Supplementary Material for: Gene Expression and Functional Analyses of Primary Rat Hepatocytes on Nanofiber Matrices

Long term culture of primary hepatocytes is valuable for diagnostic and therapeutic applications. However, standard monolayer culture of primary hepatocytes on tissue culture plastic (TCP) �C either uncoated or coated with a biological material such as collagen or laminin �C is problematic. Thus, novel support matrices are under development to better maintain gene expression and differentiated function of primary hepatocytes in vitro. In this study, a fabricated nanofiber matrix was compared to control conditions of uncoated and laminin-coated TCP. Gene expression and biochemical analyses were performed to compare functional abilities of the hepatocytes in the different conditions. Hepatocytes cultured on nanofibers maintained higher cytochrome P450 1A activity (0.49 �� 0.08 ng resorufin/ml/min) compared to hepatocytes on laminin (0.11 �� 0.05 ng resorufin/ml/min). In addition, albumin production of hepatocytes on nanofibers was greater than twice the production of hepatocytes on laminin (day 14, 34.4 �� 1.8 vs. 15.9 �� 4.5 ��g albumin/ml/day). Hepatocytes demonstrated the ability to generate urea from ammonia in all conditions; however, hepatocytes performed ureagenesis more effectively on nanofibers than on laminin (0.55 �� 0.25 ��M vs. 0.36 �� 0.24 ��M urea, day 14). Gene expression of hepatocytes cultured on nanofiber and laminin conditions were similar on a per cell basis determined by analysis using a custom microarray of 250 genes expressed in hepatocytes. Similar cell attachment data between conditions and similar numbers of cells expressing the hepatocyte marker hepatocyte nuclear factor 4��indicates that hepatocytes grown on nanofibers only marginally display improved hepatic functions compared to laminin control conditions.原代肝细胞的长期培养在诊断与治疗领域具有重要应用价值。然而，在组织培养塑料（tissue culture plastic, TCP）表面进行的标准原代肝细胞单层培养——无论未包被还是经胶原蛋白、层粘连蛋白等生物材料包被——均存在一定缺陷。因此，研究人员正开发新型支撑基质，以在体外更好地维持原代肝细胞的基因表达与分化功能。本研究将制备得到的纳米纤维基质与未包被及层粘连蛋白包被的TCP对照组进行了对比。通过基因表达与生化分析，对不同培养条件下肝细胞的功能能力进行了比较。与层粘连蛋白包被组的肝细胞（0.11 ± 0.05 ng 试卤灵/毫升/分钟）相比，纳米纤维培养组的肝细胞可维持更高的细胞色素P450 1A（cytochrome P450 1A）活性（0.49 ± 0.08 ng 试卤灵/毫升/分钟）。此外，纳米纤维培养组肝细胞的白蛋白分泌量是层粘连蛋白包被组的两倍以上（第14天：34.4 ± 1.8 μg 白蛋白/毫升/天 vs. 15.9 ± 4.5 μg 白蛋白/毫升/天）。所有培养条件下的肝细胞均具备将氨转化为尿素的能力；但纳米纤维培养组肝细胞的尿素合成效率更高（第14天：0.55 ± 0.25 μM 尿素 vs. 0.36 ± 0.24 μM 尿素）。通过针对肝细胞表达的250个基因定制的微阵列分析，以每细胞为基准进行比较，纳米纤维组与层粘连蛋白包被组肝细胞的基因表达水平相似。不同组的细胞粘附数据相似，且表达肝细胞标志物肝细胞核因子4α（hepatocyte nuclear factor 4α）的细胞数量也无显著差异，这表明与层粘连蛋白对照组相比，纳米纤维培养的肝细胞仅在肝功能方面存在小幅提升。