Small scale rat hepatocyte primary culture with applications for screening hepatoprotective substances.

Isolated hepatocytes are known to maintain their physiological functions for over a week when cultured on Matrigel, artificially reconstituted from basement membrane components. Although this culture technique has been frequently used in research on hepatocyte functions, there has been a limitation on its application for small scale experiments due to some technical problems. By using micro-culture plates with 96 round-bottom wells, we succeeded in coating the wells uniformly with Matrigel. When the cultured hepatocytes were treated with either 10 mM, 15 mM, or 20 mM of acetaminophen or 1 mM, 10 mM, or 20 mM of D-galactosamine, the viability of the hepatocytes became 91.1%, 75.3%, 64.7%, and 79.0%, 43.8%, 26.2% of the non-treated control at 48 hours, respectively. Fractionated extracts of Glycyrrhiza glabra L. and Schisandra chinensis Baillon inhibited the action of acetaminophen or D-galactosamine in this model. From these results, we concluded that the microculture system presented here is capable of maintaining the in vivo characteristics of hepatocytes and is suitable for the screening of hepatoprotective substances.