Viability and differential function of rainbow trout liver cells in primary culture: Coculture with two permanent fish cells

The study investigates the influence of different culture conditions on att achment, viability and functional status of rainbow trout (Oncorhynchus myk iss) liver cells in primary culture. Cells were isolated by a two-step coll agenase perfusion and incubated in serum-free, chemically defined minimal e ssential medium (MEM), (a) as a monolayer on uncoated PRI-MARIA(R) dishes, (b) as a monolayer on culture dishes coated with calf collagen type 1, anti (c) in coculture with the established fish cell lines RTH-149 or RTG-2. Ce ll attachment was assessed from DNA and protein concentrations per dish: vi ability was estimated from cellular lactate dehydrogenase release, and the metabolic status was investigated by measuring activities of the phosphoeno lpyruvate carboxykinase and biotransformation enzymes as well as the total cytochrome P450 contents. Seeding of hepatocytes on collagen-coated dishes dill not alter cell attachment or detachment from the culture substrate, bu t had a small, but not significant effect on cell viability and metabolic p arameters. Coculture of liver cells and RTG-2, cells reduced hepatocyte det achment from the culture substrate, and it was associated with a significan t elevation of 7-ethoxyresorufin-O-deethylase activities in the hepatic cel ls. Cytochrome P450 contents, however, were not altered. The coculture effe ct on liver cell physiology clearly depended on the type of cell line, beca use coculture with RTH-149 cells led to similar, but much weaker effects th an obtained in cocultures with RTG-2 cells. Electron microscopical observat ions re revealed the existence of gap junctions and possible exocytosis-lik e transport between cell lines and hepatocytes. The results point to the po tential of coculture systems to improve physiological parameters of trout l iver cells in primary culture.