Culture of Corneal Limbal Epithelial Stem Cells: Experience From Benchtop to Bedside in a Tertiary Care Hospital in India

Purpose: To standardize the technique of ex vivo culture of limbal epithelial stem cells (LESCs) using locally available adapted methods and evaluate the outcome of transplantation in patients with limbal stem cell deficiency. Methods: Limbal tissue specimens were isolated from cadaveric corneoscleral rims, living related donors, or contralateral eye of the patients. Harvested tissue was cultured on denuded human amniotic membrane (dHAM) using various techniques to stabilize dHAM. The optimization of in vitro culture conditions was achieved by modifications in culture media (culture media 1 and 2). The LESCs were cultured in both types of media for 2 weeks, and growth patterns were observed. Expanded cells were further characterized by immunocytochemistry (K3/12, K19, and ABCG2) and reverse transcriptase polymerase chain reaction (K12, Cx43, Pax6, ABCG2, p63, and K19). The cultivated epithelium was transplanted in 50 patients with total and partial limbal stem cell deficiencies. Results: Stabilization of dHAM was successfully achieved using coverslips. The outgrowth was observed within 1-3 days of culture using both types of culture media (P = 0.20), but cultures in culture medium 1 reached confluency faster than cultures in culture medium 2 (P = 0.0004). Histopathological analysis showed multilayer formation and immunostaining, and reverse transcriptase polymerase chain reaction data confirmed the expression of both stem cell markers (K19, p63, and ABCG2) and differentiation markers (K3, K12, and Cx43). Patients who had undergone limbal stem cell transplantation showed a stable ocular surface with improved visual acuity over a long-term follow-up period. Conclusions: LESCs were successfully cultured using locally available adapted methods, and their clinical benefits verified by transplantation.