Cryoprotective Mechanism Research Articles

Lipid membrane modulation and pigmentation: A cryoprotection mechanism in Arctic pigmented bacteria.

The present study aims to address the effect of gradual change in temperature (15-4 °C) followed by freeze-thaw on pigmented bacterial strains - Leeuwenhoekiella aequorea, Pseudomonas pelagia, Halomonas boliviensis, Rhodococcus yunnanensis, and Algoriphagus ratkwoskyi, isolated from Kongsfjorden (an Arctic fjord) to understand their survival in present climate change scenario. The total cell count and retrievability of the isolates were not affected despite the variation in temperature. In all the isolates, the saturated fatty acids, particularly stearic and palmitic acid were predominant at higher temperature, while at 4 °C, the unsaturated fatty acids, primarily cis-10-pentadecenoic, palmitoleic, and oleic acid, were major constituents, confirming homeoviscous adaptation. Even after freeze-thaw, the unsaturated fatty acid composition was retained in all the isolates except A. ratkwoskyi. The increase in unsaturated fatty acids was at the expense of their saturated analogs, probably by desaturase activity. The major pigment in the isolates resembled Zeaxanthin, whose concentration was found to be 26-65% higher after freeze-thaw, suggesting its vital role as a cryoprotective agent in regulating membrane fluidity. Such experimental simulations related to freeze-thaw in polar bacterial isolates are helpful in understanding the physiological plasticity adaptations, which could be critical for survival in harsh and rapidly changing polar environments.

Cryoprotective roles of trehalose and alginate oligosaccharides during frozen storage of peeled shrimp (Litopenaeus vannamei).

Cryoprotective saccharides are widely accepted additives that reduce thawing loss, maintain texture, and retard protein denaturation in the frozen seafood. The present study aimed to investigate the roles of trehalose and alginate oligosaccharides on cryoprotection of frozen shrimp, primarily focusing on the interactions between myosin and saccharide molecules using a molecular dynamics (MD) simulation analysis. The results indicated that soaking in the trehalose and alginate oligosaccharides solutions markedly reduced thawing and cooking losses in frozen shrimp, with respective values decreasing to 6.02%, 8.14%, and 5.99%, 8.19% after 9weeks of storage, which were significantly lower than that of fresh water treatment (9.75% and 15.09%). Our assumption was that water replacement played a leading role in cryoprotection, as shown in previous experimental results and reports. Furthermore, homology modeling and MD simulations confirmed that trehalose and alginate oligosaccharides substituted the water molecules around the myosin surface by forming hydrogen bonds with polar residues of amino acids, thereby stabilizing the structures in the absence of water during frozen storage. These conditions affected the flexibility of particular amino acid residues, enhanced the residue cross correlations within the two chains of myosin, and also increased the total interaction energy between myosin and water/saccharide molecules, thereby leading to an increase in protein stability. Finally, by comparing the experimental results to that of MD simulation, significant positive correlation existed between saccharides and the stabilization of myosin in shrimp muscle. The findings of the present study may help better understand the cryoprotective mechanisms of saccharides in frozen shrimp, and the two saccharides may be potentially used as alternative additives in seafood to maintain better quality during frozen storage.

Theoretical and experimental study of the antifreeze protein AFP752, trehalose and dimethyl sulfoxide cryoprotection mechanism: correlation with cryopreserved cell viability.

In this work the physico-chemical properties of selected cryoprotectants (antifreeze protein TrxA-AFP752, trehalose and dimethyl sulfoxide) were correlated with their impact on the constitution of ice and influence on frozen/thawed cell viability. The freezing processes and states of investigated materials solutions were described and explained from a fundamental point of view using ab-initio modelling (molecular dynamics, DFT), Raman spectroscopy, Differential Scanning Calorimetry and X-Ray Diffraction. For the first time, in this work we correlated the microscopic view (modelling) with the description of the frozen solution states and put these results in the context of human skin fibroblast viability after freezing and thawing. DMSO and AFP had different impacts on their solution's freezing process but in both cases the ice crystallinity size was considerably reduced. DMSO and AFP treatment in different ways improved the viability of frozen/thawed cells.

Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation.

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10% and 5h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

Characterization of cold-associated microRNAs in the freeze-tolerant gall fly Eurosta solidaginis using high-throughput sequencing

Significant physiological and biochemical changes are observed in freeze-tolerant insects when confronted with cold temperatures. These insects have adapted to winter by retreating into a hypometabolic state of diapause and implementing cryoprotective mechanisms that allow them to survive whole body freezing. MicroRNAs (miRNAs), a family of short ribonucleic acids, are emerging as likely molecular players underlying the process of cold adaptation. Unfortunately, the data is sparse concerning the signature of miRNAs that are modulated following cold exposure in the freeze-tolerant goldenrod gall fly Eurosta solidaginis. Leveraging for the first time a next-generation sequencing approach, differentially expressed miRNAs were evaluated in 5°C and −15°C-exposed E. solidaginis larvae. Next-generation sequencing expression data was subsequently validated by qRT-PCR for selected miRNA targets. Results demonstrate 24 differentially expressed freeze-responsive miRNAs. Notable, miR-1-3p, a miRNA modulated at low temperature in another cold-hardy insect, and miR-14-3p, a miRNA associated with stress response in the fruit fly, were shown to be significantly up-regulated in −15°C-exposed larvae. Overall, this work identifies, for the first time in a high-throughput manner, differentially expressed miRNAs in cold-exposed E. solidaginis larvae and further clarifies an emerging signature of miRNAs modulated at low temperatures in cold-hardy insects.

Distribution of Solute Molecules in Bilayer Stacks by Medium Angle Diffraction

Previously we determined using neutron membrane diffraction the density profile of solute molecules confined between lipid bilayers. This was to understand the mode of action of small solutes (sugars) as cryoprotectants (1, 2). The interlamellar profile of sugars differs with those determined indirectly from molecular dynamics simulations(3) but the result reinforces a view of the mechanism of cryoprotection(4) where specific interactions between sugar molecules and the bilayer play a secondary role. This work is extended to the interaction of another cryoprotectant, dimethylsulfoxide (DMSO), with lipid bilayers made from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The results are discussed in terms of the effects of DMSO on the stability and structure of the fluid lamellar phase. We also discuss the limitations of using fully hydrated single bilayer molecular dynamics simulations(5) to characterise the interactions of cryosolvents with phospholipid membranes and we compare findings with experiments with multiple stacked bilayer systems.1. Kent, B., T. Haus, B. Deme, V. Cristiglio, T. Darwish, T. Hunt, G. Bryant, and C. J. Garvey. 2015. Direct Comparison of Disaccharide Interaction with Lipid Membranes at Reduced Hydrations. Langmuir 31:9134-9141.2. Kent, B., T. Hunt, T. A. Darwish, T. Haus, C. J. Garvey, and G. Bryant. 2014. Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms. Journal of The Royal Society Interface 11: 95-100.3. Pereira, C. S., and P. H. Hunenberger. 2006. Interaction of the sugars trehalose, maltose and glucose with a phospholipid bilayer: A comparative molecular dynamics study. Journal of Physical Chemistry B 110:15572-15581.4.Bryant, G., and J. Wolfe. 1992. Interfacial forces in cryobiology and anhydrobiology. Cryo-Lett. 13:23-36.5.Hughes, Z. E., A. E. Mark, and R. L. Mancera. 2012. Molecular Dynamics Simulations of the Interactions of DMSO with DPPC and DOPC Phospholipid Membranes. Journal of Physical Chemistry B 116:11911-11923.

