Data-driven discovery of potent small molecule ice recrystallisation inhibitors.

Controlling the formation and growth of ice is essential to successfully cryopreserve cells, tissues and biologics. Current efforts to identify materials capable of modulating ice growth are guided by iterative changes and human intuition, with a major focus on proteins and polymers. With limited data, the discovery pipeline is constrained by a poor understanding of the mechanisms and the underlying structure-activity relationships. In this work, this barrier is overcome by constructing machine learning models capable of predicting the ice recrystallisation inhibition activity of small molecules. We generate a new dataset via experimental measurements of ice growth, then harness predictive models combining state-of-the-art descriptors with domain-specific features derived from molecular simulations. The models accurately identify potent small molecule ice recrystallisation inhibitors within a commercial compound library. Identified hits can also mitigate cellular damage during transient warming events in cryopreserved red blood cells, demonstrating how data-driven approaches can be used to discover innovative cryoprotectants and enable next-generation cryopreservation solutions for the cold chain.

Design and Mechanistic Insights into α-Helical p-Terphenyl Guanidines as Potent Small-Molecule Antifreeze Agents.

Ice formation is a critical challenge across multiple fields, from industrial applications to biological preservation. Inspired by natural antifreeze proteins, we designed and synthesized a new class of small-molecule antifreezes based on α-helical p-terphenyl scaffolds with guanidine side chains. These p-terphenyl guanidines 1, among the smallest molecules that mimic α-helical structures, exhibit potent ice recrystallization inhibition (IRI) activity, similar to that of existing large α-helical antifreeze compounds. The most effective compound, 1a, with four C1-carbon guanidine moieties, demonstrated a superior IRI activity of 0.46 (1 mg/mL). Using molecular dynamics simulations with density-functional theory and separate pKa calculations, we elucidated the mechanisms underlying their antifreeze properties.

Biophysical evidence that frostbite is triggered on nanocrystals of biogenic magnetite in garlic cloves (Allium sativum).

Trace levels of biologically precipitated magnetite (Fe3O4) nanocrystals are present in the tissues of many living organisms, including those of plants. Recent work has also shown that magnetite nanoparticles are powerful ice nucleation particles (INPs) that can initiate heterogeneous freezing in supercooled water just below the normal melting temperature. Hence there is a strong possibility that magnetite in plant tissues might be an agent responsible for triggering frost damage, even though the biological role of magnetite in plants is not understood. To test this hypothesis, we investigated supercooling and freezing mortality in cloves of garlic (Allium sativum), a species which is known to have moderate frost resistance. Using superconducting magnetometry, we detected large numbers of magnetite INPs within individual cloves. Oscillating magnetic fields designed to torque magnetite crystals in situ and disturb the ice nucleating process produced significant effects on the temperature distribution of supercooling, thereby confirming magnetite's role as an INP in vivo. However, weak oscillating fields increased the probability of freezing, whereas stronger fields decreased it, a result that predicts the presence of magnetite binding agents that are loosely attached to the ice nucleating sites on the magnetite crystals.

Influence of acute heat mitigation strategies on core temperature, heart rate and aerobic performance in females: A systematic literature review.

This review examined the effect of acute heat mitigation strategies on physiological strain and exercise performance in females exercising in the heat. Three databases were searched for original research with an acute heat mitigation (intervention) and control strategy in active females and reporting core temperature, heart rate and/or aerobic exercise performance/capacity with ≥ 24°C wet bulb globe temperature. Hedges' g effect sizes were calculated to evaluate outcomes. Thirteen studies (n = 118) were included. Most studies that applied an acute heat mitigation strategy to females did not reduce thermal (9/10) or cardiovascular (6/6) strain or improve exercise performance/capacity (8/10). The most effective strategies for attenuating thermal strain were pre-cooling with ice-slurry (effect size = -2.2 [95% CI, -3.2, -1.1]) and ice-vests (-1.9 [-2.7, -1.1]), and pre- and per-cooling with an ice-vest (-1.8 [-2.9, -0.7]). Only pre-cooling with an ice-vest improved running performance (-1.8 [-2.9, -0.7]; ~0.43 min) whilst sodium hyperhydration improved cycling capacity at 70% V O2peak (0.8 [0.0, 1.6]; ~20.1 min). There is currently limited research on acute heat mitigation strategies in females, so the evidence for the efficacy is scarce. Some studies show beneficial effects with ice-slurry, ice-vests and sodium hyperhydration, which can guide future research to support female exercise performance in the heat.

