Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to −196 °C

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ?6 molal) and cooled at very high rates (i.e., ?1000 °C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500 °C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.