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Immunoglobulin, which is widely used in the formulation of protein drugs, is prone to aggregation due to freezing. The aggregated immunoglobulin exhibits decreased immune-reactivity and increasing immunogenicity. Accordingly, large amounts of excipients are added to immunoglobulin drugs to prevent aggregation. In the present study, we found that an Arabidopsis dehydrin (AtHIRD11), which is a stress-related intrinsically disordered protein, could efficiently inhibit the cryoaggregation of bovine gamma globulin (BGG). AtHIRD11 was 3 to 4 orders of magnitude more efficient than general protectants such as sugars and amino acids at the molar levels. The K-segment, which is a conserved sequence of dehydrin, was one of the protective sites of AtHIRD11. Amino acid substitution analysis indicated that the hydrophobic amino acids contributed to the cryoprotective activity of the K-segment. Moreover, the activity was roughly correlated with the hydropathy scores of hydrophobic amino acids. BGG and the K-segment individually migrated in size exclusion chromatography, showing that the K-segment did not bind to BGG in solution. This suggests that dehydrin may prevent the cryoaggregation of BGG via the K-segment through a transient hydrophobic interaction. Dehydrin may be utilized as an effective stabilizer of immunoglobulin to minimize aggregation under freezing conditions.

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Carbohydrates are a class of macromolecules that has significant potential across several domains, including the organisation of genetic material, provision of structural support, and facilitation of defence mechanisms against invasion. Their molecular diversity enables a vast array of essential functions, such as energy storage, immunological signalling, and the modification of food texture and consistency. Due to their rheological characteristics, solubility, sweetness, hygroscopicity, ability to prevent crystallization, flavour encapsulation, and coating capabilities, carbohydrates are useful in food products. Carbohydrates hold potential for the future of therapeutic development due to their important role in sustained drug release, drug targeting, immune antigens, and adjuvants. Bio-based packaging provides an emerging phase of materials that offer biodegradability and biocompatibility, serving as a substitute for traditional non-biodegradable polymers used as coatings on paper. Blending polyhydroxyalkanoates (PHA) with carbohydrate biopolymers, such as starch, cellulose, polylactic acid, etc., reduces the undesirable qualities of PHA, such as crystallinity and brittleness, and enhances the PHA's properties in addition to minimizing manufacturing costs. Carbohydrate-based biopolymeric nanoparticles are a viable and cost-effective way to boost agricultural yields, which is crucial for the increasing global population. The use of biopolymeric nanoparticles derived from carbohydrates is a potential and economically viable approach to enhance the quality and quantity of agricultural harvests, which is of utmost importance given the developing global population. The carbohydrate biopolymers may play in plant protection against pathogenic fungi by inhibiting spore germination and mycelial growth, may act as effective elicitors inducing the plant immune system to cope with pathogens. Furthermore, they can be utilised as carriers in controlled-release formulations of agrochemicals or other active ingredients, offering an alternative approach to conventional fungicides. It is expected that this review provides an extensive summary of the application of carbohydrates in the realms of food, pharmaceuticals, and environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Polysaccharides are becoming potential candidates for developing food-grade cryoprotectants due to their extensive accessibility and health-promoting effects. However, unremarkable ice recrystallization inhibition (IRI) activity and high viscosity limit their practical applications in some systems. Our previous study found a galactoxyloglucan polysaccharide from tamarind seed (TSP) showing moderate IRI activity. Herein, the enhancement of the IRI performance of TSP via enzymatic depolymerization and degalactosylation-induced self-assembly was reported. TSP was depolymerized and subsequently removed ∼40 % Gal, which induced the formation of supramolecular rod-like fiber self-assembles and exhibited a severalfold enhancement of IRI. Ice shaping assay did not show obvious faceting of ice crystals, indicating that both depolymerized and self-assembled TSP showed very weak binding to ice. Molecular dynamics simulation confirmed the absence of molecular complementarity with ice. Further, it highlighted that degalactosylation did not cause significant changes in local hydration properties of TSP from the view of a single oligomer. The inconsistency between molecular simulation and macroscopic IRI effect proposed that the formation of unique supramolecular self-assemblies may be a key requirement for enhancing IRI activity. The findings of this study provided a new opportunity to enhance the applied potential of natural polysaccharides in food cryoprotection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

