The Preparation and Assessment of the Physiological Competence of Isolated Chicken Hepatocytes

Abstract

Liver cells are notoriously difficult to maintain in culture in vitro. The media used for tissue culture contain relatively low concentrations of coenzymes or of the various vitamins of the B group required for the biosynthesis of coenzymes (usually less than 1 mg each/litre) (Eagle & Levintow, 1965). When on transplantation cells are washed, they may lose the water-soluble vitamins and cofactors, unless the solutions used for this purpose contain appropriate concentrations of these substances. When cells cultured in oitro are exposed to alkylating agents, additional loss of cofactors will occur, as a result of alkylation of their constituents, and may result in cell death. Hamster kidney cells, BHK 21/13, cultured in Eagle’s medium etc. (Schoental, 1967), when exposed to about 1 mM-N-methyl-N-nitrosourethane, a carcinogen with potential alkylating action, became ‘mummified’. The cultures could be kept in sterile medium at 37°C for many weeks, but the cells retained their structures and did not undergoautolysis. Evidently all the vital cellular processes had stopped. The concentrations of NAD coenzymes in the kidneys and in the liver are higher than in some other tissues (about 500mg/kg) (cf. Weinhouse, 1955). The liver is particularly vulnerable to the depletion of coenzymes. It would appear advisable to use media with appropriately higher content of nicotinamide (and probably of certain other vitamins of the B group) when hepatocytes are cultured in tiitro, especially when they are treated with potential alkylating agents, so that the non-specific cytotoxic effects due to depletion of coenzymes may be avoided.