Potential of fructans as natural cryoprotectant agents in plant cryopreservation: concept validation on Arabidopsis thaliana L.

BACKGROUND: Today, synthetic chemicals are used in vitrification solutions for cryopreservation studies to mimic natural cryoprotectants that supply tolerance to organisms in nature against freezing stress. In the case of plants, PVS2, containing glycerol, dimethyl sulfoxide (Me2SO), ethylene glycol and sucrose, is considered as the golden standard for successful cryopreservation. However, Me2SO can generally cause toxicity to certain plant cells, adversely affecting viability after freezing and/or thawing. Hence, the replacement (or substantial reduction) of Me2SO by cheap, non-toxic and natural cryoprotectants became a matter of high priority to vitrification solutions or reducing their content gained escalating importance for the cryopreservation of plants. Fructans, sucrose derivatives mainly consisting of fructose residues, are candidate cryoprotectants. OBJECTIVE: Inspired by their protective role in nature, we here explored, for the first time, the potential of an array of 8 structurally different fructans as cryoprotectants in plant cryopreservation. MATERIALS AND METHODS: Arabidopsis thaliana L. seedlings were used as a model system with a one-step vitrification method. PVS2 solutions with different Me2SO and fructan contents were evaluated. RESULTS: It was found that branched low DP graminan, extracted from milky stage wheat kernels, led to the highest recovery (85%) among tested fructans with 12.5% Me2SO after cryopreservation, which was remarkably close to the viability (90%) observed with the original PVS2 containing 15% Me2SO. Moreover, its protective efficacy could be further optimized by addition of vitamin C acting as an antioxidant. CONCLUSION: Such novel formulations offer great perspectives for cryopreservation of various crop species. Doi.org/10.54680/fr24410110512.

Influence of hydrogen bonds on state diagrams of cryoprotectant solutions.

BACKGROUND: Transformation of state diagrams of cryoprotectant solutions under the influence of weak intramolecular interactions was considered. MATERIALS AND METHODS: Phase states of aqueous glycerol and DMSO solutions within temperature range +25 to -150 degree С were studied using method of volumetric scanning tensodilatometry. Temperatures below which hydrogen bonds significantly affect crystallization-melting kinetics of such solutions were determined. RESULTS: Principles for plotting of state diagram for binary solutions with weak intermolecular interaction of the components were set up. The study demonstrates that in such solutions formation of clusters based on ice microcrystals and cryoprotectant occurs. Based on the obtained results, state diagrams for glycerol and DMSO aqueous solutions were plotted. These diagrams include area of cluster phase existence and differ fundamentally from those describing eutectic crystallization. CONCLUSION: Nanostructures occurring in cryoprotectant solutions during their cooling were analyzed. Difference between these structures and classical solid phase eutectics were demonstrated. Doi.org/10.54680/fr24410110712.

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves.

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation. Sperm data were analysed using an ANOVA or Friedman test (results mean ± SD). Paired comparison between the two freezing curves was analysed using the Wilcoxon test. Total and progressive motility were significantly higher (P<0.05) in the EY and CE-3 samples using the slow curve, whereas for the fast curve, total and progressive motility were significantly higher (P<0.05) in the EY samples compared to all the extenders and the samples frozen in CE-3 were significantly higher than the remaining extenders (P<0.05). The percentages of live acrosome intact sperm and of live non-peroxidized sperm were significantly higher (P<0.05) in the EY extender when using either of the freezing curves and in turn, were significantly higher (P<0.05) in samples frozen in CE-3 compared to the remaining extenders. Intact DNA was significantly lower (P<0.05) in the BIO extender, using the rapid curve. To conclude, the commercial equine extender with 3% dimethylformamide, without egg-yolk, could be a suitable alternative for extenders with egg-yolk.

Cryoprotective Polysaccharides with Ordered Gel Structures Induce Ice Growth Anticipation and Survival Enhancement during Cell Cryopreservation.