Microbiota diversity of three Brazilian native fishes during ice and frozen storage.

This study aimed to assess the bacterial microbiota involved in the spoilage of pacu (Piaractus mesopotamics), patinga (female Piaractus mesopotamics x male Piaractus brachypomus), and tambacu (female Colossoma macropomum × male Piaractus mesopotamics) during ice and frozen storage. Changes in the microbiota of three fish species (N = 22) during storage were studied through 16S rRNA amplicon-based sequencing and correlated with volatile organic compounds (VOCs) and metabolites assessed by nuclear magnetic resonance (NMR). Storage conditions (time and temperature) affected the microbiota diversity in all fish samples. Fish microbiota comprised mainly of Pseudomonas sp., Brochothrix sp., Acinetobacter sp., Bacillus sp., Lactiplantibacillus sp., Kocuria sp., and Enterococcus sp. The relative abundance of Kocuria, P. fragi, L. plantarum, Enterococcus, and Acinetobacter was positively correlated with the metabolic pathways of ether lipid metabolism while B. thermosphacta and P. fragi were correlated with metabolic pathways involved in amino acid metabolism. P. fragi was the most prevalent spoilage bacteria in both storage conditions (ice and frozen), followed by B. thermosphacta. Moreover, the relative abundance of identified Bacillus strains in fish samples stored in ice was positively correlated with the production of VOCs (1-hexanol, nonanal, octenol, and 2-ethyl-1-hexanol) associated with off-flavors. 1H NMR analysis confirmed that amino acids, acetic acid, and ATP degradation products increase over (ice) storage, and therefore considered chemical spoilage index of fish fillets.

Formation of methylglyoxal (CH3C(O)CHO) in interstellar analog ices - a key intermediate in cellular metabolism.

Ketoaldehydes are key intermediates in biochemical processes including carbohydrate, lipid, and amino acid metabolism. Despite their crucial role in the interstellar synthesis of essential biomolecules necessary for the Origins of Life, their formation mechanisms have largely remained elusive. Here, we report the first bottom-up formation of methylglyoxal (CH3C(O)CHO)-the simplest ketoaldehyde-through the barrierless recombination of the formyl (HCO) radical with the acetyl (CH3CO) radical in low-temperature interstellar ice analogs upon exposure to energetic irradiation as proxies of galactic cosmic rays. Utilizing vacuum ultraviolet photoionization reflectron time-of-flight mass spectrometry and isotopic substitution studies, methylglyoxal and its enol tautomer 2-hydroxypropenone (CH3C(OH)CO) were identified in the gas phase during the temperature-programmed desorption of irradiated carbon monoxide-acetaldehyde (CO-CH3CHO) ices, suggesting their potential as promising candidates for future astronomical searches. Once synthesized in cold molecular clouds, methylglyoxal can serve as a key precursor to sugars, sugar acids, and amino acids. Furthermore, this work provides the first experimental evidence for tautomerization of a ketoaldehyde in interstellar ice analogs, advancing our fundamental knowledge of how ketoaldehydes and their enol tautomers can be synthesized in deep space.

Protein inclusion into ice can dissociate subunits.

An antifreeze protein's inclusion into ice can be used to purify it from other proteins and solutes. Domains that are covalently attached to the antifreeze protein are also drawn into the ice such that the ice-binding portion of the fusion protein can be used as an affinity tag. Here we have explored the use of ice-affinity tags on multi-subunit proteins. When an ice-binding protein was attached as a tag to multisubunit complexes a substantial portion of each multimer dissociated during overgrowth by the ice. The protein subunit attached to the affinity tag was enriched in the ice and the other subunit was appreciably excluded. We suggest that step growth of the advancing ice front generates shearing forces on the bound complex that can disrupt non-covalent protein-protein interactions. This will effectively limit the use of ice-affinity tags to single subunit proteins.

Nitrite facilitated transformation of halophenols in ice.

