Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm

Purpose To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm.Methods Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 degree celsius with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-mu m CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360 degrees , 270 degrees 180 degrees, 120 degrees, 90 degrees, less than 90 degrees) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm.Results The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time x size of the corneoscleral ring (p = 0.007).Conclusions The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro .