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Elfren Ray Baclagon, Sheyla Gonzalez, Sophie Xiaohui Deng; Expansion of Human Limbal Epithelial Stem/Progenitor Cells in xenobiotic-free condition using CnT-Prime Medium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3451.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether CnT-Prime (CnT-Pr) medium can substitute the supplemented hormonal epithelial medium (SHEM) for the expansion of human limbal epithelial stem/progenitor cells (LSCs) in xenobiotic-free conditions.
Limbal explants were cultured on denuded amniotic membrane in SHEM supplemented with 5% human serum (HS) and CnT-Pr with 1%, 5% or 10% HS. Single LSCs cultured on 3T3 feeder cells in SHEM were used as a control. Cell morphology, cell size, cell growth rate, outgrowth size and expression level of putative stem cell markers were analyzed.
Cell growth rate in CnT-Pr medium was lower than that in SHEM (range, 2.1-8.9 folds) and control (range, 2.1-9.2 folds) for all the conditions tested (all p<0.05). LSC cultured in CnT-Pr with 10% HS contained the highest amount of small cells (≤8 µm; 3% ± 1.4%; all p<0.05). No significant differences were found in the medium size (8-15 µm) LSC population in all the conditions tested (all p>0.05). The expression of cytokeratin (K) 14 was not significantly different among all the LSC cultures (p>0.05). The amount of K12+ cells was low and comparable in all the conditions tested (range, 0.9-2.7%; p>0.05). The vimentin+ population in the CnT-Pr medium (0.8%) was lower than that in SHEM (1.3%); however the difference was not significant (p>0.05). The amount of cells expressing high levels p63α was lower in all the CnT-Pr medium conditions (range, 5.2-17.8%) than in SHEM (29.2% ± 5.2%) and control (26% ± 2.7%). CnT-Pr supplemented with 5% HS generated the highest amount of these p63α bright cells (17.8% ± 5.7%) from all CnT-Pr conditions (p>0.05).
CnT-Pr could maintain the LSC phenotype. However, CnT-Pr was not as efficient as SHEM to expand the LSC population.
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