Cryopreservation studies with porcine corneas

Purpose. A new technique for the cryopreservation of rabbit corneas in 20% w/w dimethylsulfoxide, which has been shown to preserve significant structu ral and functional integrity of the endothelium, was tested in porcine corn eas. Methods. The characteristics of uptake of dimethylsulfoxide into porcine co rneas were measured using proton (H-1) nuclear magnetic resonance (NMR) spe ctroscopy. The effect on structural integrity of exposure to 20% w/w dimeth ylsulfoxide without freezing was first assessed using vital staining (acrid ine orange and propidium iodide), and optimum temperature conditions for ad dition and removal of the cryoprotectant were derived. The effects on struc tural integrity of cryopreservation in 15% and 20% w/w dimethylsulfoxide, a nd of reducing the degree of cell swelling during cryoprotectant removal fo llowing cryopreservation, were then evaluated. Results. The characteristics of uptake of dimethylsulfoxide from a 10% w/w solution fitted a single exponential, resulting in a maximum tissue concent ration of 14.6% when the addition occurred on ice, and 18.5% when the addit ion took place at room temperature. The toxic effects of dimethylsulfoxide in porcine corneas were highly temperature dependant and only evident after removal of the cryoprotectant. Unlike rabbit corneas. cryopreservation of porcine corneas in 15% and 20% w/w dimethylsulfoxide induced substantial en dothelial injury which was not improved by reducing the degree of cell swel ling that occurred during removal of the cryoprotectant. Conclusions. Porcine corneas were substantially more susceptible to the tox ic effects of dimethyl sulfoxide, and to cryopreservation injury, than rabb it corneas. These results underline the importance of species variation in animal studies aimed at the cryopreservation of human tissue for transplant ation.