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, colony-forming 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.
['Stasi|Kalliopi|K|', 'Goings|DaVida|D|', 'Huang|Jiayan|J|', 'Herman|Lindsay|L|', 'Pinto|Filipa|F|', 'Addis|Russell C|RC|', 'Klein|Dahlia|D|', 'Massaro-Giordano|Giacomina|G|', 'Gearhart|John D|JD|']
D000328:Adult D000368:Aged D000818:Animals D002454:Cell Differentiation D049109:Cell Proliferation D002478:Cells, Cultured D018920:Coculture Techniques D017077:Culture Media, Conditioned D005260:Female D005500:Follow-Up Studies D006801:Humans D007150:Immunohistochemistry D016850:Limbus Corneae D008297:Male D051379:Mice D008875:Middle Aged D010100:Oxygen D013234:Stem Cells D015262:Xenobiotics
xeno-free limbal stem cell culture oxygen