The growth rates of ice crystals are measured by cryomicroscopic observations for various glass-forming aqueous solutions. The size of ice crystals after cooling and their maximum size during warming is deduced from these rates by integration over the considered thermal range and is expressed as a function of the cooling and warming rates used. The analysis is however limited to the determination of the heterogeneous ice hucleation thermal range which is the critical step in the calculation of the critical cooling and warming rates. The expression of the ice crystal growth rates for glass-forming aqueous solutions is approximated to the expression previously reported by Doremus for crystal growth rates in glasses with a dependence on the viscosity of the solution for high supercoolings. The integration leads to expression involving exponential functions.

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Shoot apices of asparagus (Asparagus officinalis L.) can be successfully cryopreserved (by freezing at a rate of 0.5℃/min in the presence of 8% DMSO and 0.2 M sorbitol) after pretreatments either by precultufe in media containing 0.5 M glucose for 2 days or by cold acclimation at 0℃ for 20 days. In the present study, we examined the effects of pretreatments for subsequent successful cryopreservation of the shoot apices. Freezing tolerance, as revealed by LT_<50> under equilibrium-freezing, was -20℃ in both specimens with pretreatment by 0.5 M glucose and by cold acclimation, while LT_<50> was -7℃ in control specimens. Both pretreatments produced distinct cellular ultrastructural changes as observed with ultra-thin section electron microscopy. Number of plastids rich in starch granules increased in precultured specimens, whereas those poor in the starch granules increased in cold acclimated specimens. Occurrence of freezing injury by equilibrium-freezing, as observed with a freeze-fracture replica technique, was associated with formation of aparticulate domains with accompanying fracture-jump lesions in the plasma membranes, not only in precultured specimens with 0.5 M glucose and in cold acclimated specimens, but also in control specimens.

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The freeze concentration processes of agarose gels with or without sucrose were observed using a Cryo-SEM and a light microscope for the purpose of understanding the behavior of a polymer network and a sucrose solution in its gel during the freezing processes. Furthermore, whether the polymer network and a sucrose solution in the frozen gels are in a crystalline state or a glassy state was investigated using differential scanning calorimetry (DSC). As a result, it was suggested that the polymer network during freezing is assembled, like a membrane, on the overall surface of ice crystals. However, the diffusion of water is thought to be not very inhibited by the membranes because the ice crystals grew very fast and the form of ice crystals became polygonal. Such behavior of the polymer network itself was independent of the presence of sucrose. The sucrose molecules added to the gel affected the ice crystal growth in the gel in the same way as in the case of a sucrose solution. The results of the DSC measurement indicated that only the sucrose solution in the gel turns into a glassy state.

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Cryosurgery, the use of freezing to destroy undesirable tissue, has experienced significant advances in the last decade, which have brought to a precipitous growth of this field in the United States. During the last two years over one hundred hospitals have introduced the use of cryosurgery for the treatment of liver and prostate cancer. This paper will present a review of advances in the areas of imaging frozen tissue with ultrasound and magnetic resonance and in the understanding of the biophysics of freezing in tissue.

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The liver is an organ with complex functions such as metabolism and synthesis of essential substances and detoxification of unnecessary or toxic substances. Sever liver dysfunctions somemtimes become irreversible and its prognosis is then poor. Two major therapies for the hepatic failure are there such as artificial liver support (ALS) and liver transplantation. The big limit of transplantation is the shortage of donor and waiting death is reported over 20%. Thus, an artificial liver is expected to treat the patients. For clinical application of ALS using isolated hepatocytes, large amounts of viable hepatocytes have to be always obtainable for urgent clinical needs. For this purpose, we have been trying to develop methods for the large-scale preparation of hepatocytes from large animal liver and for the mass cryopreservation of isolated hepatocytes. In this article, the present status of hepatocyte preparation and preservation will be reviewed and its future aspects will be discussed.

