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Gary S L Peh, Khadijah Adnan, Benjamin L George, Heng-Pei Ang, Xin-Yi Seah, Donald T H Tan, Jodhbir S Mehta; Characterizing the incorporation of rho-kinase inhibitor Y-27632 in the propagation of human corneal endothelial cells - the dual media approach. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2068.
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To characterize the effect of Y-27632 in the propagation of human (CEnCs) using the dual media approach.
Pairs of donor corneas deemed unsuitable for transplantation were procured for this study. Primary human CEnCs isolated from independent cornea-pairs were propagated using a dual media approach as described , with or without the addition of 10μm Y-27632 - a specific rho-associated protein kinase inhibitor (ROCKi). The use of Y-27632 was initially assessed for its effect at each phase of the cell culture process - for cell adherence (xCelligence assay), cell proliferation (Click-iT EdU by immunofluorescence and BrdU by flow cytometry), and during both regular passaging and cryo-preservation. Comparisons of cultures, with or without Y-27632, were also performed for CEnCs isolated from another three independent cornea-pairs to the third passage.
The use ROCKi Y-27632 significantly improved cell adherence and cell proliferation of cultivated CEnCs. A pro-survival effect was also detected when Y-27632 was used during regular passaging and cryo-preservation of CEnCs. We also showed that incorporating Y27632 into the dual media approach in the expansion of human CEnCs generated a three-fold increase in overall cell yield by the third passage. Primary CEnCs cultured in the presence of Y-27632 were also shown to retain the expressions of characteristic markers indicative of the corneal endothelium.
Our study shows that the inclusion of ROCKi Y-27632 is beneficial for the propagation of primary CEnCs expanded using the dual media approach. It also significantly shortened the length of time required for the cultivation of CEnCs at each passage through the enhancement of cellular adherence, and increasing cell proliferation.
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