Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7569, USA.
Mol Pharm. 2010 Apr 5;7(2):491-500. doi: 10.1021/mp900227a.
This study was undertaken to examine the influence of seeding density, extracellular matrix and days in culture on bile acid transport proteins and hepatobiliary disposition of the model bile acid taurocholate. Mouse hepatocytes were cultured in a sandwich configuration on six-well Biocoat plates with an overlay of Matrigel (BC/MG) or gelled-collagen (BC/GC) for 3 or 4 days at seeding densities of 1.0, 1.25, or 1.5 x 10(6) cells/well. The lower seeding densities of 1.0 and 1.25 x 10(6) cells/well resulted in good hepatocyte morphology and bile canalicular network formation, as visualized by 5-(and 6)-carboxy-2',7'-dichlorofluorescein accumulation. In general, taurocholate cellular accumulation tended to increase as a function of seeding density in BC/GC; cellular accumulation was significantly increased in hepatocytes cultured in BC/MG compared to BC/GC at the same seeding density on both days 3 and 4 of culture. In general, in vitro intrinsic biliary clearance of taurocholate was increased at higher seeding densities. Levels of bile acid transport proteins on days 3 and 4 were not markedly influenced by seeding density or extracellular matrix except for multidrug resistance protein 4 (Mrp4), which was inversely related to seeding density. Mrp4 levels decreased approximately 2- to 3-fold between seeding densities of 1.0 x 10(6) and 1.25 x 10(6) cells/well regardless of extracellular matrix; an additional approximately 3- to 5-fold decrease in Mrp4 protein was noted in BC/GC between seeding densities of 1.25 x 10(6) and 1.5 x 10(6) cells/well. Results suggest that seeding density, extracellular matrix and days in culture profoundly influence Mrp4 expression in sandwich-cultured mouse hepatocytes. Primary mouse hepatocytes seeded in a BC/MG configuration at densities of 1.25 x 10(6) cells/well and 1.0 x 10(6), and cultured for 3 days, yielded optimal transport based on the probes studied. This work demonstrates the applicability of the sandwich-cultured model to mouse hepatocytes.
本研究旨在探讨细胞接种密度、细胞外基质和培养天数对胆汁酸转运蛋白的影响，以及对模型胆汁酸牛磺胆酸钠的肝胆处置的影响。将小鼠肝细胞以三明治构型培养在六孔 Biocoat 板上，在其上覆盖 Matrigel（BC/MG）或胶凝胶原（BC/GC），接种密度分别为 1.0、1.25 或 1.5 x 10(6)个细胞/孔，培养 3 或 4 天。较低的接种密度 1.0 和 1.25 x 10(6)个细胞/孔可导致良好的肝细胞形态和胆小管网络形成，如 5-(和 6)-羧基-2',7'-二氯荧光素积累所可视化的。一般来说，牛磺胆酸钠的细胞内积累随着 BC/GC 中的接种密度而增加；与 BC/GC 相比，在培养的第 3 天和第 4 天，在相同的接种密度下，BC/MG 中培养的肝细胞中的细胞内积累显著增加。一般来说，牛磺胆酸钠的体外内在胆汁清除率随着接种密度的增加而增加。在第 3 天和第 4 天，胆汁酸转运蛋白的水平不受接种密度或细胞外基质的显著影响，除了多药耐药蛋白 4（Mrp4），其与接种密度呈反比。无论细胞外基质如何，Mrp4 水平在接种密度为 1.0 x 10(6)和 1.25 x 10(6)个细胞/孔之间降低约 2-3 倍；在 BC/GC 中，在接种密度为 1.25 x 10(6)和 1.5 x 10(6)个细胞/孔之间，Mrp4 蛋白进一步降低约 3-5 倍。结果表明，接种密度、细胞外基质和培养天数对三明治培养的小鼠肝细胞中 Mrp4 表达有深远影响。以 1.25 x 10(6)个细胞/孔和 1.0 x 10(6)个细胞/孔的密度接种在 BC/MG 配置中的原代小鼠肝细胞，并培养 3 天，可根据研究的探针获得最佳的转运。这项工作证明了三明治培养模型在小鼠肝细胞中的适用性。
