Abstract
When cells are vitrified with a minimum degree of supercooling, i.e., in near equilibrium, no ice would form
in the cell. However, in most cases the degree of supercooling is not minimal, and minute invisible ice
crystals will form in cells during the vitrification process. During warming, differences in surface free energy
can cause them to recrystallize into larger lethal crystals, which will be lethal to the cells. It is commonly
believed that both the cooling rate and the concentration of glass-inducing solutes (cryoprotectants) has to be
high during the vitrification process. However, the results that we present here, using mouse oocytes,
contradict these beliefs. First, rather than maintaining a high cooling rate during vitrification, our data
actually demonstrate that it is the warming rate that should be high. Moreover, our results also contradict the
second belief that a high concentration of cryoprotectant is preferred. We show that if the warming rate is
extremely rapid, oocyte survival is high even when the vitrification solution contains only a half the normal
concentration of cryoprotectants. Therefore, we conclude that rapid warming, rather than cooling or high
concentration of cryoprotectant, is essential to prevent intracellular ice formation (recrystallization).
