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Kalliopi Stasi, Mina Massaro-Giordano, Jiayan Huang, John Gearhart; Comparison of Human and Mouse Feeders, Xeno-free and Standard Media in Different Oxygen Tensions for Preservation of Human Limbal Progenitor Cells during Culture. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1004.
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© ARVO (1962-2015); The Authors (2016-present)
To identify culture conditions that allow preservation of limbal stem cells in culture with human feeders and xeno-free media for potential clinical use.
Limbal epithelial cells were isolated from 82 cadaveral donors or 29 cataract patients with trypsin or dispase/trypsin in 13 variations. Feeders used: 3T3-J2, adult or neonatal or fetal human dermal fibroblasts, lung fibroblasts MRC-5 and human limbal fibroblasts. Media used: Xeno media per Pellegrini group, MCBD 151 (Schotzer-Schrehardt), or SHEM (Tseng), and Xeno-free media with Calcium 1.3mM, 1.05mM, 0.4mM and 0.1mM, and EGF 0, 2.5, 5, 10 or 20ng/ml, under O2 20%, 14% or 5%. Statistical analysis was performed on outcomes: quantitative immunocytochemistry (Q-ICC) for p63α (cytospins), RT-PCR for p63α, Δp63 and K12, Colony Forming Efficiency (CFE), Holoclone Forming Efficiency, Percentage of Aborted Colonies, and number of passages. ICC: p63α, p63 (4A4), ABCG2, Bmi1, c/EBPδ, K12, K15 and MUC1 on cytospins and coverslips. Statistical analysis: ANOVA/MANOVA with SAS 9.2
Isolation methods (cadaveral donors) evaluated for yield, viability and CFE. Yield was correlated with dispase (+0.58, p=0) and days in preservation (-0.52, p=0) and affected by trypsin (p=0) and days in preservation (p=.0006), viability was affected by trypsin (p=0.0269), and CFE was correlated with days in preservation (-0.47, p=0) and yield (+0.43, p=.0004) and affected by donor age (p=.0001) and trypsin (p=.04). Isolation method selected: dispase 2.4U/ml x2 hrs and TLE x10 min. Multivariate analysis of effect of variables on number of passages (max 10) from 15 patients cultured on 3T3-J2 showed significant effect of donor age (0.032, p= 0.002) and medium [SHEM (-2, p=0), Xeno-Free Ca0.01 with EGF10 (0.67, p=.04) or EGF20 (1.67, p=0)], and no effect of O2 14% vs. 20%. ANOVA of 34 combinations of feeder, medium and O2 conditions was significant for Q-ICC (p=.007) and CFE (p=.0002) but not significant for age, gender, feeders, medium or O2 alone. MRC-5 feeders with Xeno-free Calcium 0.01mM and EGF10ng/ml at 20% O2 were effective in Q-ICC (x4.9 fold) and p63α PCR (x1.4 fold) compared to baseline 3T3 feeders with Pellegrini medium.
Xeno-Free media and human feeders may effectively preserve limbal progenitors in culture as measured with multiple outcomes in head-to-head comparisons.
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