Corneal cryopreservation with chondroitin sulfate

In an experimental study using porcine corneas we investigated the influence of several variables of freeze-thaw trauma on survival of corneal endothelial cells after corneal cryopreservation employing chondroitin sulfate as a cryoprotectant. We examined the influence of: (1) the concentration of chondroitin sulfate in the cryopreservation medium, (2) the concentration of fetal calf serum in the cryopreservation medium, (3) the cooling rate, and (4) the preincubation period in the cryopreservation medium before freezing. Controls consisted of corneas cryopreserved in culture medium without cryoprotectants and corneas frozen in dimethyl sulfoxide (Me2SO) by the method of Capella et al. (Preservation of viable corneal tissue. Cryobiology, 2, 116-121, 1965). Morphological evaluation was performed by determining endothelial cell density after staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent storage period in organ culture at 31 degrees C in order to detect latent cell damage after freeze-thaw trauma. The group that yielded the best endothelial cell density was evaluated in perfusion chamber experiments in order to measure the functional integrity of the tissue. Controls included corneoscleral rims from freshly slaughtered pigs, corneas stored in organ culture for 1 day, and fresh corneas mechanically denuded of endothelium. It was demonstrated that corneas that had been cryopreserved in MEM medium containing 2% chondroitin sulfate and 20% fetal calf serum with a cooling rate of 1 degree C/min displayed the highest endothelial cell density (2430 cells/mm2, SO = 383, n = 15) compared with freshly dissected corneas (3395 cells/mm2, SD = 200, n = 48). Control corneas frozen by the method of Capella et al. demonstrated only a poor outcome. We conclude that in our experimental environment, corneal cryopreservation with chondroitin sulfate provides higher corneal endothelial cell densities than corneas preserved by conventional methods. Moreover, even slight changes in variables that affect the tissue in the freeze-thaw cycle have a major impact on corneal endothelial cell survival after cryopreservation.