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Gary S. Peh, Kah-Peng Toh, Deepashree Balehosur, Heng-Pei Ang, Man-Xin Lee, Donald T. Tan, Jodhbir Mehta; Isolation and Propagation of Human Corneal Endothelial Cells Using a Dual Media Culture System. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6013.
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© ARVO (1962-2015); The Authors (2016-present)
A novel culture system for the in vitro propagation of isolated human corneal endothelial cells (hCECs) using a dual media approach.
Cultures of primary hCECs isolated from pairs of donor corneas deemed unsuitable for transplantation were established and propagated using a novel dual media culture system. Isolated cells were expanded in a medium that supported the proliferation of hCECs, identified and coded in our previous study as M4 - a 1:1 mixture of the basal media Ham’s F12 and M199 (F99). The effect of incorporating a second medium, coded here as M5, into the abovementioned culture condition at least 24 hours before and after cellular passage, was examined in this study. Primary hCECs were established and cultured in each of these conditions for five passages: (A) M4 only, (B) M5 only, or (C) using M5 to stabilize and maintain the cultivated hCECs before and after the proliferative phase, with M4 as the medium utilized to promote the proliferation of hCECs. The cultured hCECs from each condition were analyzed for (1) their cellular morphology, specifically their circularity; (2) their propensity to proliferate in each of these conditions; and (3) their expression of markers indicative of corneal endothelium such as Na+K+ATPase and ZO-1.
Established primary hCECs cultures propagated using the dual media culture system could be serially passaged and were able to retain the unique polygonal morphology of the hCECs. Cultured cells also retained markers indicative of corneal endothelium. In contrast, most hCECs expanded in M4 alone appeared to have lost the unique polygonal morphology of hCECs and turned fibroblastic-like, some as early as the third passage, and the usage of M5 only were not able to support the active proliferation of hCECs.
The unique morphology of cultured hCECs can be maintained using the novel dual media culture system described in this study. We propose the use of the proliferative medium M4 followed by the maintenance medium M5 for the extended propagation of hCECs.
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