This work cross-correlated rheological, thermodynamic, and conformational features of several natural polysaccharides to their cryoprotective performance. The basis of cryoprotection of FucoPol, pectin, and agar revealed a causal combination of (i) an emerging sol-gel transition (p = 0.014) at near-hypothermia (4 °C), (ii) noncolligative attenuated supercooling of the kinetic freezing point of water (p = 0.026) supporting ice growth anticipation, and (iii) increased conformational order (p < 0.0001), where helix-/sheet-like features boost cryoprotection. FucoPol, of highest cryoprotective performance, revealed a predominantly helical structure (α/ß = 1.5) capable of forming a gel state at 4 °C and the highest degree of supercooling attenuation (TH = 6.2 °C). Ice growth anticipation with gel-like polysaccharides suggests that the gel matrix neutralizes elastic deformations and lethal cell volumetric fluctuations during freezing, thus preventing the loss of homeostasis and increasing post-thaw viability. Ultimately, structured gels capable of attenuated supercooling enable cryoprotective action at the polymer-cell interface, in addition to polymer-ice interactions. This rationale potentiates implementing alternative, biobased, noncytotoxic polymers in cryobiology.

In situ monitoring of loading and elution processes of cryoprotectants at the single-cell level with Raman spectroscopy.

BACKGROUND: The analysis of cell membrane permeability plays a crucial role in improving the procedures of cell cryopreservation, which will affect the specific parameter settings in loading, removal and cooling processes. However, existing studies have mostly focused on deriving permeability parameters through osmotic theoretical models and cell volume response analysis, and there is still a lack of the direct experimental evidence and analysis at the single-cell level regarding the migration of cryoprotectants. RESULTS: In this work, a side perfusion microfluidics chips combined with Raman spectroscopy system was built to monitor in situ the Raman spectroscopy of extracellular and intracellular solution during loading and elution process with different cryoprotectant solution systems (single and dual component). And it was found that loading a high concentration cryoprotectant solution system through a single elution cycle may result in significant residual protective agent, which can be mitigated by employing a multi-component formula but multiple elution operations are still necessary. Furthermore, the collected spectral signals were marked and analyzed to was perform preliminary relative quantitative analysis. The results showed that the intracellular concentration changes can be accurately quantified by the Raman spectrum and are closely related to the extracellular solution concentration changes. SIGNIFICANCE AND NOVELTY: By using the method of small flow perfusion (≤20 µL/min) in the side microfluidic chip after the gravity sedimentation of cells, the continuous loading and elution process of different cryoprotectants on chip and the spectral acquisition can be realized. The intracellular and extracellular concentrations can be quantified in situ based on the ratio of spectral peak intensities. These results indicate that spectroscopic analysis can be used to effectively monitor intracellular cryoprotectant residues.

Cryo-storage of porcine hides at the industrial scale for tissue engineering and regenerative medicine application.

BACKGROUND: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process. OBJECTIVE: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine. MATERIALS AND METHODS: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants. RESULTS: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage. CONCLUSION: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.

Inhibitive effect of trehalose and sodium pyrophosphate on oxidation and structural changes of myofibrillar proteins in silver carp surimi during frozen storage.

This work investigated the cryoprotective effect of trehalose (TH) and sodium pyrophosphate (SPP) alone and in combination on myofibrillar protein (MP) oxidation and structural changes in silver carp surimi during 90 days of frozen storage (-20 °C). TH combined with SPP was significantly more effective than single TH or SPP in preventing MP oxidation (P < 0.05), showing a higher SH content (6.05 nmol/mg protein), and a lower carbonyl (4.24 nmol/mg protein) and dityrosine content (1280 A.U.). SDS-PAGE results indicated that TH combined with SPP did not differ significantly from TH and SPP in inhibiting protein degradation but was more effective in inhibiting protein crosslinking. Moreover, all cryoprotectants could stabilise the secondary and tertiary structures and inhibit unfolded and aggregation of MP, with the combination of TH and SPP being the best. It's worth noting that TH combined with SPP had a synergistic effect on inhibiting the decrease in α-helix content and gel-forming ability, and the increase in surface hydrophobicity. Overall, TH combined with SPP could significantly inhibited MP oxidation and structural changes in surimi during frozen storage and improve the gel-forming ability, which was significantly better than single TH or SPP.

Composition-antifreeze property relationships of gelatin and the corresponding mechanisms.

