What Is Vitrification and How Can It Extend Life?(Papers presented at the 39th Annual Meeting)

Abstract

Vitrification, or the conversion of fluid materials to a noncrystalline glass at low temperatures, is currently considered the most promising approach to cryopreservation because it limits the amount of injurious ice formed in the material. Glasses form when solutions are cooled to supersaturation. If these mixtures fail to separate into their pure phases, rather than crystallize, they become more viscous as the temperature falls and reach a "glass transition temperature" (T_g). Though glasses are physical solids, they are thermodynamic liquids whose viscosity has exceeded some arbitrary value. This apparent paradox that glasses are simultaneously solids and liquids is explained when one takes into consideration the time scale on which T_g was determined: to a good approximation in many well studied glasses, T_g is a function of the logarithm of the time period over which the viscosity is measured. The important implication for cryobiology is that the inherent instability of glasses makes the storage temperature a critical issue. Storing at liquid nitrogen temperature is convenient but may be impracticable as aqueous glasses are fragile and fracture if cooled far below T_g. However, if the storage temperature is increased toward T_g, glasses will decompose at exponentially increasing rates. Little systematic research has been devoted to determining the constants in the equations, though we have begun to discern what the relevant equations are. The Arrhenius equation appears not to be appropriate: all the decay processes which we have examined in glass and in cryopreserved materials are hyperexponential, of the Johnson-Mehl-Avrami type. Fortunately, T_g is easy to manipulate, as mixtures of glasses have intermediate T_g values, in proportion to their mass fraction. Changing T_g in this manner is called "plasticizing", and one of the best plasticizers is water. Removing this water elevates T_g, eventually above ambient temperatures, and is secondary drying component of freeze-drying. In seeds, intracellular glasses are an important element of storage stability and can be formed by drying at ambient temperatures without the necessity of an intermediate freezing step. Seeds are a valuable model for the relationship between storage stability and the vitreous state as the data extend back over a century.