Cryoprotective Mechanism Research Articles

Insights into tail-belting by wild mice encourages fresh perspectives on physiological mechanisms that safeguard mammal tissues from freezing

We investigated tail-belting (TB), the newly-discovered freeze avoidance behavior among wild rodents. When temperatures dropped to -6 °C, wild mice (Apodemus agrarius and Apodemus flavicollis) were observed curling their tails inward and positioning it on the back. A literature search suggested TB had never been documented, presumably because rodents, especially in the laboratory, are seldomly assayed under cold stress. Due to the infrequent occurrence of the behavior, we used infrared and thermal cameras to confirm observations. We also collected tail-skin samples to investigate whether any physiological mechanisms might co-occur with TB. If such mechanisms were found, they could inform wider debate involving freeze protection among mammals, and could potentially lead to understanding mammal susceptibility or resilience to sudden temperature changes such as those associated with climate change. Lastly, we scored behaviors by bank voles (Myodes glareolus) which unexpectedly visited chambers. Across four winters, we observed TB in both Apodemus species during subzero conditions, but bank voles never performed the behavior. We also confirmed that TB occurs as an adaptive reflex which warms the tail. From tissue samples, we found that free amino acids, peptides, and glycoproteins were significantly higher during cold-stress. Thus, TB may have been accompanied by the expression of cold-protective proteins which ostensibly enable the peripheral body parts of mammals to survive temperatures well below 0 °C. These findings should inspire new dialogue regarding the role of lipids in tissues of peripheral organs in mammals. By extension, our findings may lead to the discovery of a putative cryoprotection mechanism among mammals.

Mitigating the Effects of Starch Derivatives on Cold Denaturation of Gluten Protein: Insights from Hydration Capacity and Conformation Behavior.

Mitigating the cold denaturation of gluten protein during frozen storage is crucial for the quality improvement of frozen cereal products. Our previous study observed that starch derivatives, especially short-clustered maltodextrin (SCMD), could significantly improve frozen dough quality, alleviating the deterioration of gluten-network structure. To further reveal the cryoprotection mechanism of SCMD on gluten protein during frozen storage, the modulatory roles of SCMD in the hydration capacity and conformation behavior of gluten protein were explored, in comparison with DE2 maltodextrin (MD) and pregelatinized starch (PGS). Results demonstrated that SCMD significantly facilitated the reservation of bound water and decreased the surface hydrophobicity of gluten protein after 8 weeks of frozen storage. Remarkable effects of SCMD on stabilizing the secondary structure and microenvironment of aromatic amino acids of gluten protein were observed. Further mechanistic investigation showed that when the temperature dropped from 300 to 250 K, the short-clustered structure could stabilize the α-helixes more evidently than linear structures through hydrogen bonds with water and steric hindrance effect, rather than directly with protein. Our findings will provide novel insights into the cold denaturation of gluten protein and useful guidance in selecting the optimum structure to suppress this denaturation, improving the quality of frozen cereal products.

Effect of pectin with different esterification degree on the freeze-thaw stability of gluten protein: Structures, functional properties, and Cryoprotective mechanism

The effect of degree of esterification (DE) on the cryoprotective function of pectin against the frozen deterioration of gluten protein is still unclear. Here, NMR, HPSEC-MALLS, HPGPC and optical microscrope were applied to determine the structure and ice recrystallization inhibition (IRI) activity of pectin with varying DE. The effect on structure, physicochemical properties and molecular interaction of gluten supplemented with pectin were investigated. Results suggested that low-ester pectin (LEP) exhibited higher IRI activity than that of high-ester pectin (HEP), and reduced the damage of gluten during freezing storage. Moreover, SEM and low field-NMR revealed that LEP effectively helped to maintain the network structure and inhibited the water migration of gluten. Besides, molecular docking results suggested higher affinity between LEP and gluten protein than that of HEP. These results illustrated better cryoprotective effects of LEP than that of HEP through pathways including IRI activity, water migration, and possible molecular interaction.