The prospects of the application of gases and gas hydrates in cryopreservation

In the present review, we attempt to evaluate the known properties of gas hydrates and gases that form them from the point of view of the mechanisms of cryoinjury and cryoprotection, to consider the publications on the freezing of biological materials in the presence of inert gases, and to assess the prospects of development in this field. For this purpose, we searched for information on the physical properties of gases and gas hydrates, compared the processes that occur during the formation of gas hydrates and water ice, and analyzed the effects of the formation and growth of gas hydrates on the structures of biological objects. We prepared a short review on the biological effects of xenon, krypton, argon, carbon dioxide, hydrogen sulfide, and carbon monoxide with emphasis on hypothermal conditions and the probable application of these properties in cryopreservation technology. The descriptions of the existing experiments on cryopreservation of biological objects using gases are analyzed. Based on the information we found, the most promising directions of work in the field of cryopreservation of biological objects using gases are outlined; potential problems in this field are anticipated.

Embryo and gamete cryopreservation for genetic resources conservation of laboratory animals

Article reviews the use of embryos and gametes cryopreservation for cryobanking the laboratory animal species. The special emphasis is made on the mechanisms of cryoinjury and cryoprotection during program freezing and vitrification. The species specific cryobanking problems are discussed and the prospects to overcome these problems are outlined.

C-2007: Towards understanding the protection afforded by cryoprotectants commonly used in the cryopreservation of zygotic explants of recalcitrant seeds

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. The post-cryo success achieved with the excised zygotic explants used for cryopreservation is variable, and for some species dependent on the use of cryoprotectants and rapid drying. However, the efficacy of different cryoprotectants seems to be species dependent and there is a paucity of information on the mechanisms via which particular cryoprotectants protect zygotic embryos from recalcitrat seeds during partial drying and cryogenic cooling. The present study used Differential Scanning Calorimetry (DSC) and Cryo-Scanning Electron Microscopy (Cryo-SEM) to characterise the behaviour of selected cryoprotectants in zygotic embryos during drying and cooling to cryogenic temperatures. These studies were carried out on a range ofrecalitrant-seeded species ( Castanospermum australe, Trichilia dregeana, Amaryllis belladonna, Strychnos gerrardii ) that show differential tolerance to cryoprotectants and cryopreservation. Exposure to glycerol or sucrose did not change the behaviour of water in zygotic explants of most of the species studied. DSC revealed little to no difference between fresh samples and those cryoprotected, showing very similar enthalpy of melting transitions (∼300 J/gH 2 O) and amount of unfrozen water (∼0.30 gH 2 O/gdm); except for Atropa belladonna treated with glycerol that showed a slightly higher amount of unfrozen water (∼0.45 gH 2 O/gdm). However, Cryo-SEM images showed important changes in the mechanical properties of the cryoprotected tissue during rapid drying. In general, glycerol allowed for less destructive compaction of the zygotic tissues during desiccation, particularly in A. belladonna . Glycerol was also observed to help in membrane stabilization of Corynosoma australe , particularly during rehydration of the tissue. The results are discussed in relation to other studies on the mechanisms of cryoprotection in shoot tips and will inform the future cryopreservation protocols for zygotic germplasm of recalcitrant-seeded species.

Low-temperature molecular motions in lipid bilayers in the presence of sugars: insights into cryoprotective mechanisms.

Sugars and sugar alcohols can stabilize biological systems under extreme conditions of desiccation and freezing. Phospholipid bilayers solvated by aqueous solutions of sucrose, trehalose, and sorbitol at concentrations of 0.2 and 1 M and containing incorporated spin-labeled stearic acids were studied by electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR). The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and the stearic acids were labeled with nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th carbon positions. The ratio of the echo time traces for the two field positions in the EPR spectrum possessing the largest and smallest anisotropies gave the anisotropic contribution to the echo decay, which obeys exponential time dependence with good accuracy. At low temperatures, the anisotropic contribution is induced by stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations), with the exponential decay rate Wanis proportional to <α(2)>τc, where <α(2)> is the mean angular amplitude of the motion and τc is the correlation time. In all cases, it was found that Wanis begins to increase sharply above 170-200 K, which was ascribed to the dynamical transition known for biological systems at these temperatures. For hydration by the sucrose and trehalose solutions, Wanis was found to increase noticeably also above ∼120 K, which was explained by bilayer expansion due to direct bonding of sugar molecules to the bilayer surface. The Wanis temperature dependencies were found to be close to those obtained for the simple systems of the nitroxide spin probe TEMPONE in aqueous sorbitol and sugar 1 M solutions. This correlation suggests a possible mechanism of cryoprotective action of sorbitol and sugars due to the similarity of low-temperature motions in the membrane and in the cryoprotectant-containing surrounding liquid.

