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Ray J. Tsai, Ryan Y. Tsai; Human Corneal Endothelial Stem Cells Could Be Preserved In The Long-term Ex Vivo Cultured System. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6597.
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Previously, we had developed an ex vivo culture system for human corneal endothelial cells (HCECs) on amniotic membrane and preserved the stem cell property in primary culture. To further explore human corneal endothelial stem cells that could be preserved in long-term cultured periods; serial passages and long-term cultured studies of HCEC cultures have been designed.
HCECs were cultured on amniotic membrane for one month to three months and serially passaged. During culture, BrdU was added in the culture medium for two weeks, followed by a chasing period for another two weeks. Then the cells were terminated and labeled for Anti-BrdU. Markers for stem cells, ABCG2, and differentiation marker for human corneal endothelial cells, Na/K ATPase, were studied by immunochemistry. To confirm the results, RT-PCR with total RNAs extracted from cultured human corneal endothelial cells from each culture periods and passages were performed, using Na/K ATPase and ABCG2 primers.
Serial passages from each HCECs were performed up to three passages and each passage was cultured from thirty days to 106 days. BrdU labeling retention cells were detected on p0 up to D53 cultures. Positive staining of ABCG2 could be detected up to P3 D36 culture period and Na/K ATPase could be detected up to P3 D47 culture period. Transcript expression of ABCG2 was detected on p0 D55,and p0 D106; p1 D33,and p1 D101; p2 D51 and became week positive on p3 D49. Expression of Na/K ATPase was also detected up to p3 D49.
In this ex vivo culture system, HCECs could be cultured in serial passages and long-term culture periods while maintaining the stem cells properties. These techniques will allow us to further study HCECs for cell banking.
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