Exploring the Cryopreservation Mechanism and Direct Removal Strategy of TAPS in Red Blood Cell Cryopreservation.

Cryopreservation of red blood cells (RBCs) plays an indispensable role in modern clinical transfusion therapy. Researchers are dedicated to finding cryoprotectants (CPAs) with high efficiency and low toxicity to prevent RBCs from cryopreservation injury. This study presents, for the first time, the feasibility and underlying mechanisms of a novel CPA called tris(hydroxymethyl)aminomethane-3-propanesulfonic acid (TAPS) in RBCs cryopreservation. The results demonstrated that the addition of TAPS achieved a post-thaw recovery of RBCs at 79.12 ± 0.67%, accompanied by excellent biocompatibility (above 97%). Subsequently, the mechanism for preventing RBCs from cryopreservation injury was elucidated. On one hand, TAPS exhibits a significant amount of bound water and effectively inhibits ice recrystallization, thereby reducing mechanical damage. On the other hand, TAPS demonstrates high capacity to scavenge reactive oxygen species and strong endogenous antioxidant enzyme activity, providing effective protection against oxidative damage. Above all, TAPS can be readily removed through direct washing, and the RBCs after washing showed no significant differences in various physiological parameters (SEM, RBC hemolysis, ESR, ATPase activity, and Hb content) compared to fresh RBCs. Finally, the presented mathematical modeling analysis indicates the good benefits of TAPS. In summary, TAPS holds potential for both research and practical in the field of cryobiology, offering innovative insights for the improvement of RBCs cryopreservation in transfusion medicine.

A comparative study of tissue protein synthesis rates in an Antarctic, Harpagifer antarcticus and a temperate, Lipophrys pholis teleost.

The affect of temperature on tissue protein synthesis rates has been reported in temperate and tropical, but not Antarctic fishes. Previous studies have generally demonstrated low growth rates in Antarctic fish species in comparison to temperate relatives and elevated levels of protein turnover. This study investigates how low temperatures effect tissue protein synthesis and hence tissue growth in a polar fish species. Groups of Antarctic, Harpagifer antarcticus and temperate, Lipophrys pholis, were acclimated to a range of overlapping water temperatures and protein synthesis was measure in white muscle (WM), liver and gastrointestinal tract (GIT). WM protein synthesis rates increased linearly with temperature in both species (H. antarcticus 0.16-0.23%.d-1, L. pholis, 0.31-0.76%.d-1), while liver (H. antarcticus 0.24-0.27%.d-1, L. pholis, 0.44-1.03%.d-1) and GIT were unaffected by temperature in H. antarcticus but increased non-linearly in L.pholis (H. antarcticus 0.22-0.26%.d-1, L. pholis, 0.40-0.86%.d-1). RNA to protein ratios were unaffected by temperature in H. antarcticus but increased weakly, in L.pholis WM and liver. In L.pholis, RNA translational efficiency increased significantly with temperature in all tissues, but only in liver in H. antarcticus. At the overlapping temperature of 3 °C, protein synthesis (WM 26%, Liver, 39%, GIT, 35%) and RNA translational efficiency (WM 273%, Liver, 271%, GIT, 300%) were significantly lower in H. antarcticus than L.pholis, while RNA to protein ratios were significantly higher (WM 270%, Liver 170%, GIT 186%). Tissue specific effects of temperature are detectable in both species. This study provides the first evidence, that tissue protein synthesis rates are constrained in Antarctic fishes.

Seasonal shifts in gut microbiota and cold tolerance metrics in a northern population of Reticulitermes flavipes (Blattodea: Rhinotermitidae).