This study investigated the expediated transformation of halophenols in the presence of nitrite (NO2-) under slightly acidic conditions in ice, whereas such transformation was negligible in liquid water at 4 °C. We proposed that this phenomenon was attributed to the freeze-concentration effect, incurring a pH drop and the aggregation of NO2- and halophenols within the liquid-like grain boundary layer amid ice crystals. Within this micro-environment, NO2- underwent protonation to generate reactive nitrous acid (HNO2) and nitrosonium ions (NO+) that facilitate the nitration and oxidation of halophenols. When 10 µÐœ halophenol was treated by freezing in the presence of 5 µÐœ NO2-, the total yields of nitrated products reached 2.4 µÐœ and 1.4 µÐœ within 12 h for 2-chlorophenol (2CP) and 2-bromophenol (2BP), respectively. NO+ drove oxidative coupling reactions, generating hydroxyl polyhalogenated diphenyl ethers (OH-PBDEs) and hydroxyl polyhalogenated diphenyls via C-O or C-C coupling. These two pathways were intricately intertwined. The presence of natural organic matter (NOM) mitigated the formation of nitrated products and completely suppressed the coupling products. This study offers valuable insights into the fate of halophenols in ice and suggests potential pathways for the formation of nitrophenolic compounds and OH-PBDEs in natural cold environments. These findings also open up a new avenue in environmental chemistry research.

Multiple RONS-Loaded Plasma-Activated Ice Microneedle Patches for Transdermal Treatment of Psoriasis.

Cold atmospheric plasma (CAP) is a fledgling therapeutic technique for psoriasis treatment with noninvasiveness, but clinical adoption has been stifled by the insufficient production and delivery of plasma-generated reactive oxygen and nitrogen species (RONS). Herein, patches of air-discharge plasma-activated ice microneedles (PA-IMNs) loaded with multiple RONS are designed for local transdermal delivery to treat psoriasis as an alternative to direct CAP irradiation treatment. By mixing two RONS generated by the air-discharge plasma in the NOx mode and O3 mode, abundant high-valence RONS are produced and incorporated into PA-IMNs via complex gas-gas and gas-liquid reactions. The PA-IMNs abrogate keratinocyte overproliferation by inducing reactive oxygen species (ROS)-mediated loss of the mitochondrial membrane potential and apoptosis of keratinocytes. The in vivo transdermal treatment confirms that PA-IMNs produce significant anti-inflammatory and therapeutic actions for imiquimod (IMQ)-induced psoriasis-like dermatitis in mice by inhibiting the release of associated inflammatory factors while showing no evident systemic toxicity. Therefore, PA-IMNs have a large potential in transdermal delivery platforms as they overcome the limitations of using CAP directly in the clinical treatment of psoriasis.

Ice recrystallization inhibition activity of pulse protein hydrolysates after immobilized metal affinity separation.

Creating molecules capable of inhibiting ice recrystallization is an active research area aiming to improve the freeze-thaw characteristics of foods and biomedical materials. Peptide mixtures have shown promise in preventing freezing-induced damage, but less is known about the relationship between their amino acid compositions and ice recrystallization inhibition (IRI) activities. In this article, we used Ni2+ immobilized metal affinity chromatography (IMAC) to fractionate pulse protein hydrolysates, created by Alcalase and trypsin, into mixtures lacking and enriched in His, and Cys residues. The aim of this study was to fractionate pulse protein hydrolysates based on their amino acid compositions and evaluate their resulting physicochemical and IRI characteristics. Ni2+ IMAC fractionation induced IRI activity in all of the evaluated soy, chickpea, and pea protein hydrolysates regardless of their amino acid composition. Ni2+ IMAC fractionation produced chemically distinct fractions of peptides, differing by their molecular weights, amino acid composition, and IRI activities. The resulting peptide mixtures' molecular weight, amino acid composition, secondary structure, and sodium ion levels were found to have no correlation with their IRI activities. Thus, we demonstrate for the first time the ability of Ni2+ IMAC fractionation to induce IRI activity in hydrolyzed pulse proteins.

Photolysis of dinotefuran and nitenpyram in water and ice phase: Influence mechanism of temperature over photolysis.

Neonicotinoids are widely used pesticides around the world, but the photolysis of neonicotinoids in cold agricultural region are still in blank. This paper aimed to study the influence of cold temperature over photolysis of neonicotinoids. To this end, the photolysis rates and photoproducts of dinotefuran and nitenpyram in water, ice and freeze-thawing condition were determined. Coupled with quantum chemistry calculation, the influence mechanisms of temperature and medium were investigated. The results showed the photolysis rates of neonicotinoids in water condition slightly declined with the lowered temperature due to the photolysis reactions were endothermic reactions. However, the photolysis rates increased by 89.8 %, 59.2 %, 49.4 % and 9.5 % for dinotefuran and nitenpyram in ice and thawing condition, respectively. This phenomenon was posed by the concentration-enhancing effect and change of photo-chemical properties of neonicotinoids in ice condition, which included lowered bond cleavage energy, lowered first excited singlet state energy and expanded light absorption range. The photolysis pathways of the two neonicotinoids did not change in different medium, but the concentration of carboxyl products was relatively higher than that of water condition due to the more amounts of reactive oxygen species in ice medium, which might increase the secondary pollution risk after ice-off in spring due to the higher ecotoxicity to nontarget organism of these photoproducts. The influence of cold temperature and medium change should be considered for the environmental fate and risk assessment of neonicotinoids in cold agricultural region.

