Methods and compositions for the cryopreservation of organs

Abstract

Methods and compositions are provided for the introduction and washout of vitrifiable concentrations of cryoprotectant in organs and tissues. The methods comprise cooling the organ to below −10° C. by perfusion with a solution having a freezing point below −10° C., a temperature from −10 to −40° C., and a tonicity from 1.1 to 2.0 times isotonic, after previous perfusion with said solution for a time insufficient for approximate osmotic equilibration of the organ with the solution. The methods further comprise increasing the concentration of cryoprotectant further at a temperature from −10 to −40° C. to prepare the organ or tissue for vitrification. The methods further comprise cooling and vitrifying the organ, rewarming it, and perfusing the organ with a vitrifiable concentration of cryoprotectant that is the same as or less than the concentration used for vitrification, without the addition of an osmotic buffering agent. Rewarming is accomplished either by rapid (>1°C./min, and preferably −0.2-20° C./min) elevation of arterial perfusate temperature from below −20° C. to above −15° C. during continuous perfusion of the organ or by perfusing the organ with pre-warmed arterial perfusate at >−15° C. Extraordinarily effective multicomponent compositions are also provided for the process, particularly involving a vitrification solution whose warming rate after vitrification can be <1° C./min without freezing during rewarming and a chilling injury protective solution having zero toxicity to whole organs at 0° C. and permitting almost complete avoidance of chilling injury at −20 to −25° C.

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