PERIFUSED MONOLAYER-CULTURES OF RAT HEPATOCYTES AS AN IMPROVED INVITRO SYSTEM FOR STUDIES ON UREOGENESIS

A perifusion system has been developed for cultivation of adult rat hepatocytes, permitting continuous supply of medium to the cell monolayer instead of periodical changes as used in conventional culture technique (discontinuous culture). Additionally, a modification of Waymouth's MB 752/1 medium is described, which favoured the expression of several metabolic and regulatory events mentioned below and supported the maintenance of several enzymes involved in nitrogen metabolism. The improved nutritional conditions accelerated early monolayer formation and metabolic recovery, and favoured long-term cultivation of metabolically active and hormone responsive hepatocytes. Urea formation from substrates contained in the medium was found to be 2- to 3-fold higher and preserved for a considerably longer time than with discontinuously cultured cells, and was further enhanced by addition of tryptose phosphate broth or 4 mM NH4Cl even after 10 days in culture. In the presence of glucagon (10-7 M) the urea production was more than doubled during a 24 h incubation period on the 4th day. Pretreatment with this hormone for 24 h also markedly stimulated the capacity of perifused cells for ureogenesis. Concomitantly, a rise in arginase activity up to 2-fold could be measured in response to glucagon, which was largely suppressed by simultaneous presence of leucine in concentrations between 5 and 10 mM. © 1979.