Survival of mouse oocytes after being cooled in a vitrification solution to −196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

Abstract

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196°C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95°C/min to 69,250°C/min to −196°C and for each cooling rate, subjecting them to five warming rates back above 0°C at rates ranging from 610°C/ min to 118,000°C/min. In samples warmed at the highest rate (118,000°C/min), survivals were 70 to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610°C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95°C/min to 69,000°C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.

Clinical evaluation of two formulations of slow-freezing solutions for cleavage stage embryos

“…Seki and Mazur [13,14] have shown that warming rates are more important than cooling rates for successful cryopreservation. In the slow-freezing program suggested by Testart, fast warming is required.…”

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature?

“…Table 1 represents just two examples when the Isachenkos published their paper, and other cryobiologists, who came to the same conclusions, namely: -There is no proof of the absence of vitrification inside the sperm even at quite slow cooling [Morris, 2006]; the role of intracellular ice in the death of fast cooling mouse sperm is also questioned in [Mazur & Koshimoto, 2002]. -Some cells can be vitrified in "diluted" solutions at relatively slow rate of cooling but very fast warming is essential for kinetic VF [Mazur & Seki, 2011] Thus, both cryobiologists have reported similar findings as the Isachenkos observations, but they unfortunately fell short of mentioning Isachenkos in their own publications and presentations (e.g., in Cryo-2010 in Bristol), which might have made looking their observations (that were solid, of course) for an unfamiliar reader as "pioneering" or even as "a new paradigm for cryopreservation by vitrifcation" [Mazur & Seki, 2011] ].…”

Developmental Ability of Vitrified Mouse Oocytes Expressing Water Channels

“…With this method, rapid cooling and rapid warming (especially rapid warming) prevent intracellular ice from forming and recrystallizing [22,23], thus increasing the survival of cells in which dehydration and permeation by cryoprotectants are insufficient. Therefore, this method is effective for cells that have low membrane-permeability.…”