Study on biologic viability of cultural rabbit's limbal corneal epithelial cells after cryopreservation

Objective To search for the feasibility and the effect that cultural limbal corneal epithelial cells were cryopreserved. Methods Small specimens of limbal epithelial cells were excised from the limbal regions in rabbits.and were cultivated. Some monolayer cells were digested into suspending liquid and were cryopreserved in two groups of cryoprotectant solutions respectively. Cryopreserved cells were frozen in liquid nitrogen and were thawed after half a month,a month and two month differently. The cultivated limbal epithelial cells were identified by immunohistochemical and electron microscope.The structure and activity of frozen and non-frozen cells were compared by inverted microscope, electron microscope and MTT colorimetry. Results Subculturing cells were positive staining for monoclonal antibody cytokeratin AE1 and anti-proliferating cell nuclear antigen(anti-PCNA), were rarely positive for monoclonal antibody cytokeratin AE5. The adherence and growth of cryopreserved cells delayed compared with that of the fresh cells. Electron microscope revealed that cryopreserved cells appeared different extent dropsy and the recovery of the cell ultrastructure were in 7 days. MTT colorimetry proved that the proliferation of frozen cells were lower than that of non-frozen cells after thawing( P 0.05) and after 5 days of subculturing( P 0.05).There were no significant differences among the detective values of different frozen time groups( P 0.05), whereas the difference was statistically significant between that of the dimethyl sulfoxide group and the glycerol group( P 0.05). Conclusion Limbal corneal epithelial cells frozen kept the characteristics of epithelial cells. After these thawed cells were continued cultivating, there were a lot of stem cells that had ability in proliferation in subculturing cells. The cryopreservative effect of dimethyl sulfoxide was better than that of glycerol. The approach of stage freezing was simple and feasible in order to cryopreserve cultural limbal corneal epithelial cells. These cells which were frozen by the reasonable cryoprotectant had similar stucture and function after subculturing.