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The protective agents for halophilic bacteria subjected to L-drying were screened by using Oceanospirillum multiglobuliferum IFO 13614 as an indicator and 75% seawater containing 5% monosodium glutamate, 1.5% adonitol and 1% sorbitol as a basal protective medium (BPM). Effects of 26 compounds on the survival of O. multiglobuliferum IFO 13614 were examined, and methly cellulose and L-proline were found as an effective agent. By the addition of 0.25% methyl cellulose 4000cps or 0.25% proline into BPM, the survival value of O. multiglobuliferum IFO 13614 increased to the value about 10-fold higher than that using BPM alone, and the protective effect was enhanced by using a mixture of these compounds. Furthermore, the mixture of methyl cellulose and proline was also effective for preservation of various halophilic bacteria.

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In recent years, a possibility that glass transitions occur during food processings has been realized. And devitrifications at high temperature region were observed during a process of ascending temperature of foods presented in glassy state. On the other hand, studies of fish meat powder having gel forming and biological activities have been carried out with Alaska pollack, carp and sardin. The gel forming ability of the powder was obtained by adding stabilizers like sugars and using lyophilization. These fish meat powder made of myofibrils might be presented in glassy state. In the present paper, therefore, the relationship between glass transition and food preservability of these myofibrils was determined by using the differential scanning calorimetry (DSC). Carp myofibrils were freeze-dried with sucrose, raffinose, dextrin, sorbitol, ovalbumin, etc. as a representative of each sort which is known as a material effected on transition into vitrification, and ground into powder, and then induced to DSC study. As the results any transition seemed to devitrification was not found on the DSC patterns of these powders. On the other hand endo thermic heat peaks of DSC seemed to denaturations were observed around at 110℃.

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Experiments were carried out to establish the technique of cryopreservation of animal blood vessel. Blood vessels taken out from rats, swine and dogs were used. Blood vessels of rats, which had been preserved in liquid nitrogen from 2 weeks to 16 months, were allografted to rats' abdominal arteries. 8 rats out of 32 survived more than a week. Observation of the grafts taken out again from these rats were in good condition without any pathological problem.

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Cryoprotectants are used to improve the viability of biological cells in cryopreservation of the cells. However, the details of the mechanism of protection of cells by cryoprotectants are not yet clear. In this study the process of solidification of human red blood cell suspensions was investigated microscopically. Three kinds of solutions were used: physiological saline, and physiological salines with 2.00M dimethyl sulfoxide (DMSO) and with 20.0wt% dextran (Dxt). Addition of DMSO or Dxt to physiological saline caused the cellular ice crystal to have secondary arms and a morphologically complicated structure, and simultaneously, facilitated to deform flexibly the freezing interface in response to the cells. This result suggests that DMSO and Dxt have an effect to decrease the mechanical action on the cells.

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From the Tg data for the LiCl-H_2O-C_2H_5OH solutions and aqueous R_4NX solutions (R=CH_3, C_2H_5, n-C_3H_7; X = Cl, Br and NO_3) and the Raman spectra for aqueous KCl and (C_2H_5)_4NCl solutions, it is concluded that hydrophobic interaction plays an improtant role in these solutions (except the KCl solution) even in high salt concentration regions.

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Glass-forming composition regions of aqueous alkylurea (R=CH_3, C_2H_5) solutions were determined by a simple DTA method. Aqueous solutions of methylurea, 1,3-dimethylurea, 1,1-diethylurea show the normal Tg behavior with solute concentration, but those of tetrameth-ylurea and ethylurea show anormalous Tg behavior. It is concluded that the hydrophobic interaction plays an important role in the glass-forming of these solutions.

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The volume change of aqueous solutions on freezing was measured for various alkali metal and alkaline earth metal salts. For sodium salts without the influence of anion species, sodium salts showed almost the same pattern, i.e., the change was smaller than that of water, and it became smaller with the increase in the concentration of sodium salts. For potassium salts the change did not show the same pattern according to the influence of anion species. With the increase in the concentration of potassium salts, the change for KCl became lager, the change for KNO_3 was constant, and the change for K_2CO_3 became smaller. For magnesium and calcium salts the changes did not show the same pattern according to the influence of anion species, too, and all the changes became smaller with the increase in the concentration of salts. When the changes dependent on cation species in the same anions were compared, the biggest was for potassium, next for sodium, and the smallest were for calcium and magnesium.

