Promoting effect of feeder cells in maintenance of adult rat hepatocytes.

A procedure is described for maintaining primary cultures of adult rat hepatocytes for prolonged periods of time on layer of irradiated mouse fibroblast cell line (C3H/1OT1/2) and on a secondary lung fibroblasts obtained from Sprague Dawley rats. Morphologically and ultrastructurally the cocultivated hepatocytes retained many characteristics of hepatocytes in vivo. Within 24 hours after seeding, the individual cells were attached on the feeder cell layer and the in vivo polarity of the liver cells reappeared. Electron microscope studies demonstrated the appearance of newly developed bile ducts and junctions between hepatocytes as well as between hepatocytes and feeder cells. Histochemically, these cells were positive for glucose-6-phosphatase and for glycogen. After 14 days in culture the hepatocytes could be reseeded onto fresh C3H1OT1/2 cells. In contrast, hepatocytes maintained on plastic substrate lost their glycogen content and the epithelial character of the liver cells after 5 days in culture, and by day 10 this culture became predominantly fibroblastic. It is suggested that hepatocytes maintained on an irradiated fibroblast feeder layer provide a valuable approach for studying the morphogenesis, cytotoxicity, or the metabolism of different chemicals in vitro.