16. Controlling cryoprotectant toxicity and chilling injury

Abstract

Cryoprotectant toxicity and chilling injury are the central problems blocking the full deployment of vitrification, including its use for organ cryopreservation. Controlling these sources of injury is handicapped by the fact that little information is available about the nature of either cryoprotectant toxicity or chilling injury. To date, toxicity has been controlled by following 14 largely empirical rules of toxicity suppression in vitrification, but recently both theory and empirical experiment have begun to point the way to a coming ability to transcend cryoprotectant toxicity and with it, chilling injury as well. One interpretation of a recent gene expression profiling experiment on rat liver slices is that cryoprotectant toxicity in advanced vitrification solutions is related to protein denaturation. This is consistent with independent evidence that toxicity is correlated with stronger water hydrogen bonding by the permeating cryoprotectants in such solutions, which might destabilize the protein hydration layer and thus induce denaturation. Individual cryoprotectants apparently injure cells by specific mechanisms that don’t include denaturation, but when individual agents are diluted and combined to form vitrification solutions of high total concentration, injury may be primarily non-specific and mediated by denaturation linked to reduced water availability. Although worrisome at first, cells have evolved many ways of coping with protein denaturation, which may be augmented to achieve a therapeutic effect. In addition, chilling injury of vitrifiable liver slices was found to mimic cryoprotectant toxicity based on additional gene expression profiling, suggesting that chilling injury during vitrification may arise from the superimposition of cold denaturation on pre-existing protein destabilization by cryoprotectants. This implies that reducing cryoprotectant toxicity will also reduce chilling injury, but other interventions for chilling injury may also be helpful, including interventions that have been aimed at preventing membrane lipid phase transitions in other systems. Interestingly, those interventions include using antifreeze proteins to stabilize the cell membrane, which raises the question of whether other ice-active agents that might be more practically incorporated into vitrification solutions would have similar protective effects in addition to their intended role of interfering with ice formation. Another factor in controlling chilling injury has been tonicity adjustment. It has been reported that setting the nominal osmolality of non-penetrating species in a vitrification solution to 1.2–1.5 times isotonic minimizes chilling injury in rabbit renal cortical slices at all temperatures from −20 °C to −80 °C, below which chilling injury no longer increases. While helpful, this intervention places constraints on the amount of impermeant that can be used in vitrification solutions for the kidney. The mechanism of this effect is unknown, although it has been speculated that hypertonicity prevents injury otherwise caused by thermal contraction of cell membranes.