Effects of cryopreservation on histology and viability of cultured corneal epithelial cell sheets in rabbit

Purpose: To increase the availability of cultured corneal epithelial cell sheets as a replacement for corneal limbal allograft, the effects of cryopreservation on viability of cultured corneal epithelial cells were investigated. Methods: Normal rabbit corneal limbal tissue was excised, and the cells were cultured with mitomycin C-treated 3T3-J2 cells as a feeder layer. Stratified corneal epithelial cell sheets were obtained within 20 days. All sheets with a diameter of 8 mm were punched out with a biopsy punch and stored in either 10% glycerol or dimethyl surfoxide (DMSO) at -80 degrees C or -196 degrees C for 4 or 12 weeks. After thawing, the sheets were evaluated histologically, and cell viability was analyzed by colorimetric cell viability assay using a tetrazolium salt. Results: Structural damage such as vacuolar degeneration was more clearly observed in the corneal epithelial cell sheets cryopreserved with DMSO than those with glycerol, especially at -80 degrees C, whereas only minor morphologic changes were observed in the corneal epithelial cell sheets cryopreserved in glycerol at both temperatures. Colorimetric cell viability assay revealed that the storage conditions at the lower temperature (-196 degrees C) showed higher cell survival than those at the higher storage temperature (-80 degrees C). The difference between the two cryoprotectants, however, was not significant. Among the conditions used in this study, the samples cryopreserved with glycerol at -196 degrees C showed the highest cell survival rate (70.3 +/- 8.3% and 66.4 +/- 14.7% for 4 and 12 weeks, respectively). The difference between this group and those stored at -80 degrees C was significant for both 4 weeks and 12 weeks of storage using either glycerol or DMSO. Although the cryopreserved cell sheets could not maintain their original layered structure after thawing, viable cell sheets could be regenerated. Conclusions: The cell survival rates obtained after freezing storage were reasonable; however, the cryopreserved sheets could not maintain their original layered structure. More work needs to be done to better preserve the corneal epithelial cell sheets.