Eastern subterranean termites, Reticulitermes flavipes (Kollar), are widely distributed across North America where they are exposed to a broad range of environmental conditions. However, mechanisms for overwintering are not well understood. Wisconsin is a unique location to study mechanisms of cold tolerance as it represents the northern boundary for persistent R. flavipes populations. In this study, we evaluated seasonal shifts in cold tolerance using critical thermal minimum (CTmin) and supercooling point (SCP) and examined how these measurements correlate to changes in the microbial community of the termite gut. Results showed seasonal acclimatization to cold, which is consistent with the use of behavioral freeze-avoidant mechanisms. However, these insects also demonstrated an increased susceptibility to freezing later in the season, which may be tied to changes in gut microbiota. Our results found shifts in the composition of the gut microbiome in R. flavipes between mid- to late summer and early to late fall. These differences may be suggestive of a change in metabolism to adjust to a period of reduced feeding and increased metabolic stress during overwintering. Specifically, results showed an increased abundance of Methanobrevibacter sp. (Euryarchaeota) associated with cold, which may be indicative of a metabolic shift from acetogenesis to methanogenesis associated with overwintering. Further work is needed focusing on specific contributions of certain gut microbes, particularly their role in metabolic adaptability and in providing protection from oxidative stress associated with changes in environmental conditions.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Polyethylene Glycol and Caspase Inhibitor Emricasan Alleviates Cold Injury in Primary Rat Hepatocytes.

Current methods of storing explanted donor livers at 4°C in University of Wisconsin (UW) solution result in loss of graft function and ultimately leads to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4°C, we investigated the effects of lowering the storage temperature to -4°C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5% PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.

The biomechanical and biological effect of supercooling on cortical bone allograft.

BACKGROUND: The need for a storage method capable of preserving the intrinsic properties of bones without using toxic substances has always been raised. Supercooling is a relatively recently introduced preservation method that meets this need. Supercooling refers to the phenomenon of liquid in which the temperature drops below its freezing point without solidifying or crystallizing. OBJECTIVES: The purpose of this study was to identify the preservation efficiency and applicability of the supercooling technique as a cortical bone allograft storage modality. METHODS: The biomechanical effects of various storage methods, including deep freezing, cryopreservation, lyophilization, glycerol preservation, and supercooling, were evaluated with the three-point banding test, axial compression test, and electron microscopy. Additionally, cortical bone allografts were applied to the radial bone defect in New Zealand White rabbits to determine the biological effects. The degree of bone union was assessed with postoperative clinical signs, radiography, micro-computed tomography, and biomechanical analysis. RESULTS: The biomechanical properties of cortical bone grafts preserved using glycerol and supercooling method were found to be comparable to those of normal bone while also significantly stronger than deep-frozen, cryopreserved, and lyophilized bone grafts. Preclinical research performed in rabbit radial defect models revealed that supercooled and glycerol-preserved bone allografts exhibited significantly better bone union than other groups. CONCLUSIONS: Considering the biomechanical and biological superiority, the supercooling technique could be one of the optimal preservation methods for cortical bone allografts. This study will form the basis for a novel application of supercooling as a bone material preservation technique.

Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

Self-pressurised rapid freezing at arbitrary cryoprotectant concentrations.