Analysis of bacterial community structure, functional variation, and assembly mechanisms in multi-media habitats of lakes during the frozen period.

Ice, water, and sediment represent three interconnected habitats in lake ecosystems, and bacteria are crucial for maintaining ecosystem equilibrium and elemental cycling across these habitats. However, the differential characteristics and driving mechanisms of bacterial community structures in the ice, water, and sediments of seasonally frozen lakes remain unclear. In this study, high-throughput sequencing technology was used to analyze and compare the structure, function, network characteristics, and assembly mechanisms of bacterial communities in the ice, water, and sediment of Wuliangsuhai, a typical cold region in Inner Mongolia. The results showed that the bacterial communities in the ice and water phases had similar diversity and composition, with Proteobacteria, Bacteroidota, Actinobacteria, Campilobacterota, and Cyanobacteria as dominant phyla. The bacterial communities in sediments displayed significant differences from ice and water, with Chloroflexi, Proteobacteria, Firmicutes, Desulfobacterota, and Acidobacteriota being the dominant phyla. Notably, the bacterial communities in water exhibited higher spatial variability in their distribution than those in ice and sediment. This study also revealed that during the frozen period, the bacterial community species in the ice, water, and sediment media were dominated by cooperative relationships. Community assembly was primarily influenced by stochastic processes, with dispersal limitation and drift identified as the two most significant factors within this process. However, heterogeneous selection also played a significant role in the community composition. Furthermore, functions related to nitrogen, phosphorus, sulfur, carbon, and hydrogen cycling vary among bacterial communities in ice, water, and sediment. These findings elucidate the intrinsic mechanisms driving variability in bacterial community structure and changes in water quality across different media phases (ice, water, and sediment) in cold-zone lakes during the freezing period, offering new insights for water environmental protection and ecological restoration efforts in such environments.

Ice formation and its elimination in cryopreservation of oocytes.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

Characterization of gelatin-oxidized riclin cryogels and their applications as reusable ice cubes in shrimp preservation.

Traditional ice is usually employed to preserve food freshness and extend shelf life. However, ice cannot bear repeated freeze - thaw cycles during the transportation and retailing process, resulting in microbial cross-contamination and spoilage of foods. Herein, succinoglycan riclin was oxidated (RO) and crosslinked with gelatin (Ge), the Ge-RO cryogels were prepared via Schiff base reaction and three freeze - thaw cycles. The Ge-RO cryogels showed improved storage modulus (G') and thermal stability compared with pure gelatin hydrogel. The polymer framework of Ge-RO gels exhibited stable properties against ice crystals destructions during nine freeze - thaw treatments. During the storage and repeated freeze - thaw treatments of shrimps, Ge-RO cryogels exhibited a remarkable preservation effect on shrimps, and their freshness was evaluated using an electronic nose technique equipped with ten sensors. The results demonstrated that the shrimp muscle preserved in ice generated off-odors and resulted in high sensor responses. The sensor responses were reduced sharply of shrimps preserved in cryogels. Moreover, 1H NMR-based metabolomics analysis revealed that shrimps in Ge-RO cryogels group reversed the metabolic perturbations compared with the traditional ice group, the metabolic pathways were related to energy metabolism, nucleotide metabolism, and amino acid metabolism, which provide new clues to the freshness of shrimps. Furthermore, RO exhibited superior antimicrobial activity against E. coli and S. aureus microorganisms. Thus, the crosslinked cryogels are potentially applicable to food preservation, offering sustainable and reusable solutions against traditional ice.

Compromised heat loss leads to a delayed ice slurry induced reduction in heat storage.