For the safety and efficacy of frozen cell therapy products, determination of cellular viability is key. However, results of cell viability measurements do not only depend on the cell line or on the inflicted stress, but also on the assay used, making inter-experimental comparisons difficult. The aim of this study was thus to assess commonly used viability assays in clinically relevant human mesenchymal/stromal stem cells and human A549 lung carcinoma cells. Post freeze-thaw stress viability and proliferation were evaluated under different conditions using trypan blue, acridine orange/DAPI stain, alamarBlue, ATP, and neutral red assays. Significant differences in cell viability between metabolic assays were observed, likely due to their distinct intrinsic detection mechanisms. Membrane-integrity based assays generally overestimated cell viabilities in this study. Furthermore, noticeable differences in inter-assay sensitivities were observed. These differences highlight that cell viability methods should be meticulously selected and their associated results carefully interpreted in a relevant context to ensure reliable conclusions. Indeed, although cell membrane integrity based assays are a popular choice to determine cellular quality attributes after freezing and thawing, we demonstrate that metabolic assays may be more suitable in this context., Competing Interests: Declaration of Competing Interest This research was funded by and conducted at Lonza Ltd., Basel. M. K. was supported by a grant from Lonza, L. S. was employed at Lonza Ltd., Basel at the time of this work. HC. M. and J. H. are not affiliated with Lonza. The authors have not received any additional benefits. None of the disclosed methods or formulations are currently commercially applied and hence there is no direct financial benefit for Lonza., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Standard freezing protocols of clinically relevant cell lines commonly employ agents such as fetal bovine serum and dimethyl sulfoxide, which are a potential concern from both a regulatory and a patient safety perspective. The aim of this work was to develop formulations with safe and well tolerated excipients for the (cryo-) preservation of cell therapy products. We evaluated the cryoprotective capabilities of urea and glucose through measurements of cell metabolic activity. Freezing of clinically relevant human mesenchymal stromal/stem cells and human dermal fibroblasts at ≤ - 65°C at equimolar ratios of urea and glucose resulted in comparable viabilities to established dimethyl sulfoxide. Pre-incubation of human mesenchymal stromal/stem cells in trehalose and addition of mannitol and sucrose to the formulation further enhanced cell viability after freeze-thaw stress. Other cell types assessed (A549 and SK-N-AS) could not satisfactorily be preserved with urea and glucose, highlighting the need for tailored formulations to sustain acceptable cryopreservation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

The wood frog, Rana sylvatica (aka Lithobates sylvaticus) is the main model for studies of natural freeze tolerance among amphibians living in seasonally cold climates. During freezing, ∼65% of total body water can be converted to extracellular ice and this imposes both dehydration and hypoxia/anoxia stresses on cells. The current study analyzed the responses of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1), a crucial oxygen-sensitive regulator of gene expression, to freezing, anoxia or dehydration stresses, examining six tissues of wood frogs (liver, skeletal muscle, brain, heart, kidney, skin). RT-PCR revealed a rapid elevation hif-1α transcript levels within 2 h of freeze initiation in both liver and brain and elevated levels of both mRNA and protein in liver and muscle after 24 h frozen. However, both transcript and protein levels reverted to control values after thawing except for HIF-1 protein in liver that dropped to ∼60% of control. Independent exposures of wood frogs to anoxia or dehydration stresses (two components of freezing) also triggered upregulation of hif-1α transcripts and/or HIF-1α protein in liver and kidney with variable responses in other tissues. The results show active modulation of HIF-1 in response to freezing, anoxia and dehydration stresses and implicate this transcription factor as a contributor to the regulation of metabolic adaptations needed for long term survival of wood frogs in the ischemic frozen state., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The present study aimed to determine the effects of sucrose on the physical stability, cellular entry pathways and functional efficacy of poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs). PLGA-NPs were synthesized in the absence or presence of 10 % sucrose, using HEI-101, an unmodified small interfering RNA (siRNA), as a drug model. The newly