Self-pressurised rapid freezing (SPRF) has been proposed as a simple alternative to traditional high-pressure freezing (HPF) protocols for vitrification of biological samples in electron microscopy and cryopreservation applications. Both methods exploit the circumstance that the melting point of ice reaches a minimum when subjected to pressure of around 210 MPa, however, in SPRF its precise quantity depends on sample properties and hence, is generally unknown. In particular, cryoprotective agents (CPAs) are expected to be a factor; though eschewed by many SPRF experiments, vitrification of larger samples notably cannot be envisaged without them. Thus, in this study, we address the question of how CPA concentration affects pressure inside sealed capillaries, and how to design SPRF experiments accordingly. By embedding a fibre-optic probe in samples and performing Raman spectroscopy after freezing, we first present a direct assessment of pressure build-up during SPRF, enabled by the large pressure sensitivity of the Raman shift of hexagonal ice. Choosing dimethyl sulphoxide (DMSO) as a model CPA, this approach allows us to demonstrate that average pressure drops to zero when DMSO concentrations of 15 wt% are exceeded. Since a trade-off between pressure and DMSO concentration represents an impasse with regard to vitrification of larger samples, we introduce a sample architecture with two chambers, separated by a partition that allows for equilibration of pressure but not DMSO concentrations. We show that pressure and concentration in the fibre-facing chamber can be tuned independently, and present differential scanning calorimetry (DSC) data supporting the improved vitrification performance of two-chamber designs. Lay version of abstract for 'Self-pressurised rapid freezing at arbitrary cryoprotectant concentrations' Anyone is familiar with pipes bursting in winter because the volume of ice is greater than that of liquid water. Less well known is the fact that inside a thick-walled container, sealed and devoid of air bubbles, this pressure build-up will allow a fraction of water to remain unfrozen if the sample is also cooled sufficiently rapidly far below the freezing point. This phenomenon has already been harnessed for specimen preparation in microscopy, where low temperatures are useful to immobilise the sample, but harmful if ice formation occurs. However, specimen preparation cannot always rely on this pressure-based effect alone, but sometimes requires addition of chemicals to inhibit ice formation. Not enough is known directly about how these chemicals affect pressure build-up: Indeed, rapid cooling below the freezing point is only possible for small sample volumes, typically placed inside sealed capillaries, so that space is generally insufficient to accommodate a pressure sensor. By means of a compact sensor, based on an optical fibre, laser and spectrometer, we present the first direct assessment of pressure inside sealed capillaries. We show that addition of chemicals reduces pressure build-up and present a two-chambered capillary to circumvent the resulting trade-off. Also, we present evidence showing that the two-chambered capillary design can avoid ice formation more readily than a single-chambered one.

Technologies for Vitrification Based Cryopreservation.

Cryopreservation is a unique and practical method to facilitate extended access to biological materials. Because of this, cryopreservation of cells, tissues, and organs is essential to modern medical science, including cancer cell therapy, tissue engineering, transplantation, reproductive technologies, and bio-banking. Among diverse cryopreservation methods, significant focus has been placed on vitrification due to low cost and reduced protocol time. However, several factors, including the intracellular ice formation that is suppressed in the conventional cryopreservation method, restrict the achievement of this method. To enhance the viability and functionality of biological samples after storage, a large number of cryoprotocols and cryodevices have been developed and studied. Recently, new technologies have been investigated by considering the physical and thermodynamic aspects of cryopreservation in heat and mass transfer. In this review, we first present an overview of the physiochemical aspects of freezing in cryopreservation. Secondly, we present and catalog classical and novel approaches that seek to capitalize on these physicochemical effects. We conclude with the perspective that interdisciplinary studies provide pieces of the cryopreservation puzzle to achieve sustainability in the biospecimen supply chain.

Assisted reproductive technologies (ARTs) in Mithun (Bos frontalis): What progress has been made so far? An overview.

Mithun, a unique bovine species, endemic to parts of North East India and plays an important role in the socioeconomic, cultural and religious fabrics of the local tribal population. To date, Mithuns are reared in a traditional free-range system by communities and increased deforestation, agricultural commercialization, disease outbreaks and indiscriminate slaughtering of elite Mithun for table purposes have significantly decreased its habitat and the elite Mithun population. Greater genetic gain is achieved with the implementation and effective use of assisted reproductive technologies (ARTs); however, presently it is limited to organized Mithun farms. At a slow pace, Mithun farmers are adopting semi-intensive rearing systems and interest in the use of ARTs is gradually escalating in Mithun husbandry. This article reviews the current status of ARTs such as semen collection and cryopreservation, estrus synchronization and timed artificial insemination (TAI), multiple ovulation and embryo transfer and in vitro embryo production and future perspectives in Mithun. Mithun semen collection and cryopreservation have been standardized, and estrus synchronization and TAI are suitable technologies that can be easily implemented under field conditions in near future. The establishment of an open nucleus-breeding system under community participatory mode along with the introduction of the ARTs is an alternative to the traditional breeding system for rapid genetic improvement of Mithun. Finally, the review considers the potential benefits of ARTs in Mithun and future research should include the use of these ARTs which will provide additional opportunities for improved breeding regimens in Mithun.

