Cells for Sight Transplantation and Research Programme Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK.
Curr Eye Res. 2012 Dec;37(12):1067-74. doi: 10.3109/02713683.2012.713155. Epub 2012 Aug 22.
Aim of this study was to evaluate the efficiency of human MRC-5 cells to act as a feeder layer for conjunctival epithelial cells in order to develop a complete xenobiotic-free culture system for the expansion of conjunctival epithelial progenitor cells for clinical applications.
Human conjunctival epithelial cells were expanded from a bulbar biopsy, in a completely xenobiotic-free culture system using growth arrested MRC-5 cells as a feeder layer, without (MRC-5/No Serum) and in the presence of 5% (MRC-5/HS 5%) or 10% (MRC-5/HS 10%) human serum. The total cell count, the surface area as well as the total colony-forming efficiency (CFE), the percentage of aborted colonies and the expression of putative progenitor cell markers p63α, ABCG2, CK15 was compared to the gold standard culture system (GS) in which growth arrested 3T3 feeder cells and feotal calf serum were used.
The epithelial cell count revealed significantly less proliferation in the MRC-5/No Serum group compared to the GS conditions. All groups showed immunoreactivity to CK19; however, more differentiated epithelial cells were observed in the MRC-5/No Serum- and MRC-5/HS 10%-group and less immunoreactivity to p63 α and ABCG2 was found in these groups compared to GS and MRC-5/HS 5% conditions. This was in accordance with CFE results, were the MRC-5/HS 5% group showed similar CFE results compared to the GS group, while in the MRC-5/No Serum- and MRC-5/HS 10%-group significantly lower CFE's were observed.
Our results indicate that a completely xenobiotic-free culture system using MRC-5 cells as a feeder layer in combination with human serum can be successfully used to expand conjunctival epithelial cells with progenitor cell characteristics and might be a useful tool for the safe expansion of these cells for clinical use.
本研究旨在评估人 MRC-5 细胞作为结膜上皮细胞饲养层的效率，以开发用于临床应用的结膜上皮祖细胞的完全无外源物培养系统。
使用生长停滞的 MRC-5 细胞作为饲养层，在完全无外源物的培养系统中从球结膜活检中扩增人结膜上皮细胞，无（MRC-5/无血清）和存在 5%（MRC-5/HS 5%）或 10%（MRC-5/HS 10%）人血清。比较总细胞计数、表面积以及总集落形成效率（CFE）、流产集落的百分比和假定祖细胞标志物 p63α、ABCG2、CK15 的表达，与使用生长停滞的 3T3 饲养细胞和胎牛血清的金标准培养系统（GS）进行比较。
与 GS 条件相比，MRC-5/无血清组的上皮细胞计数显示增殖明显减少。所有组均对 CK19 呈免疫反应性；然而，在 MRC-5/无血清组和 MRC-5/HS 10%组中观察到更分化的上皮细胞，并且这些组中 p63α 和 ABCG2 的免疫反应性较低，与 GS 和 MRC-5/HS 5%条件相比。这与 CFE 结果一致，其中 MRC-5/HS 5%组与 GS 组具有相似的 CFE 结果，而在 MRC-5/无血清组和 MRC-5/HS 10%组中观察到明显较低的 CFE。
我们的结果表明，使用 MRC-5 细胞作为饲养层的完全无外源物培养系统与人类血清相结合，可成功用于扩增具有祖细胞特征的结膜上皮细胞，可能是用于临床应用的这些细胞安全扩增的有用工具。
