Corneal Limbal Stem Cell Cryopreservation

Background: Limbal stem cell therapy has been used successfully to treat various corneal disorders. Cryopreservation of limbal stem cells for the purpose of tissue banking can provide great benefits for eye patients. Although the cryopreservants for storing limbal tissues have been developed previously, the characteristics of the limbal stem cells derived from the cryopreserved tissues were unclear, and the cell viability of limbal expansion on amniotic membranes (AMN) after cryopreservation was ~ 50%. In this study, we have applied a different culture procedure for the limbal stem cell expansion and cryopreservation to explore the feasibility of using cryopreserved limbal tissues for stem cell therapy. Methods: Corneolimbal tissue discs were dissected from human donor corneas, and explant cultures were performed on plastic or AMN. The limbal expansions were maintained in monolayer cultures (without air-lifting) for two weeks before harvest or cryopreservation, which is similar to the procedure used for the autologous transplants in humans. To investigate the effects of cryopreservation on limbal stem cell outgrowth, (1) dissected limbal tissue from each donor was also stored in liquid nitrogen with cryoprotectants prior to explant culture; (2) Limbal stem cells expanded on AMN were cryopreserved and subsequently revived to evaluate their viability after freezing and thawing by propidium iodide staining. Results: Both cryopreserved limbal tissues and limbal stem cells cultured on AMN displayed robust outgrowth after thawing. The limbal expansion on AMN remained viable after cryopreservation (87.5% cell survival). The limbal explant cultures exhibited strong positive staining for ABCG2, ΔNp63 and vimentin (stem cell markers), but weak and sporadic staining for K3 (a differentiation marker). Western blots and RT-PCR experiments confirmed the expression of these cell markers. Conclusion: Proper cryopreservation of corneal limbal tissues can be an effective method for preserving limbal stem cells and for expansion on amniotic membranes for transplantation at a later date.