Cryogenic storage increases the longevity of butternut (Juglans cinerea, L.) seed embryogenic axes.

Butternut (Juglans cinerea L.), a species listed as endangered, is currently undergoing rapid decline due to habitat loss and the introduction of Ophiognomonia clavigignenti-juglandacearum, a non-native pathogen causing butternut canker. The decline of butternut has led to the development of cryobiological methods for the ex-situ conservation of the species since viability is not maintained using conventional methods. In this study, we assess the survivability and growth of butternut embryogenic axes into plantlets after 7 years of cryopreservation. Results show that cryopreservation is a highly effective method for the long-term storage of embryogenic axes since both survival and subsequent acclimatization into plantlets was successful with an average of 76% survival and 71% acclimatization, respectively. These results surpass the actual duration viability for non-cryogenic storage of butternut seed and we hope that future testing will result in longer term success of this cryobiological method and provide much needed germplasm for future restoration.

Infrared spectroscopic analysis of hydrogen-bonding interactions in cryopreservation solutions.

BACKGROUND: In this study we investigated hydrogen bonding interactions in hydrated and frozen solutions of different cryoprotective agents (CPAs) including dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, and trehalose. We also investigated the effect of CPAs on ice crystal growth during storage and correlated this with storage stability of liposomes. METHODS: FTIR spectroscopy was used to study hydrogen bonding interactions in CPA solutions in H2O and D2O, and their thermal response was analyzed using van 't Hoff analysis. The effect of CPAs on ice crystal growth during storage was investigated by microscopy and correlated with storage stability of liposomes encapsulated with a fluorescent dye. RESULTS: Principal component analyses demonstrated that different CPAs can be recognized based on the shape of the OD band region only. Chemically similar molecules such as glycerol and ethylene glycol closely group together in a principal component score plot, whereas trehalose and DMSO appear as condensed separated clusters. The OH/OD band of CPA solutions exhibits an overall shift to higher wavenumbers with increasing temperature and changed fractions of weak and strong hydrogen interactions. CPAs diminish ice crystal formation in frozen samples during storage and minimize liposome leakage during freezing but cannot prevent leakage during frozen storage. CONCLUSIONS: CPAs can be distinguished from one another based on the hydrogen bonding network that is formed in solution. DMSO-water mixtures behave anomalous compared to other CPAs that have OH groups. CPAs modulate ice crystal formation during frozen storage but cannot prevent liposome leakage during frozen storage.

Metabolic and secretory recovery of slow frozen-thawed human ovarian tissue in vitro.

Within the options available for fertility preservation, cryopreservation of ovarian cortical tissue has become an important technique. Freezing and thawing procedures have been optimized to preserve tissue integrity and viability. However, the improvement of the tissue retransplantation is currently of great interest. Rapid angiogenesis is needed at the retransplantation site to accomplish sufficient blood supply to provide oxygen and nutrients. Many studies address this issue. However, we need to understand the physiology of the thawed tissue to gain further understanding of the complexities of the procedure. As freezing and thawing generally impairs cellular metabolism, we aimed to characterize the changes in metabolic activity and secretion of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) of frozen-thawed ovarian cortical tissue over time. Biopsy punches of ovarian cortical tissue from patients undergoing fertility preservation were maintained in culture without freezing or after a slow-freezing and thawing procedure. VEGF-A secretion was measured after 48 h by ELISA. To examine temporary changes, metabolic activity was assessed for both fresh and frozen-thawed tissue of the same patient. Metabolic activity and VEGF-A secretion were measured at 0, 24 and 48 h in culture. Thawed ovarian cortical tissue secreted significantly less VEGF-A compared to fresh ovarian cortical tissue within 48 h of culture. After thawing, metabolic activity was significantly reduced compared to fresh ovarian cortex but over the course of 48 h, the metabolic activity recovered. Similarly, VEGF-A secretion of thawed tissue increased significantly over 48 h. Here, we have shown that it takes 48 h for ovarian cortical tissue to recover metabolically after thawing, including VEGF-A secretion.