synthesized HEI-101-loaded PLGA-NPs (HEI-101-NPs) were exposed to repeated freeze-thaw cycles and iteratively tested over a six-month evaluation period. The effect of sucrose stabilization on HEI-101-NPs was independently tested in vitro for biocompatibility and cellular uptake in IMO-2B1 cells. Data analyses suggest that, without sucrose, freeze-thaw cycles of HEI-101-NPs resulted in increased particle diameter, increased polydispersity index, and reduced zeta potential. In contrast, a substantial improvement in the physical stability of HEI-101-NPs was observed in the presence of 10 % sucrose. The data revealed that the release of HEI-101 from the PLGA-NPs was governed by polymer erosion and drug diffusion. Data from cellular uptake study in IMO-2B1 cells demonstrated that, 10 % sucrose significantly reduced the inhibitory effect of nocodazole on the microtubule-dependent uptake of PLGA-NPs. In addition, the presence of 10 % sucrose seemed to lessen the inhibitory effect of sodium azide on the energy-dependent uptake of PLGA-NPs. Overall, the current data suggest that the cellular internalization of PLGA-NPs occurred through the polymerization of actin filaments under the control of the microtubules. Our findings reveal cryoprotective effect of 10 % sucrose on HEI-101-NPs that confers marked improvements in the stability, cellular uptake and efficiency for the delivery of biomolecules to inner ear cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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 (Me 2 SO) 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 Me 2 SO 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 Me 2 SO 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 Me 2 SO 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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Dehydrin is an intrinsically disordered protein involved in the cold tolerance of plants. Although dehydrins have been thought to protect biomembranes under cold conditions, the underlying protective mechanism has not been confirmed. Here we report that Arabidopsis dehydrin AtHIRD11 inhibited the aggregation of phospholipid liposomes after freezing and thawing. AtHIRD11 showed significantly greater cryoaggregation-prevention activity than cryoprotective agents such as trehalose, proline, and polyethylene glycols. Amino acid sequence segmentation analysis indicated that the K-segment of AtHIRD11 inhibited the cryoaggregation of phosphatidylcholine (PC) liposomes but other segments did not. This showed that K-segments conserved in all dehydrins were likely to be the cryoprotective sites of dehydrins. Amino acid replacement for a typical K-segment (TypK for short) sequence demonstrated that both hydrophobic and charged amino acids were required for the cryoaggregation-prevention activity of PC liposomes. The amino acid shuffling of TypK remarkably reduced cryoprotective activity. Although TypK did not bind to PC liposomes in solution, the addition of liposomes reduced its disordered content under crowded conditions. Together, these results suggested that dehydrins protected biomembranes via conserved K-segments whose sequences were optimized for cryoprotective activities., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The goldenrod gall moth (Epiblema scudderiana) is a cold hardy insect that survives subzero temperatures during the winter by supercooling bodily fluids to approximately -40 °C, allowing the insect to remain unfrozen despite the freezing temperatures. This is characterized by a drastic increase of cryoprotectant glycerol along with widespread downregulation of non-essential genes and processes to conserve cellular energy. This study examined the role of epigenetic enzymes in regulating this freeze-avoidant process across a range of freezing temperatures experienced in nature. Cold and subzero temperature exposure in E. scudderiana resulted in upregulation of select DNA methyltransferase (DNMT) enzymes with concurrent decreases in DNMT activity and no change in activity of the Ten-Eleven Translocation (TET) demethylation enzyme activities. Levels of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity decreased during cold exposures. The increase in DNMT expression and concurrent decrease in HAT activity suggests a role for DNA methylation to assist with transcriptional suppression. These findings propose that epigenetic regulation of genes and histones underpin the winter survival strategies of this insect., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A soluble protein fraction from radish (Raphanus sativus L.) taproot had cryoprotective activity for lactate dehydrogenase (LDH). The activity was found mainly in the heat-stable fractions of soluble proteins. The cryoprotective protein, whose molecular mass was 43 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis, was obtained by successive chromatographies on TOYOPEARL SuperQ and TOYOPEARL DEAE. MALDI-TOF MS/MS analysis indicated that the purified protein was a radish vacuolar calcium-binding protein (RVCaB), which is reportedly related to calcium