Oorts Marlies, Keemink Janneke, Deferm Neel, Adriaensen Robin, Richert Lysiane, Augustijns Patrick, Annaert Pieter
Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, O&N2, Herestraat 49 - Box 921, 3000 Leuven, Belgium.
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.
J Pharmacol Toxicol Methods. 2018 Mar-Apr;90:31-38. doi: 10.1016/j.vascn.2017.10.007. Epub 2017 Oct 25.
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.
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.
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.
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.
三明治培养的大鼠肝细胞（SCRH）已成为研究肝脏药物处置的一种极其重要的体外模型。SCRH维持在两层细胞外基质之间。在这种培养形式下，通常适用4天的培养周期。本研究的目的是通过额外添加一层胶原蛋白覆盖物来改良传统的SCRH，以延长SCRH的肝脏表型，从而扩大该模型的适用性。
通过检测形态、细胞功能、代谢能力和Mrp2活性，将接受额外顶层（“SCRH-plus”培养物）的培养物与传统SCRH进行比较。
在SCRH-plus培养物中，与传统培养物相比，从第5天起，通过测量尿素生成评估的细胞功能有所增强。此外，这些细胞保持了典型肝细胞的外观，而传统三明治培养物则显示出快速去分化。与传统SCRH相比，SCRH-plus的细胞色素P450 3A介导的代谢清除率显著提高，而UDP-葡萄糖醛酸基转移酶介导的代谢未受影响。在培养第4天，传统SCRH和SCRH-plus均显示出广泛的胆管网络。然而，从第4天起，在传统SCRH中观察到胆汁排泄指数（BEI）下降，而SCRH-plus培养物中的BEI值直到第7天才下降。
在SCRH上额外添加一层胶原蛋白可将其使用寿命延长至7天。因此，SCRH-plus培养物将在长期毒性评估以及测定低周转率化合物的代谢方面扩大SCRH的应用范围。
