Optimal isolation and Xeno-free culture conditions for limbal stem cell function

PURPOSE. To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions. METHODS. 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, colonyforming efficiency (CFE), p63α^bright 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. RESULTS. 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, p63α^bright 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. CONCLUSIONS. The LSC function can be maintained in vitro under appropriate Xeno-free conditions.