History of the society from 1959 to 1994 was looked back, and some view points were summarized. Freeze-drying was introduced to my first study on purification of bacterial antigen in 1955. Since then my working and research were concerned with dried biological pharmaceutical products, trials on various kind of freeze-drying machines and development of apparatus for measurement of residual moisture content in dried products. The Japanese Society for Research of Freezing and Drying was established by Professor Tokio Nei, Institute of Low Temperature Sciences, Hokkaido University, in 1955. The first reorganization by the committee report on future plan (Chairman, Prof. T. Nei) in 1968 activated the society. Prof. T. Nei contributed to the society, and his strong impact is shown in the follows: establishment of the society, the first reorganization, president for 6 years, the 15th Cryobiology Meeting in Tokyo, Japan-U.S.A. Joint Seminar, publication of papers and books, encouragement for younger researchers and so on. In 1994, by progress of research field, the second reorganization of the Society was conducted by two presidents, Mitsuo Takano and Kazuhide Yamasato. Society name is changed to "Japanese Society for Cryobiology and Cryotechnology".

View full abstract

Supercooling and vitrification were studied for aqueous polyethyleneglycol (PEG) solutions of molecular weights (Ms)=200, 300, 400, 600, 1000, 1540, 2000, and 3000. It is confirmed that additivity rule holds fairly well in the supercooling of the PEG solutions of M=1000, 2000, and 3000. Glass formation was tested for the PEG solutions at the PEG concentrations at which they give the supercooling down to-100, -90 or -80℃. The T_g variations (T_g: glass transition temperature) with M were analyzed and discussed.

View full abstract

Model freeze-dried foods which consist of polymer (polyvinylpyrrolidone), reducing sugar (arabinose, xylose, fructose, glucose, and lactose), and amino acid (lysine, glycine, glutamine, and glutamic acid) were prepared, and their glass transition temperature (T_g) was investigated. Furthermore, the samples were held at a temperature below the T_g, and the progress of non-enzymatic browning reaction (NEB) was investigated. There was a little effect of the types of reducing sugar and amino acid on the T_g the freeze-dried solids. The NEB rate, however, was affected strongly on the types of reducing sugar and amino acid. The NEB rate of the samples of varying reducing sugar was higher in order of arabinose ≈ xylose>fructose>glucose>lactose. The NEB rate of the samples of varying amino acid was higher in order of lysine>glycine>glutamine>glutamic acid.

View full abstract

We have investigated additional effect of protein denaturant agent (guanidine hydrochloride (GdnHCl)) on the hydrophobic hydration structure in the aqueous protein model (n-tetrapropylammonium chrolide (n-Pr_4NCl)) solution at 77K as a function of GdnHCl concentration. It is found that the addition of GdnHCl induces the disruption of the hydration structure in the aqueous n-Pr_4NCl solution. We suggest that the hydrophobic interaction changes to other interactions such as n-Pr_4N^+H…H_2O or n-Pr_4N^+…GdnHCl by the addition of GdnHCl.

View full abstract

We have investigated low temperature-induced secondary structural changes of polypeptide and helical rich proteins using FTIR and CD spectroscopies. It is found that the low-temperature crystallization induces the increase of the solvated α-helical structure, which is due from the hydration between the α-helix structure and water molecules, of polypeptide and helical-rich proteins. The α-helical structures of polypeptide and helical-rich proteins did not unfold in spite of the ice formation in bulk water. Our results suggest that helical proteins may have the possibility of self-depression function to the low temperature-induced protein unfolding.

View full abstract

We studied thermodynamic properties of the following three different model peptides after desiccation in pure states and in binary ones with trehalose added: 1) model peptide (Hereafter referred to as PvLEA22), which consists of 2 repeats of the 11-mer consensus motif characteristic of late embryogenesis abundant proteins from P. vanderplanki, 2) model peptide designed as the two repeating sequence of PvLEA22 (Hereafter PvLEA44), and 3) control, i.e. the peptide with the amino acid composition identical with that of PvLEA22, although its sequence is randomized. Differential scanning calorimetry (DSC) measurements exhibited that all of the three peptides were vitrified in dried states at ambient temperature, although the glassy matrix of PvLEA22 in itself was found, by enthalpy relaxation rate, to be thermodynamically more stable than that of control. Two binary mixtures, PvLEA22: trehalose and control: trehalose, were analyzed by spectroscopic measurements (FT-IR) in temperature-dependent manners, with the conclusion that the glassy matrix of trehalose was strengthened by addition of the model peptides, of which effect was larger for PvLEA22 than for control.