The inherent functional fractions (gelation and ice-affinitive fractions) of gelatin enable it as a promising cryoprotectant alternative. However, the composition-antifreeze property relationships of gelatin remain to be investigated. In this study, the HW-PSG and LW-PSG fractions of gelatin from fish scales were obtained, according to the critical gelation conditions and ice-binding measurements, respectively. Thermal hysteresis (THA) value, associated with ice nucleation, of LW-PSG was higher than that of HW-PSG. Besides, the relatively low-sized ice crystals (210-550 µm2) indicated that HW-PSG showed strong ice recrystallization inhibition (IRI) ability, compared to other groups. These results suggested that LW-PSG inhibited ice nucleation, while HW-PSG displayed the strong IRI ability. Furthermore, the antifreeze mechanisms were clarified through IRI measurements and molecular dynamics simulation. The minimum size of ice crystals was found for HW-PSG gels with dense microstructure, suggesting the HW-PSG retarded the growth of ice crystals by restricting the migration and phase transformation of water molecules. The hydrogen bond interactions between the ice crystal surface and ASN1294 and PRO1433 residues of LW-PSG, and hydrophobic interactions contributed to inhibiting the nucleation of ice crystals. This study provided some references to further enhance antifreeze performance of gelatin by modulating fragment composition.

Adenosine monophosphate boosts the cryoprotection of ultrasound-assisted freezing to frozen surimi: Insights into protein structures and gelling behaviors.

Ultrasound-assisted freezing (UAF) is a clean technique for meat cryoprotections; however, its effectiveness is still limited compared to conventional cryoprotectants, e.g., sugars, polyols, especially at high dosages. To resolve this problem, a synergistic cryoprotection strategy was developed in this study. Adenosine monophosphate (AMP), an adenosine-type food additive, was introduced into frozen surimi at a considerably reduced content (0.08%), yet substantially enhanced the efficiency of UAF to comparable levels of commercial cryoprotectant (4% sucrose with 4% sorbitol). Specifically, UAF/AMP treatment retarded denaturation of surimi myofibrillar protein (MP) during 60-day frozen storage, as evidenced by its increased solubility, Ca2+-ATPase activity, sulfhydryl content, declined surface hydrophobicity, particle size, and stabilized protein conformation. Gels of UAF/AMP-treated surimi also demonstrated more stabilized microstructures, uniform water distributions, enhanced mechanical properties and water-holding capacities. This study provided a feasible approach to boost the cryoprotective performance of UAF, thus expanding its potential applications in frozen food industry.

27 MHz constant field dielectric warming of kidneys cryopreserved by vitrification.

Organs cryopreserved by vitrification are exposed to the lowest possible concentration of cryoprotectants for the least time necessary to successfully avoid ice formation. Faster cooling and warming rates enable lower concentrations and perfusion times, reducing toxicity. Since warming rates necessary to avoid ice formation during recovery from vitrification are typically faster than cooling rates necessary for vitrification, warming speed is a major determining factor for successful vitrification. Dielectric warming uses an oscillating electric field to directly heat water and cryoprotectant molecules inside organs to achieve warming that's faster and more uniform than can be achieved by heat conduction from the organ surface. This work studied 27 MHz dielectric warming of rabbit kidneys perfused with M22 vitrification solution. The 27 MHz frequency was chosen because its long wavelength and penetration depth are suitable for human organs, because it had an anticipated favorable temperature of maximum dielectric absorption in M22, and because it's an allocated frequency for industrial and amateur use with inexpensive amplifiers available. Previously vitrified kidneys were warmed from -100 °C by placement in a 27 MHz electric field formed between parallel capacitor plates in a resonant circuit. Power was varied during warming to maintain constant electric field amplitude between the plates. Maximum power absorption occurred near -70 °C, with a peak warming rate near 150 °C/min in 50 mL total volume with approximately 500 W power. After some optimization, it was possible to warm ∼13 g vitrified kidneys with unprecedentedly little injury from medullary ice formation and a favorable serum creatinine trend after transplant. Distinct behaviors of power absorption and system tuning observed as a function of temperature during warming are promising for non-invasive thermometry and future automated control of the warming process at even faster rates with user-defined temperature dependence.

