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Cryopreservation of rabbit corneas in dimethyl sulfoxide
M C Wusteman1, S Boylan, D E Pegg
1Department of Biology, University of York, Heslington, United Kingdom.
Purpose:
To minimize the injury to endothelial cells during cryopreservation of rabbit corneas with dimethyl sulfoxide.
Methods:
Rabbit corneas were cryopreserved using 20% wt/wt dimethyl sulfoxide (Me2SO), added and removed in stages to maintain the osmotically induced excursions in cell volume to within +/-40% of their isotonic volume. The vehicle solution, cooling rate, and conditions of storage used were those already reported to be optimal for endothelial cell survival after exposure to low temperatures. Survival was assessed by confocal microscopy with vital staining and by the ability of the endothelium to control stromal hydration during 3 hours of normothermic perfusion. The effect of temperature of addition and removal of Me2SO (room temperature [RT] or 2 degrees C) on endothelial viability also was measured.
Results:
After thawing, all the cryopreserved corneas appeared structurally intact when assessed by vital staining and could limit stromal swelling during subsequent normothermic perfusion. Analysis of the rate of stromal swelling during the first 1.5 hours of normothermic perfusion indicated a substantial benefit when the Me2SO was removed at RT. Adding and removing the Me2SO at RT, which allowed a briefer exposure to Me2SO before cooling, resulted in better structural integrity of the endothelial layer than when the addition of cryoprotectant took place on ice.
Conclusions:
These results demonstrate the importance of osmotic stresses in the generation of injury to corneal endothelium during cryopreservation and the possibility of eventual successful cryopreservation of this tissue.