[Freeze preservation of swine corneas with combinations of intra- and extracellular cryoprotective agents].

Clinically employed methods of corneal cryopreservation usually use the intracellular cryoprotectant dimethyl sulfoxide (DMSO). However, it has been demonstrated that extracellular cryoprotectants such as chondroitin sulfate (ChS) also display effective cryoprotection. The purpose of our study was to investigate the effect of combinations of intra- and extracellular cryoprotectants in corneal cryopreservation. Porcine corneas were cryopreserved in a cryopreservation medium consisting of MEM-medium containing 20% fetal calf serum and 2% chondroitin sulfate. The medium was varied by the addition of 2%, 4% and 8% DMSO. Sixty corneas were cryopreserved at -196 degrees C and thawed at 37 degrees C in a water bath. Morphometric evaluation was not performed directly after thawing but after a 24-h storage period in organ culture. Cryopreservation in medium without DMSO revealed the best results concerning endothelial cell density (2581 cells/mm2). Addition of 2% or 4% DMSO revealed no significant changes in endothelial cell density. Addition of 8% DMSO, however, resulted in a significant decrease (1312 +/- 319 cells/mm2) combined with a significantly higher amount of necrotic areas in the central corneal surface. We conclude that combining intra- and extracellular cryoprotectants does not enhance endothelial cell density after corneal cryopreservation. Higher concentrations of DMSO added to the cryopreservation medium appear to have a negative impact on endothelial cell viability.