Transmembrane Water Transport and Intracellular Ice Formation of Human Umbilical Vein Endothelial Cells During Freezing.

Long-term cryopreservation of human umbilical vein endothelial cells (HUVECs) is important and beneficial for a variety of biomedical research and applications. In this study, we investigated HUVEC's cryobiological characteristics and parameters that are indispensable for predicting and determining an optimal cooling rate to prevent lethal intracellular ice formation (IIF) and severe cell dehydration during the cryopreservation processes. The parameters include cell membrane hydraulic conductivity (i.e., cell membrane water permeability), Lp, cell membrane water permeability activation energy, Elp, and osmotically inactive volume of a cell Vb. Cryomicroscopy was used to study the IIF phenomena and cell volume excursion at various cooling rates, 1, 10, and 20°C/min, respectively, based on which the cryobiological parameters were determined using biophysical and mathematical models. Results from this research work laid an important cryobiological foundation for the optimization of HUVEC's cryopreservation conditions.

Enhanced Control over Ice Nucleation Stochasticity Using a Carbohydrate Polymer Cryoprotectant.

Metastable supercooling has emerged as a transformative technique for ice-free biopreservation, but issues of stability inherent to the stochastic nature of ice formation have thus far limited its translation out of the laboratory. In this work, we explore the influence of the bio-based carbohydrate polymer FucoPol on aqueous supercooling using an isochoric nucleation detection technique. We show that FucoPol, a high-molecular-weight, fucose-rich polysaccharide, which has previously been shown to reduce average ice crystal sizes after nucleation, also induces a concentration-dependent stabilization of metastable supercooled water, as evidenced by both a significant reduction in nucleation stochasticity (i.e., the spread in temperatures over which the system will nucleate upon cooling) and a corresponding increase in the predicted induction time of nucleation. FucoPol is found to confine the stochasticity of ice nucleation to a narrow, well-defined band of temperatures roughly one-third as wide as that of pure water under identical conditions. Importantly, this substantial reduction in stochasticity is accompanied by only a minimal (<1 °C) change in the average nucleation temperature, suggesting that this effect is distinct from colligative freezing point depression. Reducing and characterizing the stochasticity of aqueous supercooling is essential to the engineering design of practical biopreservation protocols, and the results reported herein suggest that high-viscosity polymer systems may provide a powerful and largely unexplored lever by which to manipulate metastable-equilibrium phase change kinetics at subzero temperatures.

Life in the freezer: protein metabolism in Antarctic fish.

Whole-animal, in vivo protein metabolism rates have been reported in temperate and tropical, but not Antarctic fish. Growth in Antarctic species is generally slower than lower latitude species. Protein metabolism data for Antarctic invertebrates show low rates of protein synthesis and unusually high rates of protein degradation. Additionally, in Antarctic fish, increasing evidence suggests a lower frequency of successful folding of nascent proteins and reduced protein stability. This study reports the first whole-animal protein metabolism data for an Antarctic fish. Groups of Antarctic, Harpagifer antarcticus, and temperate, Lipophrys pholis, fish were acclimatized to a range of overlapping water temperatures and food consumption, whole-animal growth and protein metabolism measured. The rates of protein synthesis and growth in Antarctic, but not temperate fish, were relatively insensitive to temperature and were significantly lower in H. antarcticus at 3°C than in L. pholis. Protein degradation was independent of temperature in H. antarcticus and not significantly different to L. pholis at 3°C, while protein synthesis retention efficiency was significantly higher in L. pholis than H. antarcticus at 3°C. These results suggest Antarctic fish degrade a significantly larger proportion of synthesized protein than temperate fish, with fundamental energetic implications for growth at low temperatures.

