ABSTRACT
PURPOSE: To assess freeze-thaw-induced endothelial cell loss by using phase-contrast microscopy and early morphologic changes within each layer of human donor corneas by using confocal microscopy. METHODS: Twenty-eight human corneas were cryopreserved in minimum essential medium containing 10% dextran with a molecular weight (MW) of 500,000 as an extracellular cryoprotectant, at a cooling rate of 1 degrees C/min and stored in liquid nitrogen at -196 degrees C. After thawing, the tissue was organ cultured to detect latent cell damage. In 22 of the corneas, the endothelial layer was subjected to routine phase-contrast microscopy after 24 hours of organ culturing. The other six specimens were evaluated layer by layer in a scanning slit confocal microscope after 6, 24, and 48 hours of organ culturing. RESULTS: Before cryopreservation, the mean +/- SD numerical density of endothelial cells was 1940 +/- 220 cells/mm(2). After cryopreservation and subsequent organ culturing, the endothelial cell density decreased to 1300 +/- 360 cells/mm(2), and two of the corneas had a completely necrotic endothelium (P = 0.001). Confocal microscopy revealed all corneal layers in each of the six specimens examined to be structurally integral after 48 hours of organ culturing. Although the reflectivity of some of the keratocytes was enhanced, there were no signs of keratolysis. CONCLUSIONS: The present study demonstrates that each corneal layer is capable of regaining its structural integrity after cryopreservation in the presence of dextran. Because the freeze-thaw-induced endothelial cell loss is still highly variable, the technique must be further refined before it can be applied clinically.
