Primary cultures of adult mouse and rat hepatocytes for studying the metabolism of foreign chemicals

Abstract

A primary hepatocyte culture was developed as a model system to investigate the metabolism of foreign chemicals. Hepatocytes were prepared from adult male Charles River CD-1 mice and adult male Sprague-Dawley rats by in situ pre-perfusion of the liver with ethyleneglycol-bis-(β-amino-ethyl ether) N, N′-tetra-acetic acid (EGTA) followed by perfusion with calcium and collagenase. The digested liver was dispersed, and hepatocytes were isolated by filtration and differential centrifugation yielding 10 8 hepatocytes per mouse liver and 5 × 10 8 hepatocytes per rat liver. More than 90 per cent of the hepatocytes excluded trypan blue. Hepatocytes were prepared aseptically, plated on tissue culture dishes coated with rodent tail collagen (2.5 × 10 6 cells/60 mm dish), and cultured in serum-free modified Waymouth's medium. Within 4hr the hepatocytes attached to the collagen, and by 24 hr they had flattened and formed a monolayer. A non-metabolizable alanine analog, α-aminoisobutyric acid, accumulated in mouse hepatocytes with peak incorporation occurring at 24 hr. Cultured mouse and rat hepatocytes were able to N-demethylate para-chloro- N-methylaniline (PCMA). An NADPH-generating system stimulated N-demethylation 2.75-fold in freshly isolated mouse hepatocytes, but did not stimulate metabolism in cultured mouse hepatocytes. SKF 525-A inhibited PCMA N-demethylation in cultured mouse hepatocytes with an I 50 of 3.75 × 10 −5 M. Hormonal supplementation of the culture medium stimulated PCMA metabolism measured in 24- and 48-hr cultures. These studies demonstrate the utility of rodent hepatocyte cultures as models of hepatic metabolism of foreign chemicals.