Interactions between ions and water-DMSO mixed solvent

As a classical cryoprotectant, the cryoprotective mechanism of DMSO remains elusive. In this study, we explore the capability of DMSO to hinder the formation of salt hydrates in the water-DMSO-salt ternary system, given that, at low temperatures, salt hydrates are more prone to attach to surfaces than ice, potentially preventing mechanical damage to cell membranes. Using THz-TDS, we validate the ability of DMSO to suppress salt hydrate formation. Combining IR spectroscopy, we conclude that this inhibitory effect partly arises from the interaction between cations and the S=O groups in DMSO molecules. Semi-quantitative DR spectroscopy provides similar insights from the perspective of molecular reorientation dynamics. Taking into account the intensified ion-dipole interactions due to a higher ion surface charge density, we focus our discussion on MgCl2, while NaCl, more akin to biological processes, serves as a reference. Delving into the specific molecular mechanisms, we put forth conjectures regarding the direct interaction between cations and DMSO or the indirect influence of cations on DMSO through the modulation of hydrogen bonds in surrounding water molecules. These research findings contribute to a better understanding and mastery of cryoprotectants.

Development of Macromolecular Cryoprotectants for Cryopreservation of Cells.

Cryopreservation is a common way for long-term storage of therapeutical proteins, erythrocytes, and mammalian cells. For cryoprotection of these biosamples to keep their structural integrity and biological activities, it is essential to incorporate highly efficient cryoprotectants. Currently, permeable small molecular cryoprotectants such as glycerol and dimethyl sulfoxide dominate in cryostorage applications, but they are harmful to cells and human health. As acting in the extracellular space, membrane-impermeable macromolecular cryoprotectants, which exert remarkable membrane stabilization against cryo-injury and are easily removed post-thaw, are promising candidates with biocompatibility and feasibility. Water-soluble hydroxyl-containing polymers such as poly(vinyl alcohol) and polyol-based polymers are potent ice recrystallization inhibitors, while polyampholytes, polyzwitterions, and bio-inspired (glyco)polypeptides can significantly increase post-thaw recovery with reduced membrane damages. In this review, the synthetic macromolecular cryoprotectants are systematically summarized based on their synthesis routes, practical utilities, and cryoprotective mechanisms. It provides a valuable insight in development of highly efficient macromolecular cryoprotectants with valid ice recrystallization inhibition activity for highly efficient and safe cryopreservation of cells.

The cage effect of electron beam irradiation damage in cryo-electron microscopy

Electron beam irradiation can cause damage to biological and organic samples, as determined via transmission electron microscopy (TEM). Cryo-electron microscopy (cryo-EM) significantly reduces such damage by quickly freezing the environmental water around organic molecules. However, there are multiple hypotheses about the mechanism of cryo-protection in cryo-EM. A lower temperature can cause less molecular dissociation in the first stage, or frozen water can have a “cage” effect by preventing the dissociated fragments from flying away. In this work, we use real-time time-dependent density functional theory molecular dynamics(rt-TDDFT-MD) simulations to study the related dynamics. We use our recently developed natural orbital branching (NOB) algorithm to describe the molecular dissociation process after the molecule is ionized. We find that despite the difference in surrounding water molecules at different temperatures, the initial dissociation process is similar. On the other hand, the dissociated fragments fly away at room temperature, while they remain in the same cage when frozen water is used. Our results provide direct support for the cage effect mechanism.

Reconfiguring the Electrolyte Network Structure with Bio‐Inspired Cryoprotective Additive for Low‐Temperature Aqueous Zinc Batteries

AbstractDespite promising performance at ambient temperature, the development of aqueous zinc batteries is jeopardized by the freeze of aqueous electrolytes and deteriorative electrode‐electrolyte interphase at low temperatures. Herein, inspired by the cryoprotective mechanism of extracellular polysaccharides in biological organisms, a quaternized galactomannan polysaccharide (q‐GPA) is proposed as a cryoprotective additive for improving low‐temperature performance of aqueous zinc batteries. Mechanistic studies revealed that a multi‐hydroxyl galactomannan backbone can substantially attenuate the activity of water molecules through the reconfiguration of the hydrogen bond network, which inhibits ice crystal formation at subzero temperatures and thus depress the freezing point of the electrolyte. Meanwhile, the quaternary ammonium groups tethered on the q‐GPA skeleton are intended to neutralize the interfacial electric field through electrostatic repulsion, thereby accelerating Zn2+ deposition kinetics and prohibiting zinc dendrite growth. Impressively, the q‐GPA–modified electrolyte enables an extended lifespan of over 1700 h in Zn||Zn symmetric battery at a high current density of 3 mA cm−2 and an ultralong cycle life of 5000 cycles with a capacity retention of 99.2% in the Zn||Na2V6O16·1.5H2O (NVO) full battery at −30 °C. This work provides unprecedented possibilities for optimizing the electrolyte formulation of low‐temperature aqueous batteries.

