Application of primary hepatocyte sandwich cultures for predictive studies of drug metabolism and precarcinogen activation

Abstract

Loss of cytochrome P450 content is a common feature in conventional culture systems of primary hepatocytes. In vivo each hepatocyte is exposed to an extracellular matrix (space of Disse) at two opposing basolateral surfaces. This in vivo symmetry can be reconstructed by culturing hepatocytes within two layers of collagen, thus forming a sandwich configuration. Methods: Metabolism of various model drugs (tacrolimus, Urapidil) and of the environmental pollutant dimethylbenzanthracene (DMBA) was studied in rat and human hepatocytes. Mutagenic effects of precarcinogen activation were studied in a threedimensional coculture model between sandwich hepatocytes and V79 cells using HPRT tests. Metabolites were analyzed by HPLC and LC/MS. Results: Sandwich hepatocytes generated metabolites and correctly reflected species specific metabolic patterns. Maintenance of metabolic properties in hepatocytes was dependent on extracellular matrix geometry. The amount of DMBA induced mutations tended to be higher than in standard S9 Mix assays. Conclusion: This model may be a valuable tool for predictive analysis of species dependent drug biotransformation and toxicological risk assessment of pharmaceutical and environmental compounds.