Compromised heat loss due to limited convection and evaporation can increase thermal strain. We aimed to determine the effectiveness of ice slurry ingestion to reduce thermal strain following hyperthermia in a state of compromised heat loss. Twelve healthy males (age: 25 ± 4y) underwent hot water immersion to elevate rectal temperature (Trec) by 1.82 ± 0.08°C on four occasions. In the subsequent 60-min of seated recovery, participants ingested either 6.8 g·kg-1 of ice slurry (-0.6°C) or control drink (37°C) in ambient conditions (21 ± 1°C, 39 ± 10% relative humidity), wearing either t-shirt and shorts (2 trials: ICE and CON) or a whole-body sweat suit (2 trials: ICE-SS and CON-SS). Trec and mean skin temperature (Tsk) were recorded and a two-compartment thermometry model of heat storage was calculated. Heat storage was lower in ICE compared with CON at 20-40min (p ≤ 0.044, d ≥ 0.88) and for ICE-SS compared with CON-SS at 40-60 min (p ≤ 0.012, d ≥ 0.93). Trec was lower in ICE compared with CON from 30-60min (p ≤ 0.034, d ≥ 0.65), with a trend for a reduced Trec in ICE-SS compared with CON-SS at 40min (p = 0.079, d = 0.60). A greater Tsk was found in ICE-SS and CON-SS compared with ICE and CON (p < 0.001, d ≥ 3.37). A trend for a lower Tsk for ICE compared with CON was found at 20-40min (p ≤ 0.099, d ≥ 0.53), no differences were found for ICE-SS vs CON-SS (p ≥ 0.554, d ≤ 0.43). Ice slurry ingestion can effectively reduce heat storage when heat loss through convection and evaporation is compromised, relevant to those wearing personal protective equipment or those with compromised sweat loss. Compromised heat loss delays the reduction in heat storage, possibly related to ice slurry ingestion not lowering Tsk.

The effect of a combined cooling intervention on cognitive function in the heat during an intermittent running protocol.

Despite optimal cognitive function being essential for performance, there is a lack of research on the effectiveness of combined cooling interventions on team sport athlete's cognitive function when exercising in the heat. In a randomised, crossover design, 12 unacclimatised men (age: 22.3 ± 3.0 years, body mass: 73.4 ± 5.1 kg, height: 181.0 ± 5.3 cm and V ˙ O 2 $\dot{\mathrm{V}}{\mathrm{O}}_{2}$ max: 51.2 ± 9.5 mL/kg/min) participated in a control (CON) and combined cooling trial (ice slurry and ice collar; COOL). A battery of cognitive tests were completed prior to, during (at half-time) and following a 90-min intermittent running protocol in the heat (33°C, 50% relative humidity (RH)). Perceptual and physiological measures were taken throughout the protocol. In CON, response times were quicker on the Stroop task complex level (p = 0.002) and the visual search test complex level at full-time (p = 0.014) compared to COOL. During COOL, response times were quicker at half-time on the Stroop task complex level (p = 0.024) compared to CON. Lower rectal temperatures were seen during COOL (CON: 37.44 ± 0.65°C and COOL: 37.28 ± 0.68°C) as well as lower skin, neck and forehead temperatures (main effect of trial, all p < 0.05). Lower ratings of thermal sensation and perceived exertion and enhanced thermal comfort were recorded during COOL (main effect of trial, all p < 0.05). Whilst minimal differences in cognitive function were found when using the combined cooling intervention, the findings highlight a practical and effective strategy to improving many physiological and perceptual responses to intermittent exercise in the heat.

Ice Phase Classification Made Easy with Score-Based Denoising.

Accurate identification of ice phases is essential for understanding various physicochemical phenomena. However, such classification for structures simulated with molecular dynamics is complicated by the complex symmetries of ice polymorphs and thermal fluctuations. For this purpose, both traditional order parameters and data-driven machine learning approaches have been employed, but they often rely on expert intuition, specific geometric information, or large training data sets. In this work, we present an unsupervised phase classification framework that combines a score-based denoiser model with a subsequent model-free classification method to accurately identify ice phases. The denoiser model is trained on perturbed synthetic data of ideal reference structures, eliminating the need for large data sets and labeling efforts. The classification step utilizes the smooth overlap of atomic position (SOAP) descriptors as the atomic fingerprint, ensuring Euclidean symmetries and transferability to various structural systems. Our approach achieves a remarkable 100% accuracy in distinguishing ice phases of test trajectories using only seven ideal reference structures of ice phases as model inputs. This demonstrates the generalizability of the score-based denoiser model in facilitating phase identification for complex molecular systems. The proposed classification strategy can be broadly applied to investigate structural evolution and phase identification for a wide range of materials, offering new insights into the fundamental understanding of water and other complex systems.

