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Hideyuki Miyashita, Kazuo Tsubota, Shigeto Shimmura; Structure and turnover rate of long-term cultured human limbal epithelial cell sheets. Invest. Ophthalmol. Vis. Sci. 2014;55(13):504.
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© ARVO (1962-2015); The Authors (2016-present)
To confirm whether primary cultured human limbal epithelial cell sheets can maintain homeostasis long term with KGF and rho kinase inhibitor Y-27632.
Human limbal epithelial cells (4-8 x104cells / insert) were primary cultured on plastic cell culture inserts, with a feeder layer of human mesenchymal stem cells in the bottom of a paired well. Cells were fed with supplementary hormonal epithelial medium, containing KGF and ROCK inhibitor Y-27632 but not EGF. From 5 months to 7 months, cultured medium in cell culture inserts was daily collected, and the number of cell debris was counted by hematocytometer. At 6 months, some cell sheets were treated with EdU (10 μM) for 1 day, followed by the immunostaining of cryosections and whole mount sheets with anti-EdU, antibody for immature epithelial cell marker K15, and DAPI. Nuclei in randomly selected 5 images (Obj x20, 1.48x10-3 cm2) were counted manually by using Image J and add-in cell counter software, to estimate the total cell number in each culture insert (4.67 cm2).
Each cell culture insert was estimated to contain 3.4 ± 0.1 x106 cells (mean ± S.D., n=3) at 6 month, which shed 4.0 ± 0.8 x104 cells every day from 5 to 7 months. K15 was expressed heterogeneously in the basal layer. EdU was mainly observed in K15 negative cells.
Primary human limbal epithelial cell sheets daily shed approximately 1% of total cells even after 6 months of cultivation, suggesting that these sheets maintain homeostasis.
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