Rescuing a Troubled Tolcapone with PEGylated PLGA Nanoparticles: Design, Characterization, and Hepatotoxicity Evaluation.

Tolcapone is an orally active catechol-O-methyltransferase (COMT) inhibitor used as adjuvant therapy in Parkinson's disease. However, it has a highly hepatotoxic profile, as recognized by the U.S. Food and Drug Administration. As a possible solution, nanoscience brought us several tools in the development of new functional nanomaterials with tunable physicochemical properties, which can be part of a solution to solve several drawbacks, including drug's short half-life and toxicity. This work aims to use PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a stable carrier with lower hydrodynamic size and polydispersity to encapsulate tolcapone in order to overcome its therapeutic drawbacks. Using the nanoprecipitation method, tolcapone-loaded nanoparticles with a DLC% of 5.7% were obtained (EE% of 47.0%) and subjected to a lyophilization optimization process to obtain a final shelf-stable formulation. Six different cryoprotectants in concentrations up to 10% (w/v) were tested. A formulation of PLGA nanoparticles with 3% hydroxypropyl-ß-cyclodextrin (HPßCD) as a cryoprotectant (PLGA-HP@Tolc), presenting sub-200 nm sizes and low polydispersity (PdI < 0.200) was selected. Cytotoxicity assays, namely, MTT and SRB, were used to study the metabolic activity and cell density of tolcapone and PLGA-HP@Tolc-treated cells. In both assays, a hepatocarcinoma cell line (HepG2) growing in glucose or glucose-free media (galactose-supplemented medium) was used. The results demonstrated that the treatment with the PLGA-HP@Tolc formulation led to a decrease in cytotoxicity in comparison to free tolcapone-treated cells in both media tested. Moreover, the elected formulation also counteracted ATP-depletion and excessive ROS production induced by tolcapone. The results suggest that HPßCD might have a dual function in the formulation: cryoprotectant and anticytotoxic agent, protecting cells from tolcapone-induced damage. Using an in vitro COMT inhibition assay, the PLGA-HP@Tolc formulation demonstrated to inhibit COMT as efficiently as free tolcapone. Overall, the results suggest that tolcapone-loaded PLGA NPs could be an interesting alternative to free tolcapone, demonstrating the same in vitro efficacy in inhibiting COMT but with a safer cytotoxic profile.

Targeted development and optimization of small-molecule ice recrystallization inhibitors (IRIs) for the cryopreservation of biological systems.

Despite the routine use of cryopreservation for the storage of biological materials, its outcomes are often sub-optimal (including reduced post-thaw viability, recovery, and functionality) due to the damage caused by uncontrolled ice growth. Traditional cryoprotective agents (CPAs), including dimethyl sulfoxide (DMSO), fail to prevent damage caused by ice growth and concerns over CPA cytotoxicity have fostered an increased interest in developing improved CPAs and cryoprotection strategies. The inhibition of ice recrystallization by natural antifreeze (glyco)proteins [AF(G)Ps] to improve cryopreservation outcomes has been examined; however, the ice binding properties of these substances and their challenging large-scale production make them poor CPA candidates. Therefore, the development and deployment of biocompatible, small-molecule ice recrystallization inhibitors (IRIs) for use as CPAs is a worthwhile objective. Extensive structure-activity relationship studies on AF(G)Ps revealed that simple carbohydrate derivatives could inhibit ice recrystallization. It was later discovered that this activity could be fine-tuned by delicately balancing the molecule's hydrophobicity and hydrophilicity. Current generation small-molecule IRIs have been meticulously designed to avoid binding to the surface of ice and subsequent biological testing (for both cytotoxicity and cryopreservation efficacy) has demonstrated significant improvements to the cryopreservation outcomes of several cell types. However, an individualized cell-specific approach for the simultaneous assessment of multiple cryopreservation outcomes is necessary to realize the full potential of IRIs as CPAs. This article provides a detailed overview of the development of small-molecule carbohydrate-based IRIs and highlights the crucial cell-specific biological considerations that must be taken into account when assessing cryopreservation outcomes. https://doi.org/10.54680/fr24210110112.