Can xenon in water inhibit ice growth? Molecular dynamics of phase transitions in water-Xe system.

Motivated by recent experiments showing the promise of noble gases as cryoprotectants, we perform molecular dynamics modeling of phase transitions in water with xenon under cooling. We follow the structure and dynamics of xenon water solution as a function of temperature. Homogeneous nucleation of clathrate hydrate phase is observed and characterized. As the temperature is further reduced we observe hints of dissociation of clathrate due to stronger hydrophobic hydration, pointing towards a possible instability of clathrate at cryogenic temperatures and conversion to an amorphous phase comprised of "xenon + hydration shell" Xe·(H2O)21.5 clusters. Simulations of ice-xenon solution interface in equilibrium and during ice growth reveal the effects of xenon on the ice-liquid interface, where adsorbed xenon causes roughening of ice surface but does not preferentially form clathrate. These results provide evidence against the ice-blocker mechanism of xenon cryoprotection.

Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms

Trehalose, a natural disaccharide with bioprotective properties, is widely recognized for its ability to preserve biological membranes during freezing and dehydration events. Despite debate over the molecular mechanisms by which this is achieved, and that different mechanisms imply quite different distributions of trehalose molecules with respect to the bilayer, there are no direct experimental data describing the location of trehalose within lipid bilayer membrane systems during dehydration. Here, we use neutron membrane diffraction to conclusively show that the trehalose distribution in a dioleoylphosphatidylcholine (DOPC) system follows a Gaussian profile centred in the water layer between bilayers. The absence of any preference for localizing near the lipid headgroups of the bilayers indicates that the bioprotective effects of trehalose at physiologically relevant concentrations are the result of non-specific mechanisms that do not rely on direct interactions with the lipid headgroups.

Static magnetic field increases survival rate of dental pulp stem cells during DMSO-free cryopreservation

Successful and efficient cryopreservation of living cells and organs is a key clinical application of regenerative medicine. Recently, magnetic cryopreservation has been reported for intact tooth banking and cryopreservation of dental tissue. The aim of this study was to assess the cryoprotective effects of static magnetic fields (SMFs) on human dental pulp stem cells (DPSCs) during cryopreservation. Human DPSCs isolated from extracted teeth were frozen with a 0.4-T or 0.8-T SMF and then stored at −196 °C for 24 h. During freezing, the cells were suspended in freezing media containing with 0, 3 or 10% DMSO. After thawing, the changes in survival rate of the DPSCs were determined by flow cytometry. To understand the possible cryoprotective mechanisms of the SMF, the membrane fluidity of SMF-exposed DPSCs was tested. The results showed that when the freezing medium was DMSO-free, the survival rates of the thawed DPSCs increased 2- or 2.5-fold when the cells were exposed to 0.4-T or 0.8-T SMFs, respectively (p < 0.01). In addition, after exposure to the 0.4-T SMF, the fluorescence anisotropy of the DPSCs increased significantly (p < 0.01) in the hydrophilic region. These results show that SMF exposure improved DMSO-free cryopreservation. This phenomenon may be due to the improvement of membrane stability for resisting damage caused by ice crystals during the freezing procedure.

