Nanoscopy of single antifreeze proteins reveals that reversible ice binding is sufficient for ice recrystallization inhibition but not thermal hysteresis.

Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.

Stabilization of insect cell membranes and soluble enzymes by accumulated cryoprotectants during freezing stress.

Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.

Proteomic Analysis of Differences in the Freezability of Porcine Sperm Identifies α-Amylase As a Key Protein.

To investigate the mechanisms underlying the differences in the freezability of boar semen, Yorkshire boars with freezing-tolerant semen (YT, n = 3), Yorkshire boars with freezing-sensitive semen (YS, n = 3), Landrace boars with freezing-tolerant semen (LT, n = 3), and Landrace boars with freezing-sensitive semen (LS, n = 3) were selected for this study. Their sperm was subjected to protein extraction, followed by data-independent acquisition proteomics and functional bioinformatics analysis. A total of 3042 proteins were identified, of which 2810 were quantified. Some key KEGG pathways were enriched, such as starch and sucrose metabolism, carbohydrate digestion and absorption, mineral absorption, the HIF-1 signaling pathway, and the necroptosis pathways. Through PRM verification, we found that several proteins, such as α-amylase and epididymal sperm-binding protein 1, can be used as molecular markers of the freezing resistance of boar semen. Furthermore, we found that the addition of α-amylase to cryoprotective extender could significantly improve the post-thaw motility and quality of boar semen. In summary, this study revealed some molecular markers and potential molecular pathways contributing to the high or low freezability of boar sperm, identifying α-amylase as a key protein. This study is valuable for optimizing boar semen cryopreservation technology.

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure.

BACKGROUND: The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. RESULTS: Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. CONCLUSIONS: The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

Cryopreservation of cerebrospinal fluid cells preserves the transcriptional landscape for single-cell analysis.

Cerebrospinal fluid (CSF) matrix biomarkers have become increasingly valuable surrogate markers of neuropsychiatric diseases in research and clinical practice. In contrast, CSF cells have been rarely investigated due to their relative scarcity and fragility, and lack of common collection and cryopreservation protocols, with limited exceptions for neurooncology and primary immune-based diseases like multiple sclerosis. the advent of a microfluidics-based multi-omics approach to studying individual cells has allowed for the study of cellular phenotyping, intracellular dynamics, and intercellular relationships that provide multidimensionality unable to be obtained through acellular fluid-phase analyses. challenges to cell-based research include site-to-site differences in handling, storage, and thawing methods, which can lead to inaccuracy and inter-assay variability. In the present study, we performed single-cell RNA sequencing (10x Genomics) on fresh or previously cryopreserved human CSF samples from three alternative cryopreservation methods: Fetal Bovine Serum with Dimethyl sulfoxide (FBS/DMSO), FBS/DMSO after a DNase step (a step often included in epigenetic studies), and cryopreservation using commercially available Recovery© media. In comparing relative differences between fresh and cryopreserved samples, we found little effect of the cryopreservation method on being able to resolve donor-linked cell type proportions, markers of cellular stress, and overall gene expression at the single-cell level, whereas donor-specific differences were readily discernable. We further demonstrate the compatibility of fresh and cryopreserved CSF immune cell sequencing using biologically relevant sexually dimorphic gene expression differences by donor. Our findings support the utility and interchangeability of FBS/DMSO and Recovery cryopreservation with fresh sample analysis, providing a methodological grounding that will enable researchers to further expand our understanding of the CSF immune cell contributions to neurological and psychiatric disease.

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

STUDY QUESTION: Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? SUMMARY ANSWER: LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. WHAT IS KNOWN ALREADY: Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. STUDY DESIGN, SIZE, DURATION: Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. MAIN RESULTS AND THE ROLE OF CHANCE: The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3m protein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. LIMITATIONS, REASONS FOR CAUTION: The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. WIDER IMPLICATIONS OF THE FINDINGS: LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Improved Cryopreservation of Human Induced Pluripotent Stem Cell (iPSC) and iPSC-derived Neurons Using Ice-Recrystallization Inhibitors.

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.

Impact of lower concentrations of dimethyl sulfoxide on cryopreservation of autologous hematopoietic stem cells: a systematic review and meta-analysis of controlled clinical studies.

