VIABILITY OF KERATOCYTES IN EPIKERATOPHAKIA LENTICULES

Aim-To study the influence of cryoprotectant, cooling rate, and warmin g rate on recovery and viability of keratocytes from corneas for cryol athing. Methods-Corneas were frozen at -50 degrees C for 2 minutes eit her after exposure to 10% dimethyl sulphoxide in Eagle's MEM for 15 mi nutes at room temperature (about 22 degrees C), or without earlier exp osure to the cryoprotectant. Corneas were cooled either rapidly (20 de grees C/min) or slowly (1 degrees C/min), and they were warmed either rapidly (>50 degrees C/min) by direct transfer into medium at 22 degre es C or slowly (<20 degrees C/min) in air at 22 degrees C. The cryopro tectant was removed by dilution in medium containing 0 . 5 mol/l sucro se. Recovery of keratocytes was determined by using collagenase digest ion to release the cells from the stroma and trypan blue staining. Via bility was assessed by the out-growth of cells from stromal explants i n primary tissue culture. Results-The use of a cryoprotectant before f reezing was beneficial, irrespective of the different cooling and warm ing regimens. Both collagenase digestion and tissue culture revealed t hat keratocyte survival was improved when corneas were warmed rapidly rather than slowly. The collagenase digestion assay showed an apparent ly higher recovery of keratocytes after slow cooling (54 . 3%) than af ter rapid cooling (34 . 1%), but no differences in cell viability coul d be demonstrated by primary tissue culture. Conclusion-Although in th ese experiments slow cooling apparently provided the best recovery of keratocyte numbers (though not viability), previous work had revealed some disruption of the epithelial basement membrane after slow cooling . If viable keratocytes and good preservation of epithelial basement m embrane are considered to be prerequisites for epikeratophakia lenticu les then it is suggested that corneas should be prepared for cryolathi ng by freezing rapidly after exposure to 10% dimethyl sulphoxide and, following cryolathing, they should be warmed rapidly.