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Recovery of Endothelial Function after Vitrification of Cornea at -110°C

From the Division of Ophthalmology, University of Bristol, Bristol, United Kingdom.

PURPOSE. To determine whether endothelial function is retained after ice-free cryopreservation of cornea by vitrification at -110°C.

METHODS. Rabbit corneas, mounted on support rings, were exposed to a solution containing 6.8 M propane-1,2-diol (PROH) and cooled at approximately 7°C/min to -110°C, which was below the glass transition temperature (T_g) of the solution. After rewarming at approximately 12°C/min and removal of the PROH, endothelial function was assessed by monitoring corneal thickness during perfusion at 34°C.

RESULTS. Addition and removal of 6.8 M PROH without cooling to -110°C did not markedly impair endothelial function, although corneas were thicker than control samples. There was no visible crystallization of ice during cooling to -110°C; but a few small, discrete sites of crystallization remote from the endothelium, were observed during warming. After removal of the PROH, corneas approximately doubled in thickness during the first 3 hours of perfusion, but they then started to thin, which suggested active control of stromal hydration by the endothelium. This was confirmed in a further set of experiments by removal of bicarbonate ions from the perfusate at this point, which resulted in further swelling at +58 ± 2 µm/hour (SD; n = 4). Restoring bicarbonate to the perfusate halted this swelling, and the corneas then thinned at -13 ± 2 µm/hour (n = 4). Morphologically, staining with trypan blue and alizarin red S showed an apparently intact endothelial monolayer.

CONCLUSIONS. Rabbit corneal endothelium tolerated exposure to 6.8 M PROH, and endothelial function was evident after vitrification at -110°C. Preliminary morphologic results with vitrified human cornea also showed retention of endothelium.