Cell Damage Mechanisms during Cryopreservation in a Zwitterion Solution and Its Alleviation by DMSO.

Recently, zwitterions have been proposed as novel cryoprotectants. However, some cells are difficult to cryopreserve using aqueous zwitterion solutions alone. We investigated here the reason for cell damage in such cells, and it was the osmotic pressure after freeze concentration. Furthermore, the addition of dimethyl sulfoxide (DMSO) has been reported to improve the cryoprotective effect in such cells: the zwitterion/DMSO aqueous solution shows a higher cryoprotective effect than the commercial cryoprotectant. This study also clarified the mechanisms underlying the improvement in a cryoprotective effect. The addition of cell-permeable DMSO alleviated the osmotic pressure after the freeze concentration. This alleviation was also found to be a key factor for cryopreserving cell spheroids, while there has been no insight into this phenomenon.

The crystallization properties of antifreeze GelMA hydrogel and its application in cryopreservation of tissue-engineered skin constructs.

Gelatin methacrylate (GelMA) hydrogels are expected to be ideal skin tissue engineering dressings for a wide range of clinical treatments. Herein, we report the preparation of GelMA or antifreeze GelMA hydrogel sheets with different GelMA concentrations, crosslinking times, and cryoprotectant (CPA) concentrations. The crystallization properties of GelMA or antifreeze GelMA hydrogel sheets were studied by cryomicroscopy and differential scanning calorimetry (DSC). It was found that the growth of ice crystals was slower when GelMA hydrogel concentration was more than 7%. The 10% DMSO-7% GelMA hydrogel sheets crosslinked for 60 min showed no ice crystal formation and growth during cooling and warming. The DSC results showed that the vitrification temperature of the 10% DMSO-7% GelMA hydrogel sheet was -111°C. Furthermore, slow freezing and rapid freezing of fibroblast-laden GelMA or antifreeze GelMA hydrogel sheets, and tissue-engineered skin constructs were studied. The results showed no significant difference in cell survival between slow (88.8% ± 1.51) and rapid (89.2% ± 3.00) freezing of fibroblast-loaded 10% DMSO-7% GelMA hydrogel sheets, and significantly higher than that of 7% GelMA hydrogel sheets (33.4% ± 5.46). The cell viability was higher in tissue-engineered skin constructs after slow freezing (86.34% ± 1.45) than rapid freezing (72.74% ± 1.34). We believe that the combination of antifreeze hydrogels and tissue engineering will facilitate the cryopreservation of tissue engineering constructs.

Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

Cooling dynamics of droplets exposed to solid surface freezing and vitrification.

Solid surface freezing or vitrification (SSF/SSV) can be done by depositing droplets of a sample, e.g., cells in a preservation solution, onto a pre-cooled metal surface. It is used to achieve higher cooling rates and concomitant higher cryosurvival rates compared to immersion of samples into liquid nitrogen. In this study, numerical simulations of SSF/SSV were conducted by modeling the cooling dynamics of droplets of cryoprotective agent (CPA) solutions. It was assumed that deposited droplets attain a cylindrical bottom part and half-ellipsoidal shaped upper part. Material properties for heat transfer simulations including density, heat capacity and thermal conductivity were obtained from the literature and extrapolated using polynomial fitting. The impact of CPA type, i.e., glycerol (GLY) and dimethyl sulfoxide (DMSO), CPA concentration, and droplet size on the cooling dynamics was simulated at different CPA mass fractions at temperatures ranging from -196 to 25 °C. Simulations show that glycerol solutions cool faster compared to DMSO solutions, and cooling rates increase with decreasing CPA concentration. However, we note that material property data for GLY and DMSO solutions were obtained in different temperature and concentration ranges under different conditions, which complicated making an accurate comparison. Experimental studies show that samples that freeze have a delayed cooling response early on, whereas equilibration times are similar compared to samples that vitrify. Finally, as proof of concept, droplets of human red blood cells (RBCs) were cryopreserved using SSV/SSF comparing the effect of GLY and DMSO on cryopreservation outcome. At 20% (w/w), similar hemolysis rates were found for GLY and DMSO, whereas at 40%, GLY outperformed DMSO.

