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K. Kito, K. Hata, H. Kagami, K. Hirano, H. Terasaki, M. Ueda; Histology and Viability of Rabbit Cultivated Corneal Epithelial Cells after Cryopreservation with Glycerin and Dimethyl Sulfoxide . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4711.
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Purpose: To enhance the availability of the cultivated corneal epithelial sheets in clinical use, it is important to determine the optimal condition for freezing methods and the effect of cryopectants such as gylcerin and dimethyl sulfoxide (DMSO) on the viability of cultivated corneal epithelial sheets. In addition, the effect of the storage temperature was investigated. Methods: Normal rabbit corneal limbal tissue was removed, and a cell suspension was made. The cell pellet containing corneal stem cells was obtained, and the cells were inoculated on collagen shield contact lenses with 3T3-J2 cells treated with mytomycin C. Stratified corneal epithelial sheets were obtained after 20 days of culture. The sheets were stored in either 10% gylcerin or DMSO at -80° C or -196° C for three weeks. After thawing, the sheets were evaluated histologically, and cell viability was also analyzed by colorimetric cell viability assay using a tetrazolium salt. Results: The structural damages such as vacuolar degeneration were more clearly observed in the corneal epithelial sheets cryopreserved with DMSO than with glycerin, especially at -80° C. Only minor morphological changes were observed in the corneal epithelial sheets cryopreserved in glycerin at both temperatures. Colorimetric cell viability assay revealed that cell survival rate was less than 50% when cryopreserved with DMSO at -80° C, whereas more than 50% of the cells in the rest of the groups kept their viability after freezing storage. Conclusions: These results demonstrated that DMSO might not be a good cryoprotectant for storage of corneal epithelial cells. The recommended condition for the storage of cultured corneal graft is -196° C with glycerin as a cryoprotectant.
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