Elucidate the molecular basis of ampholytic chitosan as a high-performance cryoprotectant to myosin denaturation: The importance of saccharide charges

The uses of charged poly/oligo-saccharides enable the retarding of protein denaturation against various environmental stresses during food storage and manufacturing. However, at subzero temperatures, the molecular basis of such stabilization behaviors, i.e., cryoprotections, remain less explored. In this study, we introduced an ampholytic saccharide, carboxymethyl chitooligosaccharide (CMCO) that effectively inhibited the freezing-induced myosin denaturation. The in-depth cryoprotective mechanism was systematically investigated by using molecular dynamic simulation and multispectral characterizations. Results showed that CMCO may interact with myosin through hydrogen bonding and electrostatic interactions, which caused the expelling of water at protein surfaces and the reduced conformational flexibility of myosin molecules. Due to this water replacement event, both secondary and ternary structures of myosin became freezing-resistant, leading to the inhibited protein aggregations and retained functionalities, such as solubility, Ca2+-ATPase activity, and gelling properties. Moreover, cryoprotective behaviors of CMCO were charge-dependent. CMCO with a higher degree of carboxymethyl substitution (DS: 1.2) was inclined to bind and stabilize myosin molecules better than the low-DS (DS: 0.8) one, even though both outperformed other cryoprotective saccharides. Therefore, this investigation not only introducing a high-performance myosin cryoprotectant, but also elaborated the cryoprotective mechanism of ampholytic saccharides.

Low-Molecular-Weight PEGs for Cryopreservation of Stem Cell Spheroids

Stem cell spheroids (SCSs) are a valuable tool in stem cell research and regenerative medicine. SCSs provide a platform for stem cell behavior in a more biologically relevant context with enhanced cell–cell communications. In this study, we investigated the recovery of SCSs after cryopreservation at –196 °C for 7 days. Prior to cryopreservation, the SCSs were preincubated for 0 h (no preincubation), 2 h, 4 h, and 6 h at 37 °C in the presence of low-molecular-weight poly(ethylene glycol) (PEG) with molecular weights of 200, 400, and 600 Da. The recovery rate of SCSs was markedly affected by both the PEG molecular weight and the preincubation time. Specifically, when SCSs were preincubated with a PEG200 solution for 2 to 6 h, it significantly enhanced the recovery rate of the SCSs. Internalization of PEG200 through simple diffusion into the SCSs may be the cryoprotective mechanism. The PEG200 diffuses into the SCSs, which not only suppresses osmotic pressure development inside the cell but also inhibits ice formation. The recovered SCSs demonstrated both fusibility and capabilities for proliferation and differentiation comparable to SCSs recovered after dimethyl sulfoxide 10% cryopreservation. This study indicates that PEG200 serves as an effective cryoprotectant for SCSs. A simple preincubation procedure in the presence of the polymer greatly improves the recovery rate of SCSs from cryopreservation.

Molecular modelling studies reveal cryoprotective mechanism of L-Proline during the frozen storage of shrimp (Litopenaeus vannamei)

This study aimed to investigate the cryoprotective properties of proline (1% and 3% (w/v)) on shrimp. The cryoprotective mechanism was studied using physico-chemical experiments and molecular simulations. Proline had a notable positive impact on the thawing loss and texture of shrimp in comparison to the control. The denaturation of myosin in frozen shrimp was delayed by proline. Microscopy analysis demonstrated that proline effectively lowered the harm caused by ice crystals to shrimp muscle. Molecular simulations indicated that proline potentially exerted a cryoprotective effect primarily through the “water substitution” and “glassy state” hypotheses. Proline formed hydrogen bonds with myosin to replace the water molecules around myosin. Additionally, proline interacted with water molecules to form a glassy state, impeding the growth of ice crystals. Consequently, the stability of shrimp myosin was enhanced during freezing. In conclusion, proline demonstrated promise as an efficacious cryoprotectant for aquatic products.

