Marlies Oorts1, Janneke Keemink2, Neel Deferm1, Robin Adriaensen3, Lysiane Richert4, Patrick Augustijns1, Pieter Annaert5. 1. Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49 - Box 921, 3000 Leuven, Belgium. 2. Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49 - Box 921, 3000 Leuven, Belgium; Department of Pharmacy, Uppsala University, Box 580, 75123 Uppsala, Sweden. 3. Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49 - Box 921, 3000 Leuven, Belgium; Quality Assurance, GSK Consumer Healthcare Benelux, Site Apollo, Avenue Pascal 2-4-6, 1300 Wavre, Belgium. 4. KaLy-Cell, 20A rue du Général Leclerc, 67115 Plobsheim, France; Université de Bourgogne Franche-Comté, EA 4267, Besançon, France. 5. Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49 - Box 921, 3000 Leuven, Belgium. Electronic address: pieter.annaert@kuleuven.be.
Abstract
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 4days 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 7days. 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.
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 4days 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 7days. 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.
Authors: Mark T Miedel; Dillon C Gavlock; Shanhang Jia; Albert Gough; D Lansing Taylor; Andrew M Stern Journal: Sci Rep Date: 2019-06-06 Impact factor: 4.379