View full abstract

To stabilize freeze-dried mixture of nucleoside phosphorylase (NP) and xanthine oxidase (XOD), effects of sucrose and sucrose-bovine serum albumin (BSA) mixture on the remaining activity of the enzymes were investigated. Furthermore, glass transition temperatures (T_g) of the dried samples were examined. The T_g of sucrose sample was lower than that of sucrose-BSA one. This indicates that physical stability of the dried samples was improved by the addition of BSA. The activity of NP-XOD mixture was greatly protected by sucrose. Furthermore, the combination of sucrose and BSA improved the enzymes stability.

View full abstract

The electronic dephasing times of a chromophore, oxazine 4 (Ox4), doped in low temperature saccharide glasses were investigated by femtosecond photon echo spectroscopy. It was revealed that the dephasing time is much longer in the saccharide glasses compared to hydrogen bonding polymer glass, polyvinylalcohol (PVA), when temperature was lowered to 10 K. A critically damped low frequency oscillation was observed in the signal which can be attributed to the phonon mode of the saccharide glass. The electronic coherence is preserved in the low-temperature saccharide glass due to the restraint thermal fluctuation.

View full abstract

Here the three-dimensional (3-D) structure of TRET1, a novel trehalose transporter from an anhydrobiotic insect, Polypedilum vanderplanki, was predicted by homology modeling in which the 3-D structure (1SUK) of GLUT1, glucose transporter from human was selected as a template. It was found that TRET1 has 12 transmembrane (TM) helices with an inward-facing conformation. Next, to explore the dynamics of the protein, we performed molecular dynamics (MD) simulation for the protein embedded in a hydrated phospholipid bilayer. The result of principal component analysis indicated that the protein has a hinge-bending motion, that is, the helices on the intracellular side come close or draw apart together. This dynamics may be essential for substrate uptake. Furthermore, we performed docking simulation combined with binding energy calculation to investigate the substrate selectivity of TRET1. As a result, it was found that trehalose more strongly binds to TRET1 than its isomer, isotrehalose, consistent with available experimental data.

View full abstract

Elastic properties of the three kinds of polymorphic hen egg white lysozyme crystals with tetragonal, orthorhombic and monoclinic structures are investigated by micro-Brillouin scattering. The temperature dependences of sound velocity and absorption show the variation due to the difference of crystal structure. The temperature dependence of Brillouin scattering of a tetragonal crystal is measured also in the aqueous glycerol solution. The obtained relaxation parameters suggest that hydrated water molecules around a protein molecule are substituted in part for glycerol molecules. The relaxation time obeys the Arrhenius law, and the attempt relaxation rate and the activation energy clearly show the correlation predicted by the Mayer-Neldel rule.

View full abstract

We introduce a novel technology to cryopreserve cells directly in monolayer utilizing a collagen vitrigel membrane as a scaffold. The membrane has excellent mechanical strength and can be easily handled by tweezers even after culturing cells on it. In this study, we used the membrane for direct preservation of primary hepatocytes, feeder cells, and ES cells. Freshly isolated hepatocytes (4x10^5 cells), mitomycin C treated mouse fibroblasts (feeder cells) or mouse embryonic stem (ES) cells were inoculated and cultured on a collagen vitrigel membrane (35mm in diameter) in a culture dish for 24hr to form colonies in monolayer. The cell colony-spread scaffold was transferred to a new dish with fresh preservation medium containing 10%DMSO. The dish was then gradually frozen to -80℃ and stored in liquid nitrogen for 1 week to 3 months. The cell adhesiveness and viability before and after cryopreservation were evaluated by cell morphology and calcein staining, respectively. The cells preserved by this method showed significant improvement compared to cells preserved without the collagen vitrigel membrane.

