Department of Ophthalmology, Hallym University College of Medicine, Seoul, Republic of Korea.
Department of Ophthalmology, Seoul National University Bundang Hospital, Seongnam, Gyeonggi, Korea.
Invest Ophthalmol Vis Sci. 2014 Jul 17;55(8):5099-108. doi: 10.1167/iovs.14-14564.
To investigate the most appropriate media condition for the proliferation and functional maintenance of human corneal endothelial cells (HCECs).
We cultured HCECs in traditional media (medium A or D) and in stem cell media (medium E or N). The morphology of the cells was evaluated by inverted microscopy. Collagen, type VIII, alpha 2 and sodium-potassium adenosine triphosphatase (Na(+)-K(+) ATPase) expression were analyzed as differentiation markers. Octamer-binding transcription factor 3/4, glial fibrillary acidic protein, nestin and β-catenin expression were evaluated as stem cell associated proteins. The cell proliferation rate was evaluated with a cell counting kit-8 assay. Wound healing assays were also performed. The transendothelial electrical potential difference (TEPD) value was used to estimate the endothelial cell permeability in vitro.
The proliferation and morphology analyses demonstrated that there were significant differences between the media. The expression of differentiation markers and stem cell-associated proteins was different between the media. Medium D resulted in higher proliferation rates compared with the other media, while still maintaining the differentiation potential and surface marker expression profile characteristic of HCECs. Compared with other media, TEPD was higher in medium N.
Culture medium D was superior to the other media with regard to the expression of stem cell-associated proteins, proliferation, and cell migration. However, medium N was more appropriate than the other three media with regard to maintaining the proper cell shape and function.
研究最适合人角膜内皮细胞（HCEC）增殖和功能维持的培养基条件。
我们将 HCEC 分别在传统培养基（培养基 A 或 D）和干细胞培养基（培养基 E 或 N）中进行培养。通过倒置显微镜评估细胞形态。分析胶原 VIII 型α2 和钠钾三磷酸腺苷酶（Na(+)-K(+)ATPase）的表达作为分化标志物。评估八聚体结合转录因子 3/4、胶质纤维酸性蛋白、巢蛋白和β-连环蛋白的表达作为干细胞相关蛋白。通过细胞计数试剂盒-8 测定评估细胞增殖率。还进行了划痕愈合试验。使用跨内皮电潜能差（TEPD）值来评估体外内皮细胞通透性。
增殖和形态分析表明，培养基之间存在显著差异。分化标志物和干细胞相关蛋白的表达在培养基之间也不同。与其他培养基相比，培养基 D 导致更高的增殖率，同时仍保持 HCEC 的分化潜力和表面标志物表达特征。与其他培养基相比，培养基 N 的 TEPD 更高。
就干细胞相关蛋白表达、增殖和细胞迁移而言，培养基 D 优于其他培养基。然而，与其他三种培养基相比，培养基 N 更适合维持适当的细胞形态和功能。