BACKGROUND AIMS: Cryopreservation of hematopoietic stem cells (HSCs) is crucial for autologous transplantation, cord blood banking and other special circumstances. Dimethyl sulfoxide (DMSO) is used most commonly for cryopreserving HSC products but can cause infusional toxicities and affect cell viability and engraftment after transplant. A systematic review of controlled studies using lower concentrations of DMSO to cryopreserve HSC products in clinical transplant studies is needed to determine the effect of reducing DMSO concentrations on post-thaw cell viability, initial engraftment and adverse effects on patient health. METHODS: All studies identified in our systematic search (to July 11, 2023) examining the use of cryopreserved peripheral blood stem cells (PBSCs) for autologous stem cell transplantation (AHCT) were included. Meta-analysis was performed to determine how varying the concentration of DMSO during cryopreservation effects post-thaw cell viability, initial engraftment and adverse effects on patient health. RESULTS: A total of 1547 studies were identified in our systematic search, with seven published articles meeting eligibility for inclusion in meta-analysis. All patients underwent AHCT using (PBSCs) to treat hematologic malignancies. The viability of CD34+ cells post thaw was greater when cryopreserved with 5% DMSO compared with 10% DMSO, with lower rates of adverse side effects in patients. DMSO concentration had minimal impact on rates of initial engraftment. Significant heterogeneity in outcome reporting was observed and the potential for bias was identified in all studies. CONCLUSIONS: Reducing the concentration of DMSO from 10% to 5% during cryopreservation of autologous PBSCs may improve cell viability and reduce DMSO-associated adverse effects in patients undergoing AHCT. Data from more studies with similar patients and standard outcome reporting are needed to increase confidence in our initial observations. PROTOCOL REGISTRATION: PROSPERO; registration number CRD42023476809 registered November 8, 2023.

A 3-step method for preparing cryopreserved samples of apheresis products for post-thaw analysis yields a higher percentage of viable cells.

BACKGROUND: Standard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab-induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities. STUDY DESIGN AND METHODS: We developed a novel method whereby thawed samples were diluted step-wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution. RESULTS: Twenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter. DISCUSSION: Slow, step-wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

Human Natural Killer Cells Cryopreserved without DMSO Sustain Robust Effector Responses.

Natural killer (NK) cell-based immunotherapy has benefitted from the multiple strengths that NK cells offer in adoptive transfer settings, not the least of which is their safety and potential for allogeneic use. Such use, however, necessitates the cryopreservation of NK cell-based therapy products to support logistical efforts in deploying these cells in different locations, decentralized from the point of collection or manufacturing. DMSO, the most commonly used cryoprotective agent (CPA), has been effective in protecting immune cells during freezing and thawing, but its ability to induce molecular and genetic changes to immune cells as well as its toxicity has stimulated interest in alternative CPAs. However, replacing DMSO's ability to act intracellularly has been difficult, and the sensitivity of human peripheral blood-derived NK cells to freezing and thawing-induced damage has meant that investigations into the potential of replacing DMSO are lacking. As a first step toward establishing the feasibility of cryopreserving human NK cells with CPAs' alternative to DMSO, we investigate the potential of using noncell-penetrating and cell-penetrating CPAs to recover NK cells post-thaw without DMSO. Here, we find that cryoprotection using cell-penetrating CPAs can retain the viability of human peripheral blood-derived NK cells to a comparable degree to DMSO. In addition, non-DMSO-cryopreserved human NK cells were as cytotoxic as those cryopreserved with DMSO and displayed a comparable level of surface markers of activation. In summary, we present the first example of the potential of developing non-DMSO CPA formulations that could be deployed in future cell therapy regimens.

Optimization of Annealing and Metal Films Radiofrequency Heating Procedures for Vitrified Umbilical Arteries.

Vitrification is well known for its application in the cryopreservation of blood vessels, which will address the supply-demand imbalance in vascular grafts for the treatment of cardiovascular disease. Thermal stress damage and devitrification injury in umbilical arteries (UAs) require attention and resolution during the vitrification and rewarming process. In this study, we validated several cooling annealing protocols with temperatures (-130 to -100 °C) and annealing duration durations (10-20 s). Among these, the umbilical artery subjected to annealing at -110 °C for 10 s exhibited the most favorable glass transition and retained 93% of its elastic modulus (0.625 ± 0.030 MPa) compared to the fresh group. Extended annealing temperatures and durations can effectively reduce thermal stress damage, leading to improved mechanical properties by minimizing temperature gradients during cooling. Furthermore, three metal radiofrequency methods were utilized for rewarming, including the use of additional metal films and different magnetic field strengths (20, 25 kA/m). Metal radiofrequency (adding an extra metal film for cryoprotectants rewarming, 20 kA/m) achieved faster and more uniform rewarming, preserving the extracellular matrix (ECM), collagen fibers, and elastic fibers without significant differences compared to the fresh group (P < 0.05). Moreover, its preservation of the biomechanical properties of blood vessels was better than that of water bath heating. Theoretical analysis supports these findings, indicating that radiofrequency heating (RFH) with metal films reduces temperature gradients and thermal stresses during arterial rewarming. RFH contributes to the cryopreservation and clinical application of large-lumen biomaterials, overcoming challenges associated with vascular vitrification and rewarming.

