Extra collagen overlay prolongs the differentiated phenotype in sandwich-cultured rat hepatocytes

Introduction: Sandwich-cultured rat hepatocytes (SCRH) have become an invaluable in vitro model to study hepatic drug disposition. SCRH are maintained between two layers of extracellular matrix. In this configuration, culture periods of 4 days are typically applicable. The aim of the present study was to modify conventional SCRH by applying an additional collagen overlay to prolong the hepatic phenotype in SCRH and thus to extend the applicability of the model. Methods: The cultures receiving an extra top layer ('SCRH-plus' cultures) were compared with the conventional SCRH by testing the morphology, cell functionality, metabolic capacity and Mrp2-activity. Results: In the SCRH-plus cultures, cell functionality, evaluated by measuring urea production, was increased from day 5 onwards, compared to conventional cultures. Furthermore, these cells retained the appearance of typical hepatocytes, in contrast with conventional sandwich cultures which showed rapid dedifferentiation. SCRH-plus exhibited significantly improved metabolic clearance mediated by cytochrome P450 3A compared to conventional SCRH whereas UDP-glucuronosyltransferase-mediated metabolism was unaffected. Both conventional SCRH and SCRH-plus showed extensive biliary networks at day 4 of culture. However, from day 4 onwards, a decline in biliary excretion index (BEI) was observed in the conventional SCRH, while BEI values in SCRH-plus cultures did not decrease until day 7. Discussion: The application of an extra top layer of collagen on the SCRH prolongs their useful life-span to 7 days. Therefore, SCRH-plus cultures will broaden the applications of SCRH in terms of long-term toxicity evaluation and when determining metabolism of low turnover compounds.