Peh Gary S L, Adnan Khadijah, George Benjamin L, Ang Heng-Pei, Seah Xin-Yi, Tan Donald T, Mehta Jodhbir S
1] Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore [2] Duke-NUS Graduate Medical School, Singapore.
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore.
Sci Rep. 2015 Mar 16;5:9167. doi: 10.1038/srep09167.
The global shortage of donor corneas has garnered extensive interest in the development of graft alternatives suitable for endothelial keratoplasty using cultivated primary human corneal endothelial cells (CECs). We have recently described a dual media approach for the propagation of human CECs. In this work, we characterize the effects of a Rho-kinase inhibitor Y-27632 on the cultivation of CECs propagated using the dual media culture system. Seventy donor corneas deemed unsuitable for transplantation were procured for this study. We assessed the use of Y-27632 for its effect at each stage of the cell culture process, specifically for cell attachment, cell proliferation, and during both regular passaging and cryopreservation. Lastly, comparison of donor-matched CEC-cultures expanded with or without Y-27632 was also performed. Our results showed that Y-27632 significantly improved the attachment and proliferation of primary CECs. A non-significant pro-survival effect was detected during regular cellular passage when CECs were pre-treated with Y-27632, an effect that became more evident during cryopreservation. Our study showed that the inclusion of Y-27632 was beneficial for the propagation of primary CECs expanded via the dual media approach, and was able to increase overall cell yield by between 1.96 to 3.36 fold.
全球供体角膜短缺引发了人们对开发适用于使用培养的原代人角膜内皮细胞（CEC）进行内皮角膜移植的移植替代物的广泛关注。我们最近描述了一种用于人CEC增殖的双培养基方法。在这项工作中，我们表征了Rho激酶抑制剂Y-27632对使用双培养基培养系统增殖的CEC培养的影响。本研究获取了70个被认为不适合移植的供体角膜。我们评估了Y-27632在细胞培养过程各阶段的作用，特别是对细胞附着、细胞增殖以及在常规传代和冷冻保存期间的作用。最后，还对添加或不添加Y-27632扩增的供体匹配CEC培养物进行了比较。我们的结果表明，Y-27632显著改善了原代CEC的附着和增殖。当CEC用Y-27632预处理时，在常规细胞传代期间检测到非显著的促存活作用，这种作用在冷冻保存期间变得更加明显。我们的研究表明，添加Y-27632有利于通过双培养基方法扩增的原代CEC的增殖，并且能够使总体细胞产量提高1.96至3.36倍。