View full abstract

Group-3 late embryogenesis abundant (G3LEA) proteins are expressed in anhydrobiotic organisms experiencing desiccation stress and their major segments consist of repeated and conserved 11-mer amino acid units. However, little information has been obtained with regard to the properties of these repeat units. Here, we performed replica exchange molecular dynamics (REMD) simulations for a short model peptide in which the 11-mer motif is repeated two times. And for comparison the similar simulation was performed for a control peptide without such sequence regularity. It was found that the LEA model peptide has a high propensity to form the α-helical coiled coil structure in vacuo, while such a tendency was not observed for the control peptide. These results are consistent with our previous FTIR results for the corresponding peptides in the dry state. At the global energy minimum, two LEA peptide chains were shown to form right-handed coiled coil with an antiparallel arrangement, and to come in contact with each other through the interaction of hydrophilic residues. In conclusion, the intermolecular electrostatic interactions are the main driving force of the above coiled coil formation.

View full abstract

Adipose tissue-derived stem/progenitor cells (ASCs) have attracted attention as a promising cell source that replaces marrow stromal cells (MSCs). In this study, we developed an improved method for the cryopreservation by changing the component of a freezing medium (three freezing mediums including 10% DMSO, culture medium-DMSO, CELLBANKER 2, and culture medium-DMSO (serum-free), 0.1mol/L maltose and 1% sericin). Sericin, a protein hydrolysate (with an average molecular weight of 30kDa) is very rich in serine and obtained from raw silk during the degumming process. We further measured the viability and the adipo/osteogenic potential of frozen human ASCs using three freezing mediums. After thawing of cryopreserved human ASCs in the freezing medium, the cell viabilitiy was shown to be more than 95% from the trypan blue dye exclusion test. The cell proliferation of thawed human ASCs in CELLBANKER 2, and culture medium-DMSO (serum-free), 0.1mol/L maltose and 1% sericin had higher rate in comparison to culture medium-DMSO. The adipogenic induction shows similar tendency as cell proliferation. Additionally, we investigated osteogenic differentiation of cryopreserved human ASCs for 4 weeks. The frozen human ASCs were positive for von Kossa staining, suggesting that the cells had osteogenic differentiation capabilities.

View full abstract

A Low-temperature storage system for rice in which grain temperature is maintained below 15℃ during storage has been commercially used for brown rice storage in Japan. The low-temperature storage system requires an electric cooling system and electric energy to cool rice in summer. The objective of this study was to determine whether snow removed from streets can be used to replace the electric cooling system and electric energy for cooling a rice low-temperature storehouse. About 27% of the electric energy for cooling the rice storehouse could be replaced by using snow piles removed from downtown streets. The quality of rice stored in the storehouse was preserved at a level almost similar to that of freshly harvested rice. The results of this study indicate that a snow pile can be utilized for cooling a rice storehouse as a high-quality rice storage system without electric energy consumption.

View full abstract

Plant cells or tissues are usually pre-cultured for enhancement of desiccation tolerance and/or encapsulated in gel such as arginate beads when the materials are cryopreserved in liquid nitrogen by desiccation method. We successfully cryopreserved suspension cells without any pre-culture or encapsulating in gel substance by desiccation method. Suspension cultured cells of Pogonatum inflexum and Polytichum commune were desiccated rapidly or slowly and cryopresrved. Pogotatum cells were successfully cryopreserved in liquid nitrogen at a high survival rate whichever the desiccation rate. Polytrichum cells were also cryoprederved successfully and showed a high survival rate. But most Polytrichum cells were dead when they were desiccated rapidly. The qualitative differences in desiccation tolerance between Pogonatum and Polytrichum cells were associated with the speed of dehydration.

View full abstract

Freezing tolerance of plants growing in temperate and frigid zones has a crucial feature to survive in cold winter. Interestingly, in Arabidopsis cells, about half of the freezing tolerance depends on extracellular calcium. Furthermore, extracellular calcium increases tolerance to the mechanical stress caused by electroporation, which disrupts the plasma membrane, but it does not increase tolerance to the simple osmotic stress. It has also been indicated that calcium-dependent freezing tolerance involves plasma membrane resealing via SYT1 in Arabidopsis. However, the generality of this phenomenon in freezing-tolerant plants remains unknown. Here, we hypothesize that plants which survive in cold winter generally possess the calcium-dependent freezing tolerance. To test this hypothesis, winter wheat (Triticum aestivum L. cv. Chihokukomugi) leaves were used as a representative of monocots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers were used as a dicot other than Brassica species. The calcium-dependent freezing tolerance was measured with protoplasts or tissue sections by calculating as the difference between survivals in the presence and absence of calcium. Our results indicate wheat leaves and Jerusalem artichoke tubers possess the calcium-dependent freezing tolerance. Thus, it is possible that the calcium-dependent freezing tolerance is a common mechanism for many angiosperms. On the other hand, the level of calcium-dependent freezing tolerance was quite different between wheat leaves and Jerusalem artichoke tubers. This suggests that the strategy of freezing tolerance is different among plants or their organs.