Aquaporins and Animal Gamete Cryopreservation: Advances and Future Challenges.

Cryopreservation is globally used as a method for long-term preservation, although freeze-thawing procedures may strongly impair the gamete function. The correct cryopreservation procedure is characterized by the balance between freezing rate and cryoprotective agents (CPAs), which minimizes cellular dehydration and intracellular ice formation. For this purpose, osmoregulation is a central process in cryopreservation. During cryopreservation, water and small solutes, including penetrating cryoprotective agents, cross the plasma membrane. Aquaporins (AQPs) constitute a family of channel proteins responsible for the transport of water, small solutes, and certain gases across biological membranes. Thirteen homologs of AQPs (AQP0-12) have been described. AQPs are widely distributed throughout the male and female reproductive systems, including the sperm and oocyte membrane. The composition of the male and female gamete membrane is of special interest for assisted reproductive techniques (ART), including cryopreservation. In this review, we detail the mechanisms involved in gamete cryopreservation, including the most used techniques and CPAs. In addition, the expression and function of AQPs in the male and female gametes are explored, highlighting the potential protective role of AQPs against damage induced during cryopreservation.

Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges.

Differentiation of human induced pluripotent stem cells (hiPSCs) generates cell phenotypes valuable for cell therapy and personalized medicine. Successful translation of these hiPSC-derived therapeutic products will rely upon effective cryopreservation at multiple stages of the manufacturing cycle. From the perspective of cryobiology, we attempted to understand how the challenge of cryopreservation evolves between cell phenotypes along an hiPSC-to-sensory neuron differentiation trajectory. Cells were cultivated at three different stages to represent intermediate, differentiated, and matured cell products. All cell stages remained ≥90% viable in a dimethyl sulfoxide (DMSO)-free formulation but suffered ≥50% loss in DMSO before freezing. Raman spectroscopy revealed higher sensitivity to undercooling in hiPSC-derived neuronal cells with lower membrane fluidity and higher sensitivity to suboptimal cooling rates in stem cell developmental stages with larger cell bodies. Highly viable and functional sensory neurons were obtained following DMSO-free cryopreservation. Our study also demonstrated that dissociating adherent cultures plays an important role in the ability of cells to survive and function after cryopreservation.

Natural deep eutectic systems, an emerging class of cryoprotectant agents.

This work aimed at evaluating the potential of using natural deep eutectic systems (NADES) as cryoprotectant agents (CPAs). Several combinations between natural primary metabolites that have been identified in animals that live in extreme cold climates were prepared. All systems showed very little cytoxicity towards L929 cells at concentrations high as 1-2 M. Moreover, this cell line was highly tolerant to 10% (w/v) of NADES when compared to Me2SO. To test NADES as CPAs, two cell lines were used, L929 and HacaT cells. After freeze/thawing cycle, it was possible to observe that for L929 cells, NADES presented similar behaviour to Me2SO. For Hacat cell line a significant improvement on post-thawing recovery was observed. Moreover, the results presented herein showed that NADES do not need to be removed from the freezing media after thawing the cells, which is a great advantage of these materials. Additionally, we have shown that NADES can act as CPA when cells are frozen at -20 °C. In overall, the results demonstrate the high potential of NADES to be used in cryobiology as alternative CPAs.

Winter is coming: the future of cryopreservation.

The preservative effects of low temperature on biological materials have been long recognised, and cryopreservation is now widely used in biomedicine, including in organ transplantation, regenerative medicine and drug discovery. The lack of organs for transplantation constitutes a major medical challenge, stemming largely from the inability to preserve donated organs until a suitable recipient is found. Here, we review the latest cryopreservation methods and applications. We describe the main challenges-scaling up to large volumes and complex tissues, preventing ice formation and mitigating cryoprotectant toxicity-discuss advantages and disadvantages of current methods and outline prospects for the future of the field.