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Ice banks added with synthetic smectites as ice nucleating substances were investigated for their energy-saving efficiency. Because of the high dispersibility in water as well as ice nucleating activity of the smectites, significant energy-saving was indicated by the use of the ice bank developed.

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Living xylem ray parenchyma cells of woody plants are adapted to freezing temperatures during winter either by extracellular freezing or by freezing avoidance by deep supercooling. In this study, we studied the low temperature behaviour of xylem ray parenchyma cells of M. bombycis by observation using cryo-scanning electron microscopy and freeze-fracture replica technique in addition to differential thermal analysis (DTA). DTA of xylem in M. bombycis indicated deep supercooling profiles of xylem ray parenchyma cells both in winter and summer. But observation by cryo-scanning electron microscopy and freeze-fracture replica technique showed that xylem ray parenchyma cells in M. bombycis took a variety of low temperature behaviour ; in winter, deep supercooled cells, extracellularly frozen cells, freeze-concentrated cells and dried cells were observed, while in summer deep supercooled cells and dried cells were observed.

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The processes of freezing injury in Jerusalem artichoke (Helianthus tuberosus L.) tubers were studied using protoplasts isolated from cold-acclimated and deacclimated tubers. Prior to freezing, protoplasts were pre-loaded with 10^ M PDA (fluorescein diacetate) in an isotonic sorbitol solution. After freeze-thawing cycle at various temperatures, cell viability was evaluated under a fluorescence microscope according to the fluorescence images. In cold-acclimated tubers, more than 70% of protoplasts survived freezing to -25℃. By contrast, in deacclimated tubers, only 39% of protoplasts survived freezing to -10℃. Thus, freezing tolerance of proto-plasts differed significantly between cold-acclimated and deacclimated tubers. Two distinct types of injury were observed depending on freezing temperatures, which were caused by damage to plasma membrane (cell burst type) and damage to vacuolar membrane (abnormally stained type), respectively. The decrease in freezing tolerance of protoplasts after deacclimation was suggested to be mainly due to destabilization of vacuolar membrane against freeze-induced dehydration stress.

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Freezing tolerance in shoot apices of blueberried honeysuckle (Lonicera caerulea L. var. emphyllocalyx NAKAI) was low in summer, and very high in winter. Levels of freezing tolerance increased from October to December, and decreased from March to April. Water content was low in winter, and high in summer. Six kinds of sugar were detected in shoot apices. Changes in total sugar content and sucrose content did not coincide with the changes in freezing tolerance. Periodicity of accumulation of high raffinose and stachyose is considerably correlated with the period in which shoot apices showed the high freezing tolerance. These results suggest that raffinose and stachyose serve to increase freezing tolerance, and that sugar composition was more important than total sugar content.

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The mechanism of freezing injury in wheat (Triticum monococcum L.) cells cultured on medium containing abscisic acid (ABA) was investigated. Cells which had fully developed freezing resistance on ABA-medium showed a higher rate of TTC (triphenyltetrazolium chloride) reduction than that of non-frozen control cells when frozen to lethal freezing temperatures. However, this increase in TTC reduction diminished when frozen-thawed cells were incubated at 25℃ for 48 hr, suggesting that the process of freezing injury further advances during the incubation after thawing. A very similar increment of TTC reduction was observed in ABA-cultured cells even when the cells were treated for a short time with ethanol or detergents such as Triton X-100. In ABA-cultured cells, in contrast to 2,4-D-cultured cells, a relatively higher rate of O_2-consumption of cells was maintained and a rapid influx of TTC into cells was observed after freeze-thawing or after treatment with ethanol or detergents. Therefore, these are considered to be the cause of the incremental response of TTC reduction in ABA-cultured cells after freeze-thawing, and it is suggested that some structural changes in the plasma membrane occur during freeze-thawing at lethal freezing temperatures.

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