Open-source hardware- and software-based cryomicroscopy system for investigation of phase transitions in cryobiological research.

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open-source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra- and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2 SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7M Me2 SO and 100mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2 SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics.

Untargeted Lipidomics Reveal Quality Changes in High-Moisture Japonica Brown Rice at Different Storage Temperatures.

Low temperatures are an effective way of delaying grain rancidity and deterioration. However, little is known about the difference in quality changes in high-moisture japonica brown rice at different storage temperatures. In this study, the storage quality changes in japonica brown rice with a 15.50% moisture content stored at 15 °C, 20 °C, and 25 °C were investigated. In addition, an untargeted lipidomics analysis coupled with gas chromatography and mass spectrometry (GC-MS) was applied to analyze the volatile compounds and metabolite changes in the high-moisture japonica brown rice. The results showed that storage at 15 °C could well maintain the color and aroma stability of the brown rice and delay the increase in fatty acid value (FAV). The lipidomics results showed that storage at 15 °C delayed glycerolipid and sphingolipid metabolism and reduced glycerophospholipid catabolism in the brown rice. The low-temperature environment regulated these three metabolic pathways to maintain higher contents of triglycerides (TG), phosphatidylserine (PS), abd phosphatidylethanolamine (PE), and lower contents of diglycerides (DG), OAcyl-(gamma-hydroxy) FA (OAHFA), ceramides (Cer), and glycosylceramides (Hex1Cer) in the high-moisture japonica brown rice, which maintained the storage stability of the brown rice. Our results proposed the cryoprotection mechanism of postharvest brown rice from the perspective of volatile compounds and metabolite changes, providing a foothold for the further exploration of low-temperature storage as a safe and efficient cryoprotectant in the grain storage field.

Investigation of the cryoprotective mechanisms of silver carp muscle hydrolysate for frozen dough exposed to multiple freeze-thaw cycles

This study aimed to elucidate the cryoprotective mechanisms associated with silver carp muscle hydrolysate (SCMH) in the context of frozen dough subjected to 0–6 freeze-thaw (FT) cycles. The presence of SCMH in the frozen dough matrix effectively mitigated the reduction in yeast viability, as evidenced by a mere 3.7% decrease in OD660 compared to a 7.6% decline observed in the control group after 6 FT cycles. Furthermore, the incorporation of SCMH exhibited a progressive increase in the concentration of numerous free amino acids (e.g., Threonine, Alanine, Leucine, and Lysine) throughout the FT cycles. Additionally, the introduction of SCMH into the dough formulation yielded multiple benefits. It substantially curtailed water loss and hindered the shift of water from T21 to T22 during FT cycles. Moreover, it exerted an inhibitory effect on the growth of ice crystals, as indicated by a 49% reduction in the average ice crystal area following 6 FT cycles. This multifaceted cryoprotective action contributed to a subsequent decrease in cryodamage to the dough matrix, gluten network structure, and yeast cells. In summary, these findings underscore the considerable potential of integrating SCMH as a cryoprotective agent to enhance the overall quality of frozen dough products.

Component stabilizing mechanism of membrane-separated hydrolysates on frozen surimi

This study investigated the cryoprotective mechanism of ultrafiltration membrane-separated fractions (>10 kDa, UF-1; 3–10 kDa, UF-2; and <3 kDa, UF-3) derived from silver carp hydrolysates on frozen surimi. The surimi gel incorporating UF-3 exhibited a compact, continuous structure with uniform pores, even after undergoing six freeze–thaw (F-T) cycle, with the minimal reduction in entrapped water (from 95.1 % to 91.1 %) and least increase in free water (from 4.5 % to 6.6 %) as revealed by SEM and LF-NMR analysis. Through molecular docking analysis, three major peptides in UF-3 were identified to form robust interactions with the myosin head pocket, facilitated by hydrogen bonds, electrostatic forces, and hydrophobic interactions. Furthermore, molecular dynamics simulations demonstrated that the three peptides effectively prevented myosin from unfolding and aggregating by tightly binding to basic amino acids (Arg, Lys) and hydrophobic amino acids (Phe, Leu, Ile, Met, and Val) residues in the myosin head pocket, primarily governed by electrostatic energies (−156.95, −321.38, and −267.53 kcal/mol, respectively) and van der Waals energies (−395.05, −347.46, and −319.16 kcal/mol, respectively). Notably, the key action site was identified as Lys599 on myosin. The hydrophilic and hydrophobic hotspot residues of the peptides worked synergistically to stabilize the myosin structure in frozen surimi.

