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Abstract
Attempts to improve current methods of cryopreservation of corneas, whether by conventional freezing and thawing or by vitrification in the absence of ice, will require the use of high concentrations of cryoprotectants. In this study we extend our previous investigation of the tolerance of rabbit corneas to multimolar concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) added and removed at 0 degrees C in CPTES, a hyperkalaemic preservation solution containing the impermeant anionic buffer N-Tris(hydroxymethyl)methyl-2-aminoethane sulphonate (TES). Isolated corneas were exposed to 1, 2 or 3 mol/l Me2SO at 0 degrees C to minimize any effect due to temperature-dependent chemical toxicity and attention was given to the procedure for diluting Me2SO from the tissue in order to minimize osmotic stress to the endothelium. Endothelial integrity following these procedures was assessed both by the ability to control stromal hydration during perfusion on the specular microscope and by the structural integrity when examined by light and electron microscopy. The presence of an active endothelial pump and good morphology were demonstrated in corneas exposed to 1 and 2 mol/l Me2SO; serial dilution of the cryoprotectant was more beneficial than a single-step direct dilution. Corneas immersed directly into 3 mol/l Me2SO were irreparably damaged irrespective of the method of dilution. Sequential addition of 1 M, then 2 M and finally 3 M cryoprotectant followed by serial dilution was, however, tolerated by the endothelium and minor alterations to the structural integrity of the endothelial layer were rapidly repaired. The osmotic nature of these observations are analyzed and discussed.