The Japanese Society for Research of Freezing and Drying was established in 1959 with a view to promote research in the then new field of freezing and drying in various branches of science including medicine, pharmacology, agriculture and engineering. In 1994 the name of the society was changed to the Japanese Society for Cryobiology and Cryotechnology. During all these years the society has held regular meetings, seminars and symposia on various topics. With the kind help of Professor Tokio NEI international symposia were also held. A journal has also been published regularly. All these activities have helped in the dissemination of information in the areas of cryobiology and Cryotechnology and encouraged young scientists in these fields. The present paper gives an overview of the activities of the society during the past 35 years.

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The solubilities of glucose were measured in saturated alkali metal halide aqueous solutions at 30℃. The solubilities of glucose in saturated sodium halide aqueous solutions were as same as or lower than that in water. On the contrary, the solubilities of glucose in saturated potassium halide aqueous solutions were higher than that in water. The determinations of solubilities of glucose, galactose and xylose were also carried out in the presence of NaCl/KCl at 30℃. These electrolytes have effects on sugar solubilities. The effects have varieties that depends on combinations of sugars and electrolytes, electrolytes concentrations. Sodium ion has both salting-out and salting-in effects. Potassium ion has only a salting-out effect.

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Raman OD stretching spectra and homogeneous nucleation temperatures (T_H's) were measured for aqueous solutions of sucrose, maltose and trehalose over wide sugar concentration and temperature ranges. Comparison of the Raman and T_H data indicates that there are small differences in Raman spectra and T_H values among the three aqueous saccharide solutions. It is deduced that the high activity of trehalose as a protecting agent against water stress in biological systems may be due to the direct stabilization of the biological structures to be protected.

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Many organisms are capable of surviving almost complete dehydration by synthesizing large quantities of disaccharide trehalose in cells. Trehalose has no toxicity and is currently being studied for use in processed food, cosmetics, and the storage of internal organs for transplantation. This sugar is a good glass former and has the ability of forming hydrogen bonds with membrane surfaces and proteins. These properties may contribute to the occurrence of the biological function of trehalose as a protectant against desiccation. Here, we review recent advances in the understanding of the mechanism of preservation of dried and freeze-dried biomaterials with trehalose.

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Organisms that survive extreme dehydration often contain large quantities of soluble sugars, in particular di- and oligosaccharides. The presence of these sugars is believed to be one of the adaptations that allows dehydration tolerant organisms, including many plant seeds, to survive the loss of most of their cellular water. Cellular membranes are especially sensitive to the effects of dehydration. Sugars can exert various effects on membranes and may help preserve their structural and functional stability during dehydration. This review will describe the physical effects of dehydration on phospholipid bilayers and will explain how sugars can modify these effects to help stabilize membranes. As water is removed from between membranes, the close approach of the bilayers leads to physical stresses in the membranes. These stresses cause membrane lipids to be compressed together. During the fluid-to-gel phase transition, phospholipids move closer together and decrease the average surface area per lipid molecule. Thus, the dehydration-induced compression in the membranes may result in the phospholipids' transition from the fluid to the gel phase. Sugars that remain between membranes as water is removed can limit the close approach of the bilayers and, thus, can decrease the compressive physical stress that causes the fluid-to-gel phase transition to occur during dehydration. Therefore, in the presence of interlamellar sugars, the fluid-to-gel phase transition temperature (T_m) of a dry phospholipid does not increase as much as it does in the absence of sugars. Vitrification of the interlamellar sugar solution has the additional effect of adding a mechanical resistance that hinders the lateral movement of phospholipids within the bilayer and makes it necessary to cool the lipids to a lower temperature to bring about the transition to the gel phase. Thus, when the sugar solution vitrifies between fluid phase bilayers, T_m is depressed below its fully hydrated value (T_O). Neither of these effects of sugars on phospholipid phase transitions depends upon specific interactions between the sugars and lipids; instead, they can he explained using simple thermodynamics.

