted for arginine in the medium, the transformation frequency decreased markedly while the percentage of colonies producing albumin increased from 30% to 100%. We conclude that we have defined an assay for quantifying transformation of a normal hepatocyte population and for identifying and enumerating epithelial liver cell transformants that produce albumin.