Pressure effect on the survival rate of astrocytes was investigated at room temperature and 4℃. Although tissue and cells are frozen for preservation by adding cryoprotective compound, it is clarified that the survival rate of astrocytes is increased under a pressurized state at room temperature and 4℃. The optimum pressure was 0.3-1.0MPa at both room temperature and 4℃. The prominent effect was observed at 4℃ low temperature. The astrocytes died completely under normal pressure in four days at 4℃. However, under 0.3-1.0MPa, it is clarified that the survival of astrocytes increases dramatically.

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For the establishment of successful cryopreservation of Caenorhabditis elegans (C. elegans), freezing survival of the nematode particularly in the larva L4 and adult stages, was investigated in the presence of a cryoprotectant. The highest rate of freezing survival for larva L4 was obtained in the presence of 5% (v/v) dimethyl sulfoxide (Me_2SO) dissolved in M9 buffer. The survival rate increased with the dipping time in the Me_2SO solution until 13min before freezing, which corresponded with the decrease in the nematode volume. Although the volume first decreased, it recovered from shrinkage with further dipping time, indicating that Me_2SO can permeate into the nematode, which was confirmed by the use of gas chromatograph. Glycerol, on the other hand, can hardly permeate into the nematode; the volume recovery was minimal. Therefore, the remarkable increase in the rate of freezing survival when Me_2SO was added, compared with glycerol, might be caused by its permeation into the nematode body. Cooling rate, lowest temperature of cooling and initiation temperature of freezing also affected freezing survival. Slow cooling accompanied by ice seeding at a higher subzero temperature was effective in avoiding freezing of the internal nematode. Freezing avoidance inside the nematode due to osmotic dehydration in combination with the concentration of Me_2SO in the body was found to be necessary for the cryopreservation of C. elegans in both the L4 and adult stages. Taking these results into consideration, two-step freezing was conducted in which the initial cooling down to -100℃ at the rate of 0.2℃ min^<-1> with ice seeding at -3℃ was followed by plunging into liquid N_2. A survival rate above 85% was then obtained for the L4 nematode after slow warming.

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Food and living cells are strongly affected by freezing. Ice crystal structure size in frozen food is inversely proportional to the advance rate of ice front, reflecting the important role of the molecular diffusion of water in the process of ice crystal growth. In the freezing preservation of living cells, the water permeability in the plasma membrane seriously affects freezing tolerance of cells. Plant cells, in general, have much lower water permeability in the membrane as compared with animal and microbial cells so that intracellular ice crystals are easily formed in plant cells to destroy the membrane structure. By controlling the ice crystal structure size very large, the progressive freeze-concentration becomes possible. This method is very effective to make the freeze concentration system much simpler as compared with the conventional method of suspension crystallization. The progressive freeze-concentration is expected to extend the applicability of freeze concentration widely.

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Plant freezing tolerance is one of the most important factors which determine the productivity and distribution in the world. Temperate plants including many crop species have an ability to increase their freezing tolerance when exposed to low but non-freezing temperatures for certain periods, which is known as cold acclimation. Cold-acclimation-induced increase in freezing tolerance is associated with diverse changes occurring in the plasma membrane, which ultimately results in an increase in the cryostability of the plasma membrane to withstand various abiotic stresses imposed by freezing (i.e., dehydration, high-salt, and mechanical stresses) and an acceleration of the recovery process after thawing. We have intensively investigated alterations in the plasma membrane composition and accumulated evidences that indicate dynamic responses of protein and lipid compositions in the plasma membrane to low temperatures. Recently, we have initiated analysis of microdomains in the plasma membrane during cold acclimation and found that both the protein and lipid compositions of the microdomains significantly altered after cold acclimation. A microdomain-localized protein, synaptotagmin-like protein 1 (SYT1), which becomes concentrated in microdomains after cold acclimation, is likely to functionally involve in calcium-associated membrane repair process that is essential to maintain high survival after a freeze/thaw cycle. These results confirm that plant plasma membrane is the most important factor to determine how plant cells tolerate freezing conditions.

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Xylem parenchyma cells of trees adapt to subfreezing temperatures by deep supercooling. The mechanisms of deep supercooling, except for physical isolation of water, have been reviewed especially in relation with existence of diverse kinds of supercooling-facilitating substances in the xylem parenchyma cells. Present review also suggested possibility on application of such supercooling-facilitating substances to make unfrozen water in purpose of low-temperature preservation as well as to regulate freezing conditions in purpose of cryopreservation for biological materials.

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The suitable condition for the cryopreservation of mammalian oocytes and embryos differs among their maturational/developmental stages even in the same species. The permeability of the plasma membrane to water and cryoprotectants would be one of the most important cryobiological properties because it is closely related to major types of cell injury during cryopreservation. In the mouse, morulae and early blastocysts tolerate cryopreservation better than oocytes and embryos at early cleavage stages. Accordingly, morulae and blastocysts have high permeability to water and cryoprotectants, such as glycerol, ethylene glycol, dimethylsulfoxide, and acetamide. In addition, the temperature-dependency of the permeability is low, which suggests that water and these cryoprotectants move across the plasma membrane predominantly by facilitated diffusion via channels. In oocytes and embryos at early cleavage stages, on the other hand, water and cryoprotectants would move across the plasma membrane predominantly by simple diffusion through lipid bilayer, because the permeability is low and its temperature-dependency is high. In morulae, aquaporin 3, an aquaglyceroporin, is expressed in the plasma membrane abundantly, but not in oocytes. The suppression of aquaporin 3-expression in morulae markedly decreases the water-permeability. The suppression of aquaporin 3-expression also markedly decreases the permeability of morulae to glycerol and ethylene glycol, but not that to dimethylsulfoxide and acetamide. Thus, in mouse morulae, aquaporin 3 appears to play an important role in the movement of water, glycerol, and ethylene glycol, whereas cryoprotectant channels other than aquaporin 3 appear to play a role in the movement of dimethylsulfoxide and acetamide.

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Some organisms, which are habiting under cold environment below 18℃, have developed a variety of strategies to survive extreme cold environment. In frozen environments, some organisms are exposed to conditions that necessitate the partial removal of water from the intracellular space, and posed a severe threat for the destruction by the formation of extracellular ice. Various cryoprotectants, that is, glycerol or trehalose etc, and ice crystal controlling materials for prokaryotic and eukaryotic cells have an important role of survival under frozen environment. Although ice crystal controlling materials are ice nucleating protein, anti-nucleating materials, antifreezing protein and cryoprotective materials, antifreeze proteins, which can inhibit ice formation by suppressing the growth of ice nuclei, have be expected various usages for a variety of frozen processing foods. Some success usages of antifreeze proteins are introducing in this sentence.

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