Ice-free cryopreservation of mouse embryos at −196 °C by vitrification

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Abstract
The failure of complex mammalian organs, such as the kidney, to function following freezing to low temperatures is thought to be due largely to mechanical disruption of the intercellular architecture by the formation of extracellular ice1–5. Classical approaches to the avoidance of ice formation through the imposition of ultra-rapid cooling and warming rates6–8 or by gradual depression of the equilibrium freezing point during cooling to −80 °C9–13 have not been adequate. An alternative approach14–16 relies on the ability of highly concentrated aqueous solutions of cryoprotective agents to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify without the formation of ice, a process termed vitrification. When embryo suspensions are cryopreserved using conventional procedures, this supercooling behaviour allows intracellular vitrification, even in the presence of extracellular ice17–20. We have therefore used mouse embryos to examine the feasibility of obtaining high survival following vitrification of both the intra- and extracellular solutions and report here that in properly controlled conditions embryos seem to survive in high proportions after cryopreservation in the absence of ice.