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.
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 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 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 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.
Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA. pmazur@utk.edu
Peter
P
Mazur
Shinsuke
S
Seki
D000818:Animals; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003460:Crystallization; D005260:Female; D005615:Freezing; D007053:Ice; D007424:Intracellular Fluid / Q000737:chemistry / Q000502:physiology; D051379:Mice; D008813:Mice, Inbred ICR; D009865:Oocytes / Q000737:chemistry / Q000166:cytology*; D012996:Solutions / Q000737:chemistry; D058989:Vitrification
D007053:Ice; D012996:Solutions