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Kalliopi Stasi, DaVida Goings, Jiayan Huang, Lindsay Herman, Filipa Pinto, Russell C. Addis, Dahlia Klein, Giacomina Massaro-Giordano, John D. Gearhart; Optimal Isolation and Xeno-Free Culture Conditions for Limbal Stem Cell Function. Invest. Ophthalmol. Vis. Sci. 2014;55(1):375-386. doi: https://doi.org/10.1167/iovs.13-12517.
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© ARVO (1962-2015); The Authors (2016-present)
To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions.
Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63αbright cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype.
Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63αbright cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2.
The LSC function can be maintained in vitro under appropriate Xeno-free conditions.
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