Molecular Dynamics Investigation of the Effects of Concentration on Hydrogen Bonding in Aqueous Solutions of Methanol, Ethylene Glycol and Glycerol

Hydrogen bonding interaction between alcohols and water molecules is an important characteristic in the aqueous solutions of alcohols. In this paper, a series of molecular dynamics simulations have been performed to investigate the aqueous solutions of low molecular weight alcohols (methanol, ethylene glycol and glycerol) at the concentrations covering a broad range from 1 to 90 mol %. The work focuses on studying the effect of the alcohols molecules on the hydrogen bonding of water molecules in binary mixtures. By analyzing the hydrogen bonding ability of the hydroxyl (-OH) groups for the three alcohols, it is found that the hydroxyl group of methanol prefers to form more hydrogen bonds than that of ethylene glycol and glycerol due to the intra-and intermolecular effects. It is also shown that concentration has significant effect on the ability of alcohol molecule to hydrogen bond water molecules. Understanding the hydrogen bonding characteristics of the aqueous solutions is helpful to reveal the cryoprotective mechanisms of methanol, ethylene glycol and glycerol in aqueous solutions.

Slow Freezing Coupled Static Magnetic Field Exposure Enhances Cryopreservative Efficiency—A Study on Human Erythrocytes

The aim of this study was to assess the cryoprotective effect of static magnetic fields (SMFs) on human erythrocytes during the slow cooling procedure. Human erythrocytes suspended in 20% glycerol were slowly frozen with a 0.4-T or 0.8-T SMF and then moved to a −80°C freezer for 24 hr. The changes in survival rate, morphology, and metabolites of the thawed erythrocytes were examined. To understand possible cryoprotective mechanisms of SMF, membrane fluidity and dehydration stability of SMF-exposed erythrocytes were tested. For each test, sham-exposed erythrocytes were used as controls. Our results showed that freezing coupled with 0.4-T or 0.8-T SMFs significantly increased the relative survival ratios of the frozen-thawed erythrocytes by 10% and 20% (p<0.001), respectively. The SMFs had no effect on erythrocyte morphology and metabolite levels. However, membrane fluidity of the samples exposed to 0.8-T SMF decreased significantly (p<0.05) in the hydrophobic regions. For the dehydration stability experiments, the samples exposed to 0.8-T SMF exhibited significantly lower (p<0.05) hemolysis. These results demonstrate that a 0.8-T SMF decreases membrane fluidity and enhances erythrocyte membrane stability to resist dehydration damage caused by slow cooling procedures.

Long-term cryopreservation of human mesenchymal stem cells using carboxylated poly-l-lysine without the addition of proteins or dimethyl sulfoxide

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are known for their potential to undergo mesodermal differentiation into many cell types, including osteocytes, adipocytes, and chondrocytes. Therefore, hBMSCs can be used for a variety of regenerative medicine therapies, in fact, hBMC-derived osteocytes have already been used in bone reconstruction. This study discusses the viability and the differentiation properties of hBMSCs that have been cryopreserved in the absence of proteins or dimethyl sulfoxide (DMSO) by using a novel polyampholyte cryoprotective agent (CPA). This CPA is based on carboxylated poly-l-lysine (COOH-PLL) and it was produced by a reaction between ϵ-poly-l-lysine and succinic anhydride. 1H-NMR and two-dimensional correlation (1H-13C HSQC) spectroscopy revealed that COOH-PLL did not have a special structure in solution. The hBMSCs can be cryopreserved for 24 months at −80 °C by using a 7.5% (w/w) cryopreserving solution of COOH-PLL, which introduces carboxyl groups that result in > 90% cell viability after thawing. Furthermore, the cryopreserved hBMSCs fully retained both their proliferative capacity as well as their potential for osteogenic, adipogenic, and chondrogenic differentiation. Confocal laser-scanning microscopy showed that the polyampholyte CPA did not penetrate the cell membrane; rather, it attached to the membrane during cryopreservation. These results indicate that the cryoprotective mechanisms of COOH-PLL might differ from those of currently used small molecule CPAs. These results also suggest that using COOH-PLL as a CPA for hBMSC preservation can eliminate the use of proteins and DMSO, which would be safer if these cells were used for cell transplantation or regenerative medicine.