A review of recent developments in the application of nanostructures for sperm cryopreservation.

In the 1970s, sperm cryopreservation was presented as a unique route to fertility preservation. The ability to cryopreserve sperm from all species is challenging. The sperm cryopreservation process encompasses various cellular stresses such as increased osmotic pressure, ice crystal formation, and thermal shock, therefore decreasing the quality of sperm. The nanostructures due to their inherent features such as reactivity, high uptake, active surface area, and antioxidant activity, have contributed to modifying freezing protocols. In this review, the current state of the art with regards to emerging applications of nanotechnology in sperm cryopreservation are reviewed, some of the most promising advances are summarized, and the limitations and advantages are comprehensively discussed.

On the universality of viscosity in supersaturated binary aqueous sugar solutions: Cryopreservation by vitrification.

Nowadays, the physical nature of supersaturated binary aqueous sugar solutions in the vicinity of the glass transition represents a very important issue due to their biological applications in cryopreservation of cells and tissues, food science and stabilization and storage of nano genetic drugs. We present the construction of the Supplemented Phase Diagram and the non-equilibrium nature of the undersaturated-supersaturated kinetic transition. The description of its thermodynamic nature is achieved through the study of behavior of their viscosity as temperature is lowered and concentration increased. In this work, we find a universal character for the viscosities of several sugar water solutions.

Cryoprotective effect of soluble soybean polysaccharides and enzymatic hydrolysates on the myofibrillar protein of Nemipterus virgatus surimi.

Cryoprotective effect and potential mechanism of soluble soybean polysaccharides (SSPS) and enzymatic hydrolysates on surimi was investigated. After hydrolysis, the molecular weight of SSPS significantly decreased, and the hydrolysates prepared by endo-polygalacturonase (EPG-SSPS) was the lowest (154 kDa). Infrared spectrum analysis revealed that enzymatic hydrolysis didn't alter the functional groups of SSPS, but it did augment the exposure to hydroxyl groups. Surimi containing 5 % EPG-SSPS had the lowest freezable water after 20 days of frozen storage. Furthermore, the 5 % EPG-SSPS group manifested the highest metrics in total sulfhydryl (8.0 × 10-5 mol/g), active sulfhydryl content (6.7 × 10-5 mol/g), Ca2+-ATPase activity, and exhibited the lowest level in carbonyl content, surface hydrophobicity (153 µg). Notably, the 5 % EPG-SSPS maintained the stability of protein structure. Conclusively, SSPS enzymatic hydrolysate using endo-polygalacturonase imparted superior cryoprotective effect on the myofibrillar protein of surimi, and the mechanism might be a decrease in molecular weight and exposure of hydroxyl groups.

Impact of cryopreservation on elastomuscular artery mechanics.

Low temperatures slow or halt undesired biological and chemical processes, protecting cells, tissues, and organs during storage. Cryopreservation techniques, including controlled media exchange and regulated freezing conditions, aim to mitigate the physical consequences of freezing. Dimethyl sulfoxide (DMSO), for example, is a penetrating cryoprotecting agent (CPA) that minimizes ice crystal growth by replacing intracellular water, while polyvinyl alcohol (PVA) is a nonpenetrating CPA that prevents recrystallization during thawing. Since proteins and ground substance dominate the passive properties of soft biological tissues, we studied how different freezing rates, storage temperatures, storage durations, and the presence of cryoprotecting agents (5% [v/v] DMSO + 1 mg/mL PVA) impact the histomechanical properties of the internal thoracic artery (ITA), a clinically relevant blood vessel with both elastic and muscular characteristics. Remarkably, biaxial mechanical analyses failed to reveal significant differences among the ten groups tested, suggesting that mechanical properties are virtually independent of the cryopreservation technique. Scanning electron microscopy revealed minor CPA-independent delamination in rapidly frozen samples, while cryoprotected ITAs had better post-thaw viability than their unprotected counterparts using methyl thiazole-tetrazolium (MTT) metabolic assays, especially when frozen at a controlled rate. These results can be used to inform ongoing and future studies in vascular engineering, physiology, and mechanics.