Corneal tolerance of vitrifiable concentrations of glycerol

Equilibration of corneas with sufficiently high concentrations of cryoprotectants to inhibit potentially damaging ice formation during cryopreservation has not yet been achieved. This study examined the effects on the structure and function of rabbit corneal endothelium of the low toxicity cryoprotectant glycerol. Corneas were exposed to concentrations ranging from 2.0 to 6.8 M glycerol in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mM sodium bicarbonate and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 h. Endothelial structure was observed using specular microscopy during perfusion and scanning electron microscopy after perfusion. Corneas tolerated exposure to 2.0 and 3.4 M glycerol for 20 min at 4 and -5 degrees C, respectively. Tolerance of 4.8 M glycerol for 10 min at -10 degrees C was improved by decreasing the dilution temperatures. Ten-minute exposure to 6.1 and 6.8 M glycerol was tolerated at -15 degrees C. In all cases corneas initially showed signs of damage but endothelial function was regained following structural repair. Corneas exposed to 6.8 M glycerol and cooled below the glass transition temperature were nonfunctional after warming. Ice formation during warming was believed to be the cause of injury.