Optimization of Deep Eutectic Solvents Enables Green and Efficient Cryopreservation.

Cryopreservation presents significant opportunities for biomedical applications including cell therapy, tissue engineering, and assisted reproduction. Dimethyl sulfoxide (DMSO), the most commonly used cryoprotectant (CPA), can be added to cells to prevent cryogenic damage. However, the toxicity of cryoprotectants restrains its further development in many areas with safety concerns such as clinical treatment. Therefore, the development of low-toxicity cryoprotectants is essential for medical research. This work reports deep eutectic solvents (DES) as naturally biocompatible osmoprotectants for green and efficient cryopreservation of human umbilical cord mesenchymal stem cells (HuMSC), which may be an ideal alternative to DMSO. The six types of DESs were explored for thermal properties, toxicity, and permeability in cells. Raman spectroscopy and viscosity studies showed that DES exhibited an improved hydrogen-bonding system as the temperature decreased. By optimizing the freezing process (cooling rate, incubation time, and loading procedure) of DES, the viability of mouse embryonic fibroblast cells (NIH-3T3) after thawing was significantly improved. The HuMSC were successfully preserved with no significant difference (p > 0.05) in cell viability (94.65%) after thawing compared with DMSO, which preserved the cell differentiation function and improved the cell proliferation rate. The mechanism of DES in cryopreservation was investigated, and it was found that DES could bind water molecules and effectively inhibit the growth of ice crystals during ice recrystallization, reducing mechanical damage to cells. This study highlights the excellent performance of DES as a low-toxicity CPA for stem cell preservation, which may be a significant advance for future clinical cell therapy.

Plunge-Freezing Cryopreservation of Tendons.

Allograft transplantation is an important method for tendon reconstruction after injury, and its clinical success highly relies on the storage and transportation of the grafts. Cryopreservation is a promising strategy for tendon storage. In this study, we report a novel cryopreservation agent (CPA) formulation with a high biocompatibility for tendon cryopreservation. Mainly composed of natural zwitterionic betaine and the biocompatible polymer poly(vinylpyrrolidone) (PVP), it exhibited ideal abilities to depress the freezing point and inhibit ice growth and recrystallization. Notably, after cryopreservation via plunge-freezing for 1 month, Young's modulus (144 MPa, 98% of fresh tendons) and ultimate stress (46.7 MPa, 99% of fresh tendons) remained stable, and the cross-linking of collagen microfibers, protein structures, and glycosaminoglycan (GAG) contents changed slightly. These results indicate that the formulation (5 wt % betaine and 5 wt % PVP in phosphate-buffered saline, PBS solution) effectively maintains the biomechanical properties and tissue structure. This work offers a novel cryopreservation method for tendons and may also provide insights into the long-term preservation of various other tissues.

External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres.

RESEARCH QUESTION: Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device? DESIGN: A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos. RESULTS: A series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage. CONCLUSIONS: Cryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.

Screening of Hydrophilic Polymers Reveals Broad Activity in Protecting Phages during Cryopreservation.

Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. Exceptions were poly(methacrylic acid) and poly(acrylic acid), which can inhibit phage-infection with bacteria, making post-thaw recovery challenging to assess. A particular benefit of a polymeric cryopreservation formulation is that the polymers do not function as carbon sources for the phage hosts (bacteria) and hence do not interfere with post-thaw measurements. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.

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.

Antifreeze Polysaccharides from Wheat Bran: The Structural Characterization and Antifreeze Mechanism.

Exploring a novel natural cryoprotectant and understanding its antifreeze mechanism allows the rational design of future sustainable antifreeze analogues. In this study, various antifreeze polysaccharides were isolated from wheat bran, and the antifreeze activity was comparatively studied in relation to the molecular structure. The antifreeze mechanism was further revealed based on the interactions of polysaccharides and water molecules through dynamic simulation analysis. The antifreeze polysaccharides showed distinct ice recrystallization inhibition activity, and structural analysis suggested that the polysaccharides were arabinoxylan, featuring a xylan backbone with a majority of Araf and minor fractions of Manp, Galp, and Glcp involved in the side chain. The antifreeze arabinoxylan, characterized by lower molecular weight, less branching, and more flexible conformation, could weaken the hydrogen bonding of the surrounding water molecules more evidently, thus retarding the transformation of water molecules into the ordered ice structure.

Genomic and functional evaluation of exopolysaccharide produced by Liquorilactobacillus mali t6-52: technological implications.

BACKGROUND: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. RESULTS: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain's adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. CONCLUSIONS: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain's safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.