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Objective We compared preservative effects of monosaccharide (glucose), disaccharides (trehalose, maltose, sucrose), and trisaccharide (raffinose) to investigate whether effects of saccharides on lung preservation depend on its molecular weight, energy-level maintenance and cytoprotective effects. Methods Experiment 1: The effect of trehalose on the preservation of canine lungs was studied with the use of Euro-Collins solution (ECS). Experiment 2: An ex vivo rat lung model using homologous blood as the perfusate was employed. The lungs were flushed with one of the solutions containing glucose, trehalose, maltose, sucrose or raffinose and preserved for 14 hours. Results Experiment 1: The PaO 2 values in perfused with a modified ECS in which glucose was placed by trehalose were siginificantly higher than those in perfused with ECS. Experiment 2: The glucose, maltose and raffinose groups showed significantly higher levels of shunt fraction, pulmonary artery pressure and peak inspiratory pressure compared to the Fresh and trehalose groups. After reperfusion levels of ATP became significantly lower in the glucose, sucrose, maltose and raffinose groups. Ultrastructural examination revealed endothelial cell injury in the glucose, sucrose, maltose and raffinose groups. Conclusions These results show that effects of saccharides may depend on their cytoprotective effect rather than on impermeant activity or energy-level maintenance of the preserved lung. Trehalose proved to be superior to the other saccharides.

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Thermal stabilizing effect of amorphous matrices of sugars on freeze-dried proteins is investigated, remarking on the relation between the stabilizing effect and the degree of crystallinity of sugars. Several sugars and three enzymes (alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH)) were used. Enzymes were freeze-dried with sugars and stored at 65℃. Stability of freeze-dried enzymes was remarkably improved by addition of trehalose or raffmose. By measurement of X-ray diffractometry (XRD), those sugars were found to form fully amorphous matrix in freeze-dried samples. In contrast, glucose, sucrose, and lactose, which were found to be crystallized, were inferior in stabilizing enzymes. Furthermore, though sucrose showed low stabilizing effect when it was crystalline, it stabilized the enzymes highly when it was amorphous. These results indicate that amorphous matrices of sugars have thermal stabilizing effect. By Fourier Transform Infrared (FT-IR) measurement for samples containing amorphous sucrose and LDH, it was found that hydrogen bond formation between sucrose molecules and LDH was prominent. Moreover, dependence of thermal stabilizing effect of sugars on freezing temperature is discussed. Sucrose and LDH were used, and two type samples different in freezing temperature were prepared. The type A sample frozen at -30℃ showed higher thermal stabilizing effect than the type B sample frozen at -196℃, though sucrose was amorphous in both samples. However, radial distribution function (RDF) obtained by XRD showed that sucrose molecules in the type A sample are arranged more randomly than in the type B sample. Results of FT-IR measurement suggested that the degree of hydrogen bond formation between sugar and LDH is higher in the type A sample. These results suggest that the arrangement of sugar molecules affects the thermal stabilizing effect.

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A great number of new therapeutic protein have been widely used and investigated in recent years. While protein is generally stable in aqueous solution for short periods, pharmaceutical products are required to be stable through the storage during several months to several years. For the purpose of long-term stability, the formulations are usually produced in solid forms by freeze-drying. As protein is present in the solution at a very low concentration, protein pharmaceuticals are mostly formulated to create a cake with addition of excipients. Other chemical components are usually added for specific purposes, such as pH buffering, improvement of stability and osmolarity balancing. Although there are many reports on the stabilization of proteins by excipients, the stabilization mechanism of protein is complicated. Therefore formulation study of proteins has developed empirically by trial and error. We should try to establish formulations from theoretical consideration. The purpose of this paper is to discuss the prediction of stability of protein formulations.

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Forage grasses and cereals of temperate origin accumulate fructans as the principal nonstructural carbohydrate (NSC) in late autumn and utilize them throughout winter. By altering the balance between mono- and oligosaccharides and fructans, these plants accumulate fructans in late autumn, acquire freezing tolerance in early winter, maintain homeostasis under snow and grow in early spring. The NSCs also have an important effect on snow mold-plant interactions. The present review deals with the metabolism of NSCs in forage grasses and cereals from autumn to early spring.

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Insects often accumulate sugars and/or polyols at various concentrations only in winter. Glycerol is the most common polyols in both freeze-intolerant and freeze-tolerant insects, but other sugars and polyols such as trehalose, sorbitol, mannitol, inositol and glucose have been also found. Accumulation or loss of these cryoprotectants is effectively regulated by temperature-dependent activity in various enzymes. The capacity for polyol synthesis is also often linked to the developmental stage and diapause status. In freeze-intolerant insects, polyol accumulation provides colligative depression of SCP depending on the concentration and stabilizes the supercooling state of the hemolymph, although it may be less effective in many cases because the polyol content is often relatively low. On the other hand, in freeze-tolerant insects, the colligative action of polyols would regulate cell volume during extracellular ice formation to prevent excessive freeze concentration of cell. In addition, polyols could stabilize the native state of cell membrane and proteins under non-freezing and freezing low temperature conditions. Thus, winter polyol accumulation would have an important role on cold hardiness in both freeze-intolerant and freeze-tolerant insects.

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