Influence of seeding density and extracellular matrix on bile Acid transport and mrp4 expression in sandwich-cultured mouse hepatocytes.
Influence of seeding density and extracellular matrix on bile Acid transport and mrp4 expression in sandwich-cultured mouse hepatocytes.
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DOI：
10.1021/mp900227a
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发表时间：
2010-04-05
影响因子：
4.9
通讯作者：
Brouwer KL
中科院分区：
文献类型：
--
作者：
Swift B;Brouwer KL
本研究考察了接种密度、细胞外基质和培养天数对模型胆汁酸牛磺胆酸盐的胆汁酸转运蛋白和肝胆分布的影响。小鼠肝细胞在6孔BioCoat™平板上以夹心方式培养3~4天，接种密度分别为1×10~6个/孔、1.2×10~6个/孔和1.5×10~6个/孔，表面覆盖™(BC/MG)或胶原蛋白(BC/GC)。较低的接种密度(1.0和1.25×106个/孔)可获得良好的肝细胞形态和胆小管网络的形成，如5-(和6)-羧基-2‘，7’-二氯荧光素的积累所示。总体而言，在BC/GC中，牛磺胆酸的细胞积累量随种植密度的增加而增加，在培养的第3天和第4天，BC/MG中的细胞积累量显著高于BC/GC中培养的肝细胞。一般而言，在体外，牛磺胆酸盐的内在胆汁清除率在较高的播种密度下增加。除多药耐药蛋白4(MRP4)与种植密度呈负相关外，种植密度和细胞外基质对第3天和第4天的胆汁酸转运蛋白水平无明显影响。无论细胞外基质如何，在1.0×106和1.25×106个/孔的接种密度之间，MRP4的表达水平下降了2~3倍；在BC/GC中，在1.25×106和1.5×106个/孔的接种密度之间，MRP4的蛋白水平又下降了3~5倍。结果表明，种植密度、细胞外基质和培养天数对夹心培养小鼠肝细胞MRP4的表达有显著影响。原代小鼠肝细胞接种在BC/MG结构中，密度为1.25×106个/孔或更低，培养3天后，基于所研究的探针，获得了最佳的转运。这项工作证明了三明治培养模型对小鼠肝细胞的适用性。
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 Matigel™ (BC/MG) or gelled-collagen (BC/GC) for 3 or 4 days at seeding densities of 1.0, 1.25 or 1.5 × 106 cells/well. The lower seeding densities of 1.0 and 1.25 × 106 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 ~2- to 3-fold between seeding densities of 1.0 × 106 and 1.25 × 106 cells/well regardless of extracellular matrix; an additional ~3- to 5-fold decrease in Mrp4 protein was noted in BC/GC between seeding densities of 1.25 × 106 and 1.5 × 106 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 × 106 cells/well or below, 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.
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影响因子：
2.9
作者：
DUNN, JCY;TOMPKINS, RG;YARMUSH, ML
通讯作者：
YARMUSH, ML
DOI：
10.1073/pnas.87.9.3557
发表时间：
1990-05-01
影响因子：
11.1
作者：
KITAMURA, T;JANSEN, P;ARIAS, IM
通讯作者：
ARIAS, IM
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4.5
作者：
Funk, C;Pantze, M;Gasser, R
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Gasser, R
影响因子：
3.7
作者：
Hoffmaster, KA;Turncliff, RZ;Brouwer, KLR
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Brouwer, KLR
DOI：
10.1124/jpet.106.112136
发表时间：
2007-01-01
影响因子：
3.5
作者：
Faucette, Stephanie R.;Zhang, Tong-Cun;Wang, Hongbing
通讯作者：
Wang, Hongbing