Model biological systems demonstrate the inducibility of pathways that strongly reduce cryoprotectant toxicity

Abstract

Cryoprotectant toxicity is a limiting factor for the cryopreservation of many living systems. We were moved to address this problem by the potential of organ vitrification to relieve the severe shortage of viable donor organs available for human transplantation. The M22 vitrification solution is presently the only solution that has enabled the vitrification and subsequent transplantation with survival of large mammalian organs, but its toxicity remains an obstacle to organ stockpiling for transplantation. We therefore undertook a series of exploratory studies to identify potential pretreatment interventions that might reduce the toxic effects of M22. Hormesis, in which a living system becomes more resistant to toxic stress after prior subtoxic exposure to a related stress, was investigated as a potential remedy for M22 toxicity in yeast, in the nematode worm C. elegans, and in mouse kidney slices. In yeast, heat shock pretreatment increased survival by 18-fold after exposure to formamide and by over 9-fold after exposure to M22 at 30 °C, and at 0 °C and with two-step addition, treatment with 90% M22 resulted in 100% yeast survival. In nematodes, surveying a panel of pretreatment interventions revealed 3 that conferred nearly total protection from acute whole-worm M22-induced damage. One of these protective pretreatments (exposure to hydrogen peroxide) was applied to mouse kidney slices in vitro and was found to strongly protect nuclear and plasma membrane integrity in both cortical and medullary renal cells exposed to 75–100% M22 at room temperature for 40 min. These studies demonstrate for the first time that endogenous cellular defenses, conserved from yeast to mammals, can be marshalled to substantially ameliorate the toxic effects of one of the most toxic single cryoprotectants and the toxicity of the most concentrated vitrification solution so far described for whole organs.