Cultivation of human corneal endothelial cells isolated from paired donor corneas.
Cultivation of human corneal endothelial cells isolated from paired donor corneas.
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DOI：
10.1371/journal.pone.0028310
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Mehta JS
中科院分区：
文献类型：
--
作者：
Peh GS;Toh KP;Wu FY;Tan DT;Mehta JS
人角膜内皮细胞 (hCEC) 的持续扩增对于组织工程内皮结构的开发至关重要。然而，据报道，从不同基础培养基开发的各种复杂培养基可用于 hCEC 的繁殖，其中一些比其他培养基更成功。这些结果因捐赠者之间的差异而进一步混淆。本研究的目的是评估四种培养基对从一系列配对供体角膜中分离的 hCEC 的分离和增殖，以消除供体变异性。分离的原代 hCEC 在本研究中编码为：M1-DMEM 的四种先前发表的培养基中培养； M2-OptiMEM-I； M3-DMEM/F12 和 M4-Ham 的 F12/M199。将在这些条件下建立的原代 hCEC 扩增两代，并分析 (1) 它们粘附和增殖的倾向； (2)特征性角膜内皮标志物Na+K+/ATPase和ZO-1的表达； (3) 在整个研究过程中它们的细胞形态。我们发现，在所有四种培养基中分离的 hCEC 在 FNC 包被的培养皿上培养时均表现出快速附着。然而，在四种培养基中建立的 hCEC 表现出不同的增殖特征，具有显着的形态差异。 M1和M3培养的角膜内皮细胞分别不能增殖超过第一代和第二代。在M2和M4中培养的hCEC明显具有更高的增殖性，并表达人角膜内皮的标志物特征：Na+K+/ATP酶和ZO-1。然而，培养的 hCEC 的独特形态特征在第三次传代之后在 M2 或 M4 中均不再保留。 hCEC 的增殖能力和形态受四种培养基的影响很大。为了使用源自本研究中描述的分离方法的培养 hCEC 开发组织工程移植材料，我们建议使用增殖培养基 M2 或 M4 直至第三代，或在培养的 hCEC 失去其独特的细胞形态之前。
Consistent expansion of human corneal endothelial cells (hCECs) is critical in the development of tissue engineered endothelial constructs. However, a wide range of complex culture media, developed from different basal media have been reported in the propagation of hCECs, some with more success than others. These results are further confounded by donor-to-donor variability. The aim of this study is to evaluate four culture media in the isolation and propagation of hCECs isolated from a series of paired donor corneas in order to negate donor variability. Isolated primary hCECs were cultured in four previously published medium coded in this study as: M1-DMEM; M2-OptiMEM-I; M3-DMEM/F12, & M4-Ham's F12/M199. Primary hCECs established in these conditions were expanded for two passages and analyzed for (1) their propensity to adhere and proliferate; (2) their expression of characteristic corneal endothelium markers: Na+K+/ATPase and ZO-1; and (3) their cellular morphology throughout the study. We found that hCECs isolated in all four media showed rapid attachment when cultured on FNC-coated dishes. However, hCECs established in the four media exhibited different proliferation profiles with striking morphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the first and second passage respectively. The hCECs cultured in M2 and M4 were significantly more proliferative and expressed markers characteristics of human corneal endothelium: Na+K+/ATPase and ZO-1. However, the unique morphological characteristics of cultivated hCECs were not maintained in either M2 or M4 beyond the third passage. The proliferative capacity and morphology of hCECs are vastly affected by the four culture media. For the development of tissue engineered graft materials using cultured hCECs derived from the isolation methodology described in this study, we propose the use of proliferative media M2 or M4 up to the third passage, or before the cultured hCECs lose their unique cellular morphology.
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