Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
Tissue Eng Part A. 2012 Dec;18(23-24):2487-96. doi: 10.1089/ten.TEA.2011.0669. Epub 2012 Sep 27.
Hepatocyte functionality and survival decrease rapidly in culture, and both can be improved using bone marrow-derived mesenchymal stromal cells (MSCs). We have previously described an alternative, more plentiful source of MSCs coming from the perivascular area of the umbilical cord, human umbilical cord perivascular cells (HUCPVCs). Our objective was therefore to ascertain whether HUCPVCs could serve as hepatocyte stromal cells ex vivo. For this purpose, rat hepatocytes were cocultured in contact with HUCPVCs (contact coculture). Also, HUCPVCs were cocultured separated from hepatocytes with a semipermeable membrane (noncontact coculture) to assess soluble factor interactions. Next, an HUCPVC-conditioned medium (CM) was used to investigate the possibility of HUCPVC-free support, while flash-frozen HUCPVCs were employed to investigate the effects of nonsoluble interactions. In all experiments, medium samples were taken daily to assess the production of albumin. Also, at certain days, the levels of cytochrome P450 (CYP) activity and urea secretion were tested. RNA extraction was performed at the end of experiments. Our results show that HUCPVCs in contact and noncontact cocultures with hepatocytes improve albumin gene expression and secretion compared to monoculture. Flash-frozen HUCPVCs had a late improvement in albumin secretion, while CM improved it for a short period. Ureagenesis maintenance was improved by contact coculture and flash-frozen HUCPVCs. CYP activity was significantly increased in the presence of flash-frozen HUCPVCs and in noncontact cocultures. We conclude that HUCPVCs can act as stromal cells for rat hepatocytes, and that soluble and nonsoluble factors induce differential effects on hepatocytes.
肝细胞的功能和存活率在培养中迅速下降，骨髓间充质基质细胞（MSCs）的使用可以改善这两种情况。我们之前描述了一种替代的、更丰富的 MSCs 来源，来自脐带的血管周围区域，人脐带血管周围细胞（HUCPVCs）。因此，我们的目的是确定 HUCPVCs 是否可以作为肝细胞的体外基质细胞。为此，大鼠肝细胞与 HUCPVCs 共培养（接触共培养）。此外，HUCPVCs 与肝细胞分离，用半透膜共培养（非接触共培养），以评估可溶性因子的相互作用。接下来，使用 HUCPVC 条件培养基（CM）来研究无 HUCPVC 支持的可能性，同时使用冷冻 HUCPVCs 来研究非可溶性相互作用的影响。在所有实验中，每天都取培养基样本以评估白蛋白的产生。此外，在某些日子，还测试了细胞色素 P450（CYP）活性和尿素分泌的水平。实验结束时进行 RNA 提取。我们的结果表明，与单核培养相比，HUCPVCs 与肝细胞的接触和非接触共培养可改善白蛋白基因表达和分泌。冷冻 HUCPVCs 对白蛋白分泌的改善是晚期的，而 CM 则在短时间内改善了白蛋白分泌。接触共培养和冷冻 HUCPVCs 可改善尿素生成的维持。CYP 活性在冷冻 HUCPVCs 和非接触共培养存在时显著增加。我们得出结论，HUCPVCs 可以作为大鼠肝细胞的基质细胞，可溶性和非可溶性因子对肝细胞产生不同的影响。