View full abstract

Morphological behavior of ice crystals and cells in the tissues during the constant-temperature storage after rapid-freezing was visualized in time-series using a confocal laser scanning microscope (CLSM) with a fluorescent dye. Recrystallization of intracellular ice crystals, selectively-coarsening of fine ice crystals, was investigated quantitatively. Size and number of ice crystals were measured from the CLSM images and analyzed statistically to find frequency, average, and standard deviation of the size of ice crystals, total amount and number density of ice crystals, etc. in time-series during the storage at three different constant-temperatures. Influence of the storage-temperature of frozen tissues on these characteristics was made clear.

View full abstract

Dynamic characteristics of water in organs such as roots, leaves and hypocotyls in cowpea (Vigna unguiculata (L.) Walp) under drought stress were evaluated by comparing with that of related species, common bean (Phaseolus vulgaris L.) with ^1H-NMR relaxation times (T_1 and T_2). There was no difference between T_1 and T_2 of cowpea roots under drought stress for two days while those of common bean significantly decreased. Regarding leaves, although T_2 values and water content of cowpea roots maintained the initial level, these parameters of common bean significantly decreased during four days of water deficit. Water content and T_1 of hypocotyls of drought cowpea slightly decreased on the eighth days after withholding water, while hypocotyls retained water longer compared with roots and leaves under water deficit. In addition, the electric conductivity indicated that hypocotyl tissues of cowpea under drought stress for eight days were not injured while those of common bean were partly injured under drought stress for four days. Higher water mobility indicated by long T_1 in fresh cowpea hypocotyls might play an important role in maintaining integrity of cowpea organs against drought stress.

View full abstract

Blueberry stems (ca. 20cm long) were placed at -8〜-10℃ without ice inoculation and freezing process was observed using infra-red video thermography. Differential images obtained by subtraction of the initial image clearly visualized where the first freezing events occurred and how the freezing propagated in the stems. The results maybe summarized as follows: 1) freezing pattern was different from one experiment to another even when the same stems was repeatedly used; 2) in many cases, freezing was initiated from multiple sites (not like the ones reported by other researchers); 3) the speed of ice spread was faster when the stem was supercooled more; 4) freezing was sometimes arrested or slow-downed at stem nodes. These things allow us to speculate that the ice nucleation activity should occur throughout the stems. To test this hypothesis, we determined ice nucleation activity of blueberry stems using a test tube method. The results showed that every stem segment (7.5mm long) had a high ice nucleation activity. The thermography data and test tube ice nucleation assays were in agreement and supported an idea that high intrinsic ice nucleation activities occur throughout the blueberry stems that allow the stems to readily initiate freezing upon exposure to subzero temperatures.

View full abstract

The objective of this study was to establish a cryopreservation protocol for shoot apices of cranberry (Vaccinium macrocarpon Ait. cv. Early Black) and highbush blueberryn (V. corybossum L. cv. Rancoccas). Cryopreservation was carried out via encapsulation-dehydration on shoot apices excised from in vitro cultures. In the encapsulation dehydration method of both species, the protocol leading to optimal regrowth was as follows: After cold-acclimation at 5℃ in the dark for 2 weeks, excised shoot tips were pretreated for 24hr at 25℃ on hormone-free culture medium with 0.4mol/L sucrose, then encapsulated and precultured in liquid culture medium with 0.8mol/L sucrose for 16hr. Precultured beads were dehydrated for 360-mins in the dessicator containing 50g silica gel at 18% moisture content (fresh weight basis) before cryostorage for 1hr. In addition, the effect of adding glycerol and/or salicylic acid to and loading solution was examined to enhance regrowth after cryopreservation. In the present study, it was shown that adding both 0.5mol/L (cranberry) or 1.0mol/L glycerol (highbush blueberry) and 50μmol/L resulted in high regrowth.

View full abstract