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Cryopreservation is a pivotal technique in safeguarding genetic material across diverse species, despite its inherent challenges linked to induced spermatozoa damage, notably apoptosis and lipid peroxidation (LPO). Given the insufficient antioxidant defense of spermatozoa against LPO, there is a rising interest in integrating additional additives into extenders to ameliorate mammalian semen quality. Among these additives, flavonoids have garnered considerable attention due to their potent antioxidative properties. Hence, our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH = ) supplementation in the cryopreservation medium to protect canine sperm against the damaging impacts of freezing and ensure the preservation of their reproductive potential. Semen was collected from five Beagle stud dogs and then pooled. Then, the sample was divided into 7 groups, each treated with 1) 0 mM, 2) 0.1 mM FL, 3) 0.2 mM FL, 4) 0.4 mM FL, 5) 0.1 mM 3-OH = , 6) 0.2 mM 3-OH = , 7) 0.4 mM 3-OH = . Semen samples were subjected to cryopreservation in French straws and glycerol as a cryoprotectant. In the frozen thawed semen, sperm motility parameters by CASA system and sperm membrane integrity, acrosome status, mitochondrial activity, DNA fragmentation, early apoptosis with capacitation, and LPO were assessed using flow cytometry just after thawing (0 h) and 4 h post thaw. Results reveal significant increase in the proportion of live spermatozoa with undamaged acrosomes in the FL 0.1 and 3-OH = 0.2 groups at 0 h post thaw. At this time point, 3-OH = 0.1 significantly reduced the DNA fragmentation index (DFI) compared to the FL 0.1 and 0.2 groups. However, after the next 4 h, 3-OH = 0.4 exhibited the lowest (P < 0.05) DFI compared to FL 0.2 and 3-OH = 0.1. Additionally, 3-OH = 0.4 showed the highest (P < 0.05) proportion of non apoptotic and non capacitated spermatozoa compared to FL 0.1 0 h post-thaw. Simultaneously, the same group demonstrated significant reduction in apoptotic and capacitated sperm cells, at 0 h and 4 h post-thaw. Moreover, 3-OH = at 0.1 (0 h and 4 h) and 0.2 mM (4 h) significantly enhances the proportion of live sperm without LPO post thaw. Whitin the FL groups, only 0.4 FL significantly increased the percentage of live sperm without LPO. No significant effect of the tested substances was observed on sperm motility, cell membrane integrity, or mitochondrial activity. These findings highlight the promising role of flavone and 3-hydroxyflavone in enhancing sperm resilience during cryopreservation, suggesting their protective function against acrosome damages, capacitation, apoptosis and lipid peroxidation., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to -4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Cryopreservation of oocytes is an important tool for preserving genetic resources and for farm animals breeding. Processes taking place during vitrification affect oocytes and result in their reduced developmental capacity and lower fertilisation rates of cryopreserved oocytes. Further improvement in cryopreservation techniques is still required. Several authors already summarized the actual state and perspectives of oocyte cryopreservation as well as potential approaches to improve their development after thawing. The aim of this review is to specify factors affecting cryotolerance of mammalian oocytes, especially bovine in vitro matured oocytes, and to identify the areas, where more efforts were made to improve the success of oocyte cryopreservation. These factors include oocyte lipid content, membrane composition, mRNA protection, cytoskeleton stabilization and application of such potential stimulators of cell cryotolerance as antioxidants, growth factors or antifreeze proteins., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Cryopreservation of fish gonadal tissue is an important technique for preserving genetic variability. However, this technique involves the use of cryotubes, plastic containers with low degradability that are expensive and difficult to obtain in certain parts of the world. Therefore, this study aimed to evaluate the efficiency of gelatin and hypromellose hard capsules as a sustainable and accessible alternative container to the cryotube for vitrification of zebrafish (Danio rerio) gonadal tissue. The gonadal tissues (testicular or ovarian) were vitrified in cryotubes, hard-gelatin, and hard-hypromellose capsules. Gelatin capsules exhibited comparable efficacy to cryotubes in preserving spermatogonia viability (33.03 ± 10.03 % and 37.96 ± 8.35 %, respectively), whereas hypromellose capsules showed decreased viability (18.38 ± 2.09 %). Immature oocyte viability remained unaffected by the capsule materials, with no difference compared to cryotubes at all oocyte stages (Primary Growth: p < 0.0001; Cortical Alveolar: p < 0.0001; Vitellogenic: p < 0.0001). Mitochondrial activity and lipid peroxidation demonstrated no difference among cryotubes and capsules for both gonadal tissues. However, antioxidant activity was notably higher in gelatin capsules (Testes: 147.2 ± 32.32 μg; Ovary: 87.98 ± 10.91 μg) than in cryotubes (Testes: 81.04 ± 26.05 μg; Ovary: