When cells are vitrified with a minimum degree of supercooling, i.e., in near equilibrium, no ice would form in the cell. However, in most cases the degree of supercooling is not minimal, and minute invisible ice crystals will form in cells during the vitrification process. During warming, differences in surface free energy can cause them to recrystallize into larger lethal crystals, which will be lethal to the cells. It is commonly believed that both the cooling rate and the concentration of glass-inducing solutes (cryoprotectants) has to be high during the vitrification process. However, the results that we present here, using mouse oocytes, contradict these beliefs. First, rather than maintaining a high cooling rate during vitrification, our data actually demonstrate that it is the warming rate that should be high. Moreover, our results also contradict the second belief that a high concentration of cryoprotectant is preferred. We show that if the warming rate is extremely rapid, oocyte survival is high even when the vitrification solution contains only a half the normal concentration of cryoprotectants. Therefore, we conclude that rapid warming, rather than cooling or high concentration of cryoprotectant, is essential to prevent intracellular ice formation (recrystallization).

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AntiFreeze Proteins (=AFPs) bind to a surface of ice crystals and inhibit their growth. Some living organisms; fishes, insects and fungus, at low-temperature environment have several different types of AFPs. AFPs protect their body from freezing damages. To clarify the antifreeze effect of lpAFP (isolated from longsnout poacher, a fish living in the sea of Okhotsk) at single molecular level, we performed dynamical observations by using diffracted X-ray tracking (=DXT). DXT is the method to observe single molecular motions using X-rays and gold nanocrystals as probe. Gold nanocrystals were labeled on the target proteins, and when irradiated with X-rays, we monitored diffracted spots from gold nanocrystals. We can observe motions of the proteins by tracking these x-ray diffraction spots. In this study, we used lpAFP isolated from longsnout poacher, and observed the temperature dependency of the lpAFP’s single molecular Brownian motion.

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In Japan, most domestic cattle are produced by artificial insemination using cryopreserved semen. Basic principles and protocols for cattle semen freezing have not been changed for more than 50 years. Recently, however, the improvement of semen cryopreservation has been required because of low conception and/or effective use of sex-selected sperm in dairy cows. In cattle embryos, a type of slow-freezing called “Direct method”, in which cryopreserved embryos in a straw can be transferred into a recipient directly, has been commonly used for in vivo fertilized embryos. However, low survival rates were obtained in in vitro fertilized embryos and in biopsied embryos. Vitrification has not been popular in the cryopreservation of cattle embryos because cryopreserved embryos are needed to be loaded into a straw for embryo transfer after warming. More simple and practical methods with higher efficiency are needed in the cryopreservation of cattle sperm and embryos.

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Phase transfers between the water and ice are a fundamental phenomenon in the cryobiology and cryotechnology. In order to gain insight into the systems in which ice coexists, we investigated time–temperature profiles during the freezing and thawing behaviors of aqueous solutions by a thermal arrest (TA) measurement under stirring. As a result, in addition to freezing point, Tf, an another characteristic temperature, Td, a deviating point where the experimental and calculated two profiles departed from each other, was noted in the both freezing and thawing processes. The constant Td/Tf ratios were obtained in the range from 1.43 to 1.53 for aqueous solutions of NaCl, KCl, and ethylene glycol (EG). At temperature between Tf and Td, ice grains were well-dispersed in the solution and no interference of their crystal growth or thawing occurred among ice species. By contrast, at temperatures below Td, ice grains tended to contact each other, likely inducing the thermophysical properties of the system to deviate from those at temperatures between Tf and Td. These results suggest that Td can be an important parameter to define systems in which ice coexists depending on the initial solution concentration under some specific experimental conditions.

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The boson peak (BP) dynamics of semicrystalline starch was investigated using terahertz time-domain spectroscopy (THz-TDS) and low-frequency Raman spectroscopy. The BP of starch was observed around 1.0 THz in the α(ν)/ν2 spectrum obtained by THz-TDS, where α(ν) is absorption coefficient. Comparing with the previous study of glucose glass, we found that the BP frequency of starch was about 10 % lower than that of the glucose glass, and the value of α(ν)/ν2 was about 30 % smaller than the glucose glass. The results suggest that the origin of absorption around BP frequency is the dipole moment of the hydroxyl group and the α(ν) is proportional to the number of hydroxyl groups per unit volume.

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2-(1-pyridinio)benzimidazolate (SBPa) is a betaine molecule that exhibits solvatochromic shift of the visible absorption spectrum depending on solvent polarity. The maximum of the absorption band of SBPa is located at 358 and 385 nm in aqueous and methanol solutions, while those in trehalose and glucose glasses are at 377 and 369 nm, indicating the polarity of these glasses are in between water and methanol. Two dimensional fluorescence excitation spectra were measured in the saccharide glasses and excitation wavelength dependent shift of the fluorescence maximum (Red-edge effect) was observed. The inclination of the maximum emission frequency versus excitation frequency was almost identical in both glasses (~0.10) indicating similar amount of inhomogeneity. However, the inclination was smaller compared to that observed when a fluorescent molecule, coumarin 153, was utilized as a probe (0.35). [Cryobio Cryotech, 57, 121-124 (2011)] The discrepancy could be reflecting the mobility of the glassy environment surrounding the probe molecules.

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At ambient pressure, glycerol undergoes a liquid-glass transition at Tg =187 K, and the glass state was well studied. While, at room temperature glycerol undergoes a pressure induced liquid-glass transition at about Pg= 5 GPa. The liquid-glass transition of glycerol was studied under high pressure using Raman scattering spectroscopy. According to the pressure dependence of O-H stretching mode frequency, the intermolecular hydrogen bond distance decreased gradually in a liquid state, and it becomes nearly constant in a glassy state indicating the suppression of the free volume in a glass state. The difference between the ambient pressure frozen glass state and the high pressure frozen glass state was discussed. It is found that the characteristic length of medium range order is close to the size of a cyclic trimer of glycerol molecules predicted theoretically by Uchino and Yoko. The pressure induced structural change of glycerol can be essentially the shrinkage of voids and cyclic trimers remain at least up to 11 GPa.

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Group-3 late embryogenesis abundant (G3LEA) proteins are expressed in anhydrobiotic organisms experiencing desiccation stress and their major segments consist of repeats of weakly conserved 11-mer amino acid motifs. It is known that the 11-mer motifs are disordered in aqueous solution but form -helical coiled coil structure in dry state. However, little information has been obtained with regard to the underlying mechanism of such structural change. Here, we performed replica exchange molecular dynamics (REMD) simulations for a short model peptide, called PvLEA-22, in which the 11-mer motif is repeated two times. In addition, for comparison similar simulations were performed for a scrambled peptide without such sequence regularity. The simulation successfully reproduced that the content of -helix of the PvLEA-22 increases with a decrease in water content, while the major structure of the scrambled peptide remained disordered even in the dry state. On the basis of these results, we discuss the structural change mechanism of PvLEA-22.

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Appropriate timing of deacclimation is an important component of winter survival of perennial plants in temperate and boreal zones. The relationship between ambient temperature and the manner of change in freezing resistance of cells is essential information to forecast the timing of deacclimation. In this study, Japanese white birch twigs harvested in mid-February and stored at -20°C were deacclimation-treated at a constant temperature of 0, 4, 10 or 15°C for 3 or 7 days, and then freezing resistance of the cells was determined by an electrolyte leakage test. Birch cells deacclimated in different temperature ranges depending on tissues. Apparent decreases in the freezing resistance of bark cells were observed after deacclimation treatment at 10 or 15°C for 7 days. In contrast, xylem cells initiated deacclimation at 0°C within 7 days. The difference in deacclimation manner between bark and xylem might involve in their freezing behaviors of the cells.

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The purpose of this study was to predict the viscosity of fluid food based on glass transition temperature (Tg). Effect of water content on the Tg of freeze-dried calcium maltobionate was investigated. The results were analyzed by Gordon-Taylor (GT) equation, and the Tg of calcium maltobionate aqueous solution was calculated. Effects of temperature and water content on the viscosity of calcium maltobionate aqueou solution were investigated. The results were analyzed by Vogel-Fulcher-Tammann (VFT) equation. The parameter obtained by VFT analysis could be related to the Tg calculated by GT equation. From these results, it was confirmed that effects of temperature and water content on the viscosity of calcium maltobionate aqueous solution could be predicted by the calculated Tg. A fruit juice showed similar result to calcium maltobionate, but there was a deviation at a low water content.

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We have studied the recovery of activity and structure after cryopreservation of bovine heart cytochrome c (cyt-c) in aqueous solutions of an ionic liquid (IL), choline dihydrogen phosphate (CDHP), with concentrations up to X (mol%IL) = 30 using optical spectroscopy. Although the addition of CDHP induced a decrease of cyt-c activity before and after cooling to 77 K, CDHP showed a reduction in protein damage by cooling. A remarkable result is that although the addition of CDHP caused cyt-c denaturation, >70% of activity and structure after cryopreservation (X > 10) was recovered after the removal of CDHP by dialysis. The present results indicate that concentrated aqueous IL solutions have potential as cryopreservation/refolding solvents for proteins such as recombinant proteins.

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We tested the protective effect of the following reagents on the thermal denaturation of lysozyme: 1) PvLEA-22, which consists of two tandem repeats of the 11-mer motif characteristic to LEA proteins from an African sleeping chironomid, 2) its scrambled-type peptide, which has the amino acid composition identical with that of PvLEA-22, although its sequence is scrambled, and 3) a disaccharide, trehalose. The results of turbidity, enzyme activity and circular dichroism (CD) spectrum measurements showed that the LEA model peptides can inhibit the thermal denaturation of lysozyme.

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Effect of lyoprotectants on the survival rate of freeze-dried lactic acid bacteria was investigated. Freeze-drying and preservation of the bacteria with disaccharides (sucrose and trehalose) and bovine serum albumin (BSA) achieved much higher survival rates than that with dextrin. In addidtion, the disaccharide-BSA mixtures showed synergistic effects on stabilization of freeze-dried cells. This result was similar to the stabilization for freeze-dried enzyme. It was suggested that water replacement effect of lyoprotectants was more important than glass transition effect.

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There is an urgent need to develop highly effective techniques for successful and efficient gene delivery. In this study, we demonstrated the use of cryoprotectants in the newly developed freeze concentration technique in the gene delivery system. We found that at extremely low temperatures, gene transfection was effectively enhanced. Additionally, by comparing the effects of the commercially available cryoprotectant DMSO against a synthesized polyampholyte cryoprotectant, we were able to establish that the latter was more effective at increasing the rate of successful gene transfection. This particular feature could be attributed to the higher freeze concentration factor of polyampholytes in comparison with DMSO, under frozen conditions. Therefore, the study demonstrated the unique efficacy of polyampholytes as a cryoprotectant using the freeze concentration method for efficient in vitro gene delivery.

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It is known that some polyphenols with anti-ice nucleation activity decrease freezing temperature of the solution containing ice nucleators, resulting in maintenance of supercooling state of the solution for a long periods. In the previous study, recombinant proteins of Erwinia ananas ice nucleation protein, inaA with histidine-tag (His-inaA) were expressed in Escherichia coli cells and ice nucleation activity was detected in the cell suspension. In the present study, the purification and the characterization of His-inaA proteins from transformed E. coli cells were done to study the mechanism of anti-ice nucleation of these polyphenols in solutions containing inaA. When the extracts of E. coli cells expressing His-inaA were fractionated into soluble, membrane and inclusion body fractions, ice nucleation activities were detected in all three fractions. Then, His-inaA was purified from the soluble fraction by affinity column chromatography and ice nucleation activity of the purified His-inaA fraction was detected. Further, it is confirmed that anti-ice nucleation activity of polyphenols was detected in solutions containing His-inaA.

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Plants which survive subzero temperatures in winter enhance freezing tolerance upon exposure to non-freezing temperature, a phenomenon termed cold acclimation. Although the mechanism of enhanced freezing tolerance has been examined at the molecular level, its understanding is still incomplete. Recently, Arabidopsis thaliana ecotypes are collected from all over the world. By studying these ecotypes, further knowledge on freezing tolerance could be obtained. To identify novel genes involved in the freezing tolerance after cold acclimation, we developed the screening system to measure the survival rate quantitatively based on Fv/Fm value. With this method, we isolated the ecotypes with higher or lower freezing tolerance than that of Col-0 ecotype. Our method identified ecotypes which show the lower freezing tolerance, including C24 and Cvi, which are already known as ecotypes with lower freezing tolerance.

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Leaf segments of Nymphoides indica, which is at risk of extinction, were cultured on MS medium containing 3% (w/v) sucrose and different concentrations of thidiazuron and naphthalene acetic acid. Multiple shoots were produced in the primary culture and they were subcultured. From subcultured multiple shoots, plants were regenerated. This means that making multiple shoots culture might be one of suitable ways for propagation of rare or endangered plant species. Subcultured multiple shoots were cryopreserved in liquid nitrogen after pre-freezing at -40℃. The survival rate after cryopreservation was as much as 70%, and regrowth of tissues were observed.

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We attempted to cryopreserved cultured Ceratopteris thalictroides cells in liquid nitrogen. Callus cells cryoptrotected with 5% (v/v) DMSO and 10% (w/v) glucose were cooled at a rate of 1℃/min to different temperature in a programmed freezer, then immersed in liquid nitrogen. Callus cells cooled to -10 or -20℃ survived and regrew. However, regrowth of cells cooled and preserved in liquid nitrogen was not recognized. According to TTC assay, viability of preserved cells in liquid nitrogen were high enough to survive immediately after thawing but it decreased rapidly to low level within 24 h of post thaw culture. These results suggest that further improvements are important for enhancement of survival rate after preservation.

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