040 Metastable vitrification of cryoprotectant solutions

Abstract

Vitrification is a phenomenon in which liquids take on the physical properties of a solid while retaining the structure of a liquid on a molecular scale. When aqueous solutions vitrify, a solid state is achieved without phase separation or crystallization. Cytoplasmic vitrification is believed to occur during almost all forms of cryopreservation. Metastable vitrification is a cryopreservation process in which both cells and surrounding medium are supercooled, vitrified, and then rewarmed without significant freezing of water. Metastable vitrification relies on solutes to reduce the activity of water and slow the kinetics of ice nucleation and growth so that ice avoidance can be achieved at cooling and warming rates compatible with the size of the sample being cryopreserved. For small samples that can be cooled and rewarmed at rates of thousands of degrees per minute, dilute solutions can be sufficient to achieve practical vitrification. On the other end of the metastable vitrification spectrum, organ cryopreservation has necessitated the development of solutions of low water content and low melting points sufficient for vitrification at cooling and warming rates less than one degree Celsius per minute. In any vitrification solution, the temperature of maximum ice growth rate is above the temperature of maximum ice nucleation rate. In dilute solutions, there is broad overlap between ice growth and ice nucleation temperature zones, while in concentrated solutions the zones are more narrow and separated. In either case, ice nucleation during vitrification tends to occur at cold temperatures during the later stages of cooling and early stages of warming. Consequently more ice nuclei are present during warming than cooling. Therefore ice growth (“devitrification”) during warming is a more serious problem than ice growth during cooling, making warming rate the most important rate parameter of vitrification. If a solution is only marginally vitrifiable at a given cooling rate, devitrification during warming will tend to be severe unless the warming rate is even faster. In some circumstances it may be possible to escape the adverse biological consequences of devitrification if ice recrystallization can be avoided. Successful vitrification therefore requires understanding and navigating the landscape of ice nucleation, ice growth, and ice recrystallization when applicable. A complicating factor is that solutions and cell interiors with the same water activity can have different vitrification tendencies due to different solute composition so that cells may escape ice formation even if surrounding solution does not. Source of funding: This research was supported by 21st Century Medicine, Inc. Conflict of interest: None declared. wowk@21cm.com