Microscopic mechanism of protein cryopreservation in an aqueous solution with trehalose

In order to investigate the cryoprotective mechanism of trehalose on proteins, we use molecular dynamics computer simulations to study the microscopic dynamics of water upon cooling in an aqueous solution of lysozyme and trehalose. We find that the presence of trehalose causes global retardation of the dynamics of water. Comparing aqueous solutions of lysozyme with/without trehalose, we observe that the dynamics of water in the hydration layers close to the protein is dramatically slower when trehalose is present in the system. We also analyze the structure of water and trehalose around the lysozyme and find that the trehalose molecules form a cage surrounding the protein that contains very slow water molecules. We conclude that the transient cage of trehalose molecules that entraps and slows the water molecules prevents the crystallisation of protein hydration water upon cooling.

Lyophilization of liposomal drug forms (Review)

Lyophilization is a promising approach to ensuring the long-term stability of liposomes. Decades of studies in this field have revealed various cryoprotective mechanisms such as water replacement and formation of a cryoprotector matrix. Much progress has been made in obtaining highly stable liposomes after lyophilization based on optimization of the formulation and process parameters. This paper reviews cryoprotective mechanisms, their parameters affecting the cryoprotective effect, and techniques used most often in the studies. The parameters are discussed with respect to two aspects, namely, formulation factors (choice of the lipid bilayer composition, vesicle size, selection of cryoprotectors, dry mass ratio of cryoprotector to lipid, distribution of cryoprotector on both sides of the lipid bilayer) and processing factors (freezing protocols, drying protocols, etc.).

Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen

Abstract. The ice nucleation of bioaerosols (bacteria, pollen, spores, etc.) is a topic of growing interest, since their impact on ice cloud formation and thus on radiative forcing, an important parameter in global climate, is not yet fully understood. Here we show that pollen of different species strongly differ in their ice nucleation behaviour. The average freezing temperatures in laboratory experiments range from 240 to 255 K. As the most efficient nuclei (silver birch, Scots pine and common juniper pollen) have a distribution area up to the Northern timberline, their ice nucleation activity might be a cryoprotective mechanism. Far more intriguingly, it has turned out that water, which has been in contact with pollen and then been separated from the bodies, nucleates as good as the pollen grains themselves. The ice nuclei have to be easily-suspendable macromolecules located on the pollen. Once extracted, they can be distributed further through the atmosphere than the heavy pollen grains and so presumably augment the impact of pollen on ice cloud formation even in the upper troposphere. Our experiments lead to the conclusion that pollen ice nuclei, in contrast to bacterial and fungal ice nucleating proteins, are non-proteinaceous compounds.

Osmolality and Unfrozen Water Content of Aqueous Solution of Dimethyl Sulfoxide

Dimethyl sulfoxide is one of the most popular cryoprotectants in cryobiology. It is crucial for the successful cryopreservation to quantitatively analyze the cryoprotective properties of dimethyl sulfoxide. In this study, the osmolality and the unfrozen water content of the aqueous solution of dimethyl sulfoxide are determined using a Perkin-Elmer Diamond DSC. For precisely calculating the unfrozen water content, this study takes into consideration the effect of dilution on the enthalpy of fusion in the solution which is a commonly neglected factor. The osmotic virial representation and the quantitative relationships between the unfrozen water content and the solution composition are reported. The experimental results show that the aqueous dimethyl sulfoxide solution has a greatly depressed freezing point and a large osmolality. Moreover, the water-blocking ability of dimethyl sulfoxide weakens when the solution is more concentrated. The results in this study will enlarge the database of physical and chemical properties of dimethyl sulfoxide and reinforce our understanding of the cryoprotective mechanisms of dimethyl sulfoxide in aqueous solutions.