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Changkai Jia, Sanming Li, Yi Liao, Juan Li, Liying Zhang, Wei Li, Zuguo Liu; Rock inhibitor facilitates mouse and rat corneal epithelial cells culture. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4382.
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© ARVO (1962-2015); The Authors (2016-present)
In vitro primary culture of mouse or rat corneal epithelial cells (CECs) remains as a big obstacle to be resolved, which largely hinders our understanding about the molecular mechanisms to maintain the phenotypes as well as physiological functions of corneal epithelial cells. Therefore, establishment of a feasible in vitro culture protocol for animal originated epithelial cells is urgently required.
Freshly harvested mouse or rat corneas were digested in culture medium with 2U dispase ‖overnight. Then the epithelial layer was detached from stroma, and further digested into single cells with 0.05% Trypsin-EDTA. Afterwards, single cells were plated onto the collagen IV pretreated dish, and cultured with defined keratinocyte serum free medium (D-KSFM) or supplemental hormonal epithelial medium (SHEM) with the addition of Rock inhibitor Y27632, respectively. For cultures with serum supplement, NIH-3T3 cells were used as feeder layers. When cells reached confluency, some cells were used for further passages, while the others were harvested for qRT-PCR, Immunocytochemistry and western blot to examine their phenotypical markers.
Using both D-KSFM and SHEM media supplemented with Y27632, rat corneal epithelium could be easily cultured for up to 10 passages. In comparison, the culture of mouse corneal epithelial cells required prolonged culture time. While mouse CECs could be cultured by SHEM medium for a few generations, cells maintained D-KSFM with Y27632 propagated continuously. CEC specific markers Pax6, K12, P63 and K14 were examined by qRT-PCR, Immunocytochemistry and Western blot. Although these markers were positive in primary culture, K12 was sharply decreased after passage. Moreover, Pax6, a transcription factor essential for CECs phenotype maintenance, was gradually decreased. Serving as the corneal epithelial progenitor markers, p63 was maintained at a high level and K14 expression was increased during culture. Skin epithelium marker K10 was negative for all cultured cells.
Rock inhibitor is an essential supplement to promote mouse or rat CECs adherence and proliferation in vitro, which facilitates mouse and rat corneal epithelial cells culture, while the underline mechanism needs further investigation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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