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W. John Armitage, Samantha C. Hall, Caroline Routledge; Recovery of Endothelial Function after Vitrification of Cornea at −110°C. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2160-2164.
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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.
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