Изменчивость биохимических признаков Pinus sylvestris (Pinaceae) при адаптации форм в условиях избыточного увлажнения

Conifers are characterized by a certain degree of individual variability in the content of stress metabolites, which can be caused by genotypic variations or divergence in the growth conditions of individual trees. The purpose of the study is to evaluate the variation of biochemical traits of Scots pine, which are different in the color of male strobilus, during adaptation to the conditions of constant excessive moisture in the soil of the northern taiga. The dynamics of metabolic parameters of Pinus sylvestris L. were investigated in shrubsphagnum pine stands on marshy upland soils at the mouth of the Northern Dvina River (northern taiga subzone). From July to November 2018, needle samples from shoots were collected from 10 pine trees of each of the male strobilus types, differentiated by color. The contents of photosynthetic pigments, anthocyanins, ascorbic acid, proline, water-soluble proteins, and pH were measured under laboratory conditions. The synthesis of chlorophyll in the needles in the current year of formation decreased in trees of both forms during the dry summer period. A favorable temperature in the autumn period could promote an extension of photosynthetic pigment accumulation, which might negatively affect tree preparation for overwintering. There were no significant differences between f. erythranthera Sanio and f. sulfuranthera Kozubow in the needle content of chlorophyll and carotenoids, anthocyanins, ascorbic acid, free proline, water-soluble proteins, or pH. The similarities in the adaptation of the trees to the constant excessive soil moisture in northern taiga conditions were revealed. The seasonal factor had a considerable impact on the dynamics of biochemical parameters. Ascorbic acid, proline, and anthocyanin were more actively accumulated in the needles throughout the summer due to the high air temperature, resulting in an increase in antioxidant activity and the development of protective mechanisms aimed at reducing oxidative stress. The increase in water-soluble protein content in needles from October to November is one of the cryoprotective mechanisms of the trees for the winter period. Individual differences in ascorbic acid and proline contents in yellow microspore-bearing pine needles at the establishment of minus temperatures in November were considerably higher than in red microspore-bearing pine needles. This is regarded as the norm of the reaction for different types of trees to the influence of negative temperatures. For citation: Tarkhanov S.N., Pinaevskaya E.A., Aganina Y.E., Pakhov A.S. Variability of Biochemical Properties During Adaptation of Pinus sylvestris (Pinaceae) Forms to Excessive Moisture Conditions. Lesnoy Zhurnal = Russian Forestry Journal, 2023, no. 4, pp. 58–75. (In Russ.). https://doi.org/10.37482/0536-1036-2023-4-58-75

Mechanistic insights into the improvement of yeast viability by adding short-clustered maltodextrin during long-term frozen storage

The formation and growth of ice crystals are widely acknowledged to cause damage to different substances and we found short-clustered maltodextrin (SCMD) could alleviate the quality deterioration of frozen dough in previous research. But how SCMD interacts with dough components such as yeast is poorly understood. Here, we analyze the ice-recrystallization-inhibition (IRI) activity of SCMD and provide mechanistic insights into the improvement of yeast viability by adding SCMD during long-term frozen storage. SCMD had a higher IRI activity compared with DE2 maltodextrin, trehalose and glycerin at the same dosage. Through untargeted metabolomics analysis, SCMD addition proved to be enhancing lipid metabolism, regulating amino acid metabolism and energy metabolism of yeast, making yeast cells possess more similarities to the positive control with minimal freeze injury. In contrast, no significant protective effects were observed with low-concentration trehalose and glycerin. Our reports reveal the cryoprotection mechanism of SCMD from the perspective of the cellular metabolism of yeast, providing the foothold for further exploration of SCMD as a safe and efficient cryoprotectant in different fields. • Short-clustered maltodextrin (SCMD) could change ice-crystal shapes. • SCMD had a higher ice-recrystallization-inhibition (IRI) activity than trehalose and glycerin at the same dosage. • SCMD addition could improve yeast viability and fermentation ability during long-term frozen storage. • SCMD addition enhanced lipid metabolism, regulated amino acid metabolism and energy metabolism of yeast. • SCMD cryoprotection mechanism was proposed.

