[Morphologic changes of corneal epithelium in organ culture after cryopreservation]

Previous experimental studies concerning morphological changes of corneal endothelium after cryopreservation have been performed directly or within hours after thawing. The purpose of this study was to investigate morphological alterations of corneal endothelium up to 1 week after thawing in organ culture. We evaluated (1) pig corneas that were cryopreserved within 4 h of death, (2) pig corneas that had been stored in cryopreservation medium at 31 degrees C for 4 h before cryopreservation, and (3) as a control, corneas that were preserved in organ culture until the specific time of evaluation. Corneas were frozen to -196 degrees C in an automated freezing chamber in a cryopreservation medium containing chondroitin sulfate. Morphologic evaluation after trypan blue and alizarin red staining was performed after 1 day and after 1 week of organ culture preservation after thawing. One day after thawing, computer-assisted morphometric analysis of endothelial cell density and necrotic areas revealed endothelial cell loss of 25.7% in group 1 (8% necrosis), 48.7% in group 2 (17.4% necrosis) and 0% in group 3 (0% necrosis) compared to freshly dissected corneas. After 1 week, endothelial cell density had decreased by another 29% in groups 1 and 2 and by 11% in group 3 compared to results 1 day after thawing. However, necrotic areas could no longer be detected in any of the groups. We conclude that the highest decrease in endothelial cell density can be expected within 1 day after thawing. Further cell loss seems to be due to non-mitotic wound healing resulting in a confluent endothelial monolayer.