Microplastic Particles Contain Ice Nucleation Sites That Can Be Inhibited by Atmospheric Aging.

Recent research has shown that microplastics are widespread in the atmosphere. However, we know little about their ability to nucleate ice and their impact on ice formation in clouds. Ice nucleation by microplastics could also limit their long-range transport and global distribution. The present study explores the heterogeneous ice-nucleating ability of seven microplastic samples in immersion freezing mode. Two polypropylene samples and one polyethylene terephthalate sample froze heterogeneously with median freezing temperatures of -20.9, -23.2, and -21.9 °C, respectively. The number of ice nucleation sites per surface area, ns(T), ranged from 10-1 to 104 cm-2 in a temperature interval of -15 to -25 °C, which is comparable to that of volcanic ash and fungal spores. After exposure to ozone or a combination of UV light and ozone, simulating atmospheric aging, the ice nucleation activity decreased in some cases and remained unchanged in others. Our freezing data suggest that microplastics may promote ice formation in cloud droplets. In addition, based on a comparison of our freezing results and previous simulations using a global transport model, ice nucleation by microplastics will impact their long-range transport to faraway locations and global distribution.

The low-entropy hydration shell mediated ice-binding mechanism of antifreeze proteins.

Antifreeze proteins (AFPs) can inhibit ice crystal growth. The ice-binding mechanism of AFPs remains unclear, yet the hydration shells of AFPs are thought to play an important role in modulating the binding of AFPs and ice. Here, we performed all-atom molecular dynamics simulations of an AFP from Choristoneura fumiferana (CfAFP) at four different temperatures, with a focus on analysis at 240 and 300 K, to investigate the dynamic and thermodynamic characteristics of hydration shells around ice-binding surfaces (IBS) and non-ice-binding surfaces (NIBS). Our results revealed that the dynamics of CfAFP hydration shells were highly heterogeneous, with its IBS favoring a less dense and more tetrahedral solvation shell, and NIBS hydration shells having opposite features to those of the IBS. The IBS of nine typical hyperactive AFPs were found to be in pure low-entropy hydration shell region, indicating that low-entropy hydration shell region of IBS and the tetrahedral arrangements of water molecules around them mediate the ice-binding mechanism of AFPs. It is because the entropy increase of the low-entropy hydration shell around IBS, while the higher entropy water molecules at NIBS most likely prevent ice crystal growth. These findings provide new mechanistic insights into the ice-binding of AFPs.

Analysis of cryopreservation media thermophysical characteristics after ultra-rapid cooling through differential scanning calorimetry.

Cryoprotective agents play a critical role in minimizing cell damage caused by ice formation during cryopreservation. However, high concentrations of CPAs are toxic to cells and tissues. Required concentrations of CPAs can be reduced by utilizing higher cooling and warming rates, but insight into the thermophysical properties of biological solutions in the vitrification method is necessary for the development of cryopreservation protocols. Most studies on thermophysical properties under ultra-rapid cooling conditions have been qualitatively based on visualization. Differential scanning calorimetry methods are ideal for studying the behavior of biomaterials in various freezing conditions quantitatively and accurately, though previous studies have been predominantly restricted to slower cooling rates. Here, we developed an ultra-rapid cooling method for DSC that can achieve minimal cooling rates exceeding 2000 °C/min. We investigated the thermophysical vitrification behavior of ternary solutions of phosphate buffer saline (1X), dimethyl sulfoxide or glycerol and ice blocking polymers (X-1000 or Z-1000). We quantified the impact of solute concentration on ice crystal formation during rapid cooling. Our findings support the expectation that increasing the solute concentration reduces the amount of ice formation, including devitrification. Devitrification increases from 0 % to 40 % (v/v) Me2SO and then reduces significantly. The relative amounts of devitrification to the total ice formation are 0 %, 60 %, 0 % in 20 %, 40 %, 60 % (v/v) Me2SO, and 2 %, 48 %, 49 % in 20 %, 40 %, 60 % (v/v) glycerol, respectively. The results suggest that at low concentrations, such as below 20 % (v/v) for Me2SO or glycerol, increasing the warming rate after ultra-rapid freezing is not essential to eliminate devitrification. Furthermore, ice blocking polymers do not reduce ice formation substantially and cannot eliminate devitrification under ultra-rapid cooling conditions. In conclusion, our results provide insights into the impact of solute concentration on ice formation and devitrification during rapid cooling, which can be practical for optimizing cryopreservation protocols.