Crude liver membrane fractions as substrate preserve liver-specific functions in long-term, serum-free rat hepatocyte cultures

Abstract

Over time, rat hepatocytes cultured on collagen lose the capacity to express liver-specific functions. The influence on this degradation process of an alternative substratum--crude membrane fractions prepared from the liver of the same rat strain--was investigated. Freshly isolated rat hepatocytes were cultured in serum-free Williams E medium supplemented with aprotinin, selenium, dexamethasone, and insulin in flasks coated with a mixture of rat liver crude membrane fractions:collagen type I (100:1). The cells adhered firmly, exhibiting minimal spreading and remaining grouped in columns or in cell islands, and retained their liver-specific functions for more than 1 wk. Hepatocytes secreted substantially higher amounts of albumin than cells cultured on collagen-coated dishes, and on Days 1 and 9 in culture the total P-450 content was 72 and 40%, respectively, of that of freshly isolated cells. On Day 6, the 7-ethoxyresorufin-O-deethylase and the aldrin epoxidase activities were still more than 50% that of freshly isolated hepatocytes. Exposure to phenobarbital on Days 3 to 6 increased the total cytochrome P-450 content twofold; exposure to 3-methylcholanthrene increased the activity of the corresponding cytochrome P-450 isoforms to 20 times that observed in untreated cultures and 6 times that observed in freshly isolated cells. Thus, given the ease with which they are prepared, the use of crude membrane fractions combined with culture medium supplemented with aprotinin and selenium can facilitate the preparation of reproducible cultures suitable for long-term in vitro pharmacotoxicologic studies using rat hepatocytes.