Functional Limbal Epithelial Cells Can Be Successfully Isolated From Organ Culture Rims Following Long-Term Storage

PURPOSE. Because of a shortage of fresh corneal tissue for research, it was of interest to investigate the potential of successfully isolating human limbal epithelial cells (hLECs) from organ culture corneal-scleral (OCCS) rims. METHODS. Superficial segments of corneal limbus were dissected and digested using collagenase (0.5 mg/mL, 16 hours at 378C). Cell suspensions were separated into four different growth conditions: corneal epithelial cell medium (CM); CM þ 3T3-Swiss albino cells; stromal stem cell medium (SM); and SM þ 3T3 cells. Colony number, hLEC count, cell density, and colony forming efficiency (CFE) were quantified to assess different growth conditions. The expression profile associated with basal hLECs was assessed by immunofluorescence, and epithelial integrity was measured using our real architecture for 3D tissue (RAFT) corneal tissue equivalent. RESULTS. Human limbal epithelial cells can be successfully isolated from OCCS rims following 4 weeks in storage with an 80.55% success rate with 36 corneal rims. Stromal stem cell medium þ 3T3s provided optimal growth conditions. Colony number, total cell number, and cell density were significantly higher at day 7 in cultures with SM than in CM. There were no significant differences between SM and CM when assessing CFE and the expression profile associated with basal hLECs. Cells maintained in SM were found to produce a higher quality epithelium than that cultured in CM. CONCLUSIONS. Organ culture corneal-scleral rims can be a valuable source for hLEC. Using a combination of collagenase-based isolation and medium designed for stromal stem cell isolation, a high number of good quality hLECs can be cultured from tissue that would have otherwise been ignored.