Role of serum in maintenance of functional hepatocytes in primary culture.

Abstract

Serum at 5 to 10% is required for maintenance of functional adult rat hepatocytes in primary culture. One effect of the serum is to induce attachment and spreading of hepatocytes on plates as monolayers. Another role is to maintain cell viability for over 2 days. For the first effect, serum could be replaced completely by fibronectin (Fn). The effects of Fn on attachment and spreading of cells were dose-dependent and maximum at 10 micrograms/ml. Cells in serum-free medium on Fn-coated dishes showed similar activities of glycogenolysis and glycogenesis to cells cultured in medium containing 5% calf serum on untreated dishes in response to glucagon, dibutyryl cyclic AMP (bt2c AMP), isoproterenol and insulin. The increase in alkaline phosphatase [EC 3.1.3.1] activity and induction of tyrosine aminotransferase [EC 2.6.1.5] by dexamethasone (Dex) in cells under the two conditions were also similar. However, the inductions of tryptophan oxygenase [EC 1.13.11.11] by Dex, glucagon, and bt2cAMP were 4-7 times higher in cells cultured in serum-free medium. The inductions by Dex plus glucagon in the two types of cultures were inhibited similarly by insulin. In serum-free medium containing Dex and insulin in Fn-coated dishes, the cells survived as monolayers for about 50 h without detachment from the dishes, but for longer survival it was necessary to add 5% serum to the medium. A fraction with a molecular weight of over 50,000 from serum was separated by ultrafiltration and this fraction showed a similar effect to serum in increasing survival. A similar factor, but with about 70 times higher specific activity, was found in an extract of bovine pituitary gland.