Basic aspects of vitrification of aqueous solutions are briefly reviewed in relation to cryopreservation of living cells. Vitrification is a key parameter for successful cryopreservation of living tissues and cells. Raman spectra of glassy aqueous solutions are also discussed as a structural probe for the structures of aqueous solutions.

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Cryopreservation by vitrification procedure produces vitrified state both out- and inside of cells. Although vitrified state outside the cells is constructed by solidificatiion of pure vitrification solution (VS), the vitrified state inside the cells that allows for successful cryopreservation may be constructed with different manners among individual procedures depending upon differences of interaction between biological materials and VS. In case of rye protoplasts, vitrified state inside the cells, that is produced by mere osmotic dehydration of cells by VS treatment without permeation of VS inside the cells, may allow for high survival. In case of bovine blastocysts, vitrified state inside the cells, that is produced by osmotic dehydration by VS as well as partial permeation of VS inside the cells, may be prerequisite for high survival. In case of mouse blastocysts, vitrified state inside the cells, that is produced by permeation of VS in almost equilibrated state with little effects of dehydration, may produce high survival. It is suggested that the differences of vitrified state that allows for high survival may depend upon differences of the dehydration tolerance among specimens. Thus, appropriate VS should be chosen on the basis of difference in the dehydration tolerance among the specimens.

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There are four basic methods to cryopreserve human parts:hypothermia, freezing, lyophilization, and vitrification, each using various cryoprotectants. With the exception of hypothermia, the other three methods involve preservation in a hybrid or polycrystalline state of supercooling, solid freezing and vitrification. It is very difficult to indefinitely cryopreserve large human organs, blood cells and tissue grafts by vitrification as they are not in a fixed state but in a process of relaxation except at a temperature of absolute zero (-273℃). Any ice crystals present eventually change to a damaging hexagonal form. Vitrified water relaxes to devitrify to ice crystals. Devitrification accompanies an increase of specific volume frequently resulting in a formation of cracks in the grafts. It is difficult to observe this time of phase transition. This author had spent 35 years cryopreserving human blood cells and tissue grafts at a temperature of mechanical freezer (-80℃), liquid nitrogen (-196℃), liquid helium (-273℃) and presents several observations: 1. Using an optimal preservation regimen, the frozen solid state is the safest for preservation of large volumes of blood cells for transfusion and tissue grafts for transplantation. 2. Vitrified blood cells in large volume and tissue grafts are solidified in a polycrystalline hybrid state, but contain numerous glassy transformed areas with many grain boundaries at liquid nitrogen temperature. Glass inevitably has Griffith's flaw which is a cause of cracking. Recently, cardiovascular surgeons have encountered crack formations of tissue grafts during thawing process. The more glassy areas that exist, the greater the occurrence of crack formation during devitrification results. 3. The author recommends cryopreserving cells and tissues at a temperature which is far higher or lower temperature than that of the glass transformation. Unfortunately except at zero degrees Kelvin (-273℃), devitrification will spontaneously generate due to the relaxation phenomenon, resulting in crack formation. This cracking causes cryobanked heart valves and vessels to be fatally damaged. 4.The prospect of vitrification or of freeze preservation of large human parts is still far from certain. Greater efforts for interdisciplinary studies of medicine, biology, chemistry and physics must be made to achieve successful vitrification of human organs and tissues.

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Complete vitrification can avoid the potentially damaging effects of both extracellular and intracellular crystallization. This method is very useful for long-term storage of germplasm due to minimum space and maintenance requirement. Three cryogenic procedures; vitrification, encapsulation-dehydration and encapsulation-vitrification, are suitable for cryopreservation of meristerms or somatic embryos. In this paper, the advantageous and disadvantageous between these procedures were evaluated and discussed. The vitrification method produces a high rate of shoot formation after cryopreservation, requires a minimal time for dehydration, yet poses difficult when handling a large number of meristems during the treatment phase. The encapsulation-dehydration method is easy to handle and alleviates dehydration process, however, introduces a low survival rate. It also produces a longer lag time for growth recovery and requires considerable time for dehydration. Contrarily, the encapsulation-vitrification method provides a high degree of survival, greatly shortens the dehydration time, and provides easy handling of even cells or small explants such as hairy roots.

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The effect of the addition of various monosaccharides to a vitrification solution on the survival rate of bovine blastocysts produced in vitro was investigated. In vitro matured and in vitro fertilized bovine Day 7-8 blastocysts were cassified into three developmental stages:early blastocysts (EB), blastocysts (BL), and expanded blastocysts (exB). Blastocysts were cryopreserved in one of the vitrification solutions which consists of 20% glycerol + 20% ethylene glycol + 0.3M sucrose + 3% polyethylene glycol + 0.3M (monosaccharide). As a monosaccharide, fructose, glucose, inositol, mannitol, sorbitol and xylose were respectively employed in the vitrification solution above. The basic solution was Dulbecco's PBS. Embryos were exposed to each vitrification solution in three steps, and after loading to 0.25ml straws, were plunged into liquid nitrogen. After warming in water bath at 20℃, cryoprotectants were diluted in 1/2 and 1/4M sucrose solution each for 5 min. Equilibration and dilution procedures except warming were conducted at room temperature (23 to 27℃). After dilution, embryos were cultured in Ham's F10 + 0.1mM β-mercaptoethanol + 20% fatal calf serum. Survival rates at 48 hr. (hatching rate at 96hr.) incubation of all stages of blastocysts exposed to six types of the monosaccharide additions and vitrified were 65.6 (55.5), 69.6 (56.3), 71.3 (59.1), 66.1 (56.7), 66.7 (56.7), 85.2 (79.3) % in sorbitol, mannitol, fructose, glucose, inositol and xylose respectively. Xylose-addition to the vitrification solution showed significantly higher survival rate in both 48 hr. survival rate (P<0.05) and 96 hr. hatching rate (P<0.001) than 5 other monosaccharrides addition. Within every solutions, more developed staged blastocysts showed higher survival rate. These results indicate that the addition of xylose to the vitrification solution improves the survival of vitrified bovine embryos produced in vitro.

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Since the concept of glass transition has been introduced to food science, a lot of phenomena relating with food became to be understood. In this paper, the roles of water in food as plasticizer, the effect of molecular weight of food polymer, and the dealing with the multicomponent system considering actual food are outlined. Additionally, the glass forming property of food protein during boiling and drying is discussed.

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