Cryoprotective Mechanism of DMSO Induced by the Inhibitory Effect on Eutectic NaCl Crystallization.

Cryopreservation is a critical procedure in autologous hematopoietic stem cell transplantation. Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice. Optimization of the cryopreservation protocol in the past revealed a dramatic loss of cell viability associated with a reduction of the DMSO concentration below 2 vol % in the freezing medium. The cryoprotective mechanism of DMSO is usually ascribed to the ability to suppress ice formation and reduce the adverse effects of the freeze-concentrated solution. This work proposes an alternative hypothesis considering the detrimental impact of NaCl eutectic crystallization on cell viability. Thermoanalytical and microstructural analysis of the DMSO effect on eutectic phase transformation of cryoprotective mixtures revealed a correlation between the loss of cell viability and eutectic NaCl crystallization. DMSO inhibits the eutectic crystallization of NaCl and preserves cell viability. Thermodynamic description of the inhibitory action and possible mechanism of cryoinjury are provided.

Metabolomics analysis of buck semen cryopreserved with trehalose.

Trehalose is commonly used as an impermeable cryoprotectant for cryopreservation of cells, but its cryoprotective mechanism has now not but been determined. This study investigated the cryopreservation impact of trehalose on buck semen cryopreservation and finished metabolic profiling of freeze-thawed media by way of the GC–MS-based metabolomics for the first time. Metabolic pattern recognition and metabolite identification by means of principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and metabolic pathway topology analysis revealed the results of trehalose on buck sperm metabolism at some point of cryopreservation. The results confirmed that trehalose drastically progressed sperm motility parameters and structural integrity after thawing. PCA and PLS-DA analysis discovered that the metabolic patterns of the freezing-thawing media of buck semen cryopreserved with trehalose (T group) or without trehalose (G group, Control) were certainly separated. Using screening conditions of VIP >1.5 and p vaule <0.05, a total of 48 differential metabolites have been recognized, whithin l-isoleucine, L-leucine, L-threonine, and dihydroxyacetone were notably enriched in valine, leucine and isoleucine biosynthesis, glycerolipid metabolism, and aminoacyl-tRNA biosynthesis pathways. In brief, trehalose can efficiently improve membrane structural integrity and motion parameters in buck sperm after thawing, and it exerts a cryoprotective impact with the aid of changing sperm amino acid synthesis and the glycerol metabolism pathway.

Phase transitions and mechanisms of cryoprotection of serum-/xeno-free media based on dextran and dimethyl sulfoxide

The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating polymers (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (−30, −50, −50 and −196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.

Redefining the Molecular Interplay between Dimethyl Sulfoxide, Lipid Bilayers, and Dehydration.

The potentially damaging action of dimethyl sulfoxide (DMSO) on phospholipid bilayers remains a matter of controversy. We have conducted a series of long-scale molecular dynamics simulations of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers at various levels of hydration in the presence of variable quantities of DMSO. These simulations provide evidence for a non-destructive dehydrating mechanism of action for DMSO on DOPC bilayers across a wide concentration range and levels of hydration. Specifically, under full- and low-hydration conditions, the bilayer underwent a minor lateral contraction, coinciding with surface dehydration in the presence of dilute DMSO solutions (XDMSO < 0.3). At higher DMSO concentrations, this bilayer structure was retained despite a progressive deterioration of the hydration structure at the interface. A similar convergence of bilayer structural properties was observed under dehydration conditions for 0.3 < XDMSO < 0.7. Destabilization occurred for dehydrated bilayers in the presence of XDMSO ≥ 0.7, suggesting the existence of a DMSO concentration and/or dehydration threshold. However, such DMSO concentrations far exceed those established as toxic to other cellular components. Our findings represent a computational model for DMSO-DOPC interactions that is consistent with a range of experimental characterizations, offering new molecular insights into the cryoprotective mechanisms of action of DMSO.