storage in the vacuoles of radish taproot. The purified RVCaB inhibited the cryoinactivation, cryodenaturation, and cryoaggregation of LDH. RVCaB had greater cryoprotective activity than general cryoprotectants. When RVCaB was divided into 15 segments (Seg01 to Seg15, 15 amino acids each), Seg03, which had a high hydrophobicity scale, showed remarkable cryoprotective activity. This indicated that RVCaB protected LDH from freezing and thawing damage presumably through a specific hydrophobic area (i.e., Seg03)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ice recrystallization inhibitors have emerged as novel cryoprotectants to improve cell viability for cryopreservation. Nanocelluloses were identified as new materials for ice recrystallization inhibition (IRI); however, conventional nanocelluloses aggregate and lose IRI activity at high ionic strengths, which limit their application as cryoprotectants. In this study, we synthesized a novel group of nanocelluloses - electrosterically stabilized cellulose nanocrystals (ECNCs), which remained dispersed and IRI-active at high ionic strengths. ECNCs improved the post-thaw viability of HCT-116 colorectal cancer cells in slow/fast freezing-slow thawing protocols in the presence of 1-20% v/v dimethyl sulfoxide (DMSO), as well as in slow/fast freezing-fast thawing protocols at reduced DMSO concentrations. The effectiveness in cryoprotection did not match the IRI activity in ECNCs, polyethylene glycol (PEG), and polyvinyl alcohol (PVA); and in ECNCs with different surface charge densities. Overall, ECNCs demonstrated IRI and cryoprotection activities, but the mechanism of cryoprotection remains unknown., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dehydrins are intrinsically disordered proteins which are related to cold tolerance in plants. Dehydrins show potent cryoprotective activities for freeze-sensitive enzymes such as lactate dehydrogenase (LDH). Previous studies demonstrated that K-segments conserved in dehydrins had cryoprotective activities and that K-segment activities depended on the hydrophobic amino acids in the segment. However, the cryoprotective roles of hydrophobic amino acids in dehydrin itself have not been reported. Here, we demonstrated that hydrophobic amino acids were required for the cryoprotective activity of Arabidopsis dehydrin AtHIRD11. Cryoprotective activities were compared between AtHIRD11 and the corresponding mutant in which all hydrophobic residues were changed to T (AtHIRD11Φ/T) by using LDH. The change strikingly reduced AtHIRD11 activity. A segmentation analysis indicated that the conserved K-segment (Kseg) and a previously unidentified segment (non-K-segment 1, NK1) showed cryoprotective activities. Circular dichroism indicated that the secondary structures of all peptides showed disorder, but only cryoprotective peptides changed to the ordered forms by sodium dodecyl sulfate. Ultracentrifuge analysis indicated that AtHIRD11 and AtHIRD11Φ/T had similar molecular sizes in solution. These results suggest that not only structural disorder but also hydrophobic amino acids contributed to the cryoprotective activity of AtHIRD11. A possible mechanism based on an extended molecular shield model is proposed., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rana sylvatica, known as the wood frog, can survive extremely cold temperatures during winter by undergoing full-body freezing, where it tolerates freezing of 65-70% of its total body water. During freezing, cellular dehydration decreases damage to the cell by preventing ice crystallization. Challenged with many stresses, these animals are forced to develop physiological adaptations to osmoregulation and osmoprotection that are necessary to ensure their survival. The purpose of this study was to elucidate a potential mechanism by which the transcription factor, NFAT5, regulates the expression of three osmoregulatory proteins (aldose reductase, SMIT, and BGT-1). These three proteins control cellular concentrations of the organic osmolytes: betaine (BGT-1), myo-inositol (SMIT), and sorbitol (aldose reductase). We studied this mechanism during the freeze-thaw stress in R. sylvatica liver, kidney, and skeletal muscle. Protein expression of BGT-1, SMIT, aldose reductase, and NFAT5 were examined using immunoblotting. We identified that the NFAT5 pathway facilitated osmoregulation in a tissue-specific manner during freezing. In skeletal muscle, we demonstrated that NFAT5 upregulation in thawing led to increases in the protein levels of BGT-1. In liver, NFAT5 was upregulated during freezing, along with aldose reductase. Furthermore, neither of these patterns of expression were observed in kidney as none of these four proteins showed differential expression during freezing or thawing. Therefore, the NFAT5 osmoregulatory pathway appears to be tissue-specific. Our novel findings on a mechanism of osmoregulation in R. sylvatica highlight the importance of studying naturally stress-tolerant animals to identify novel pro-survival pathways., (Copyright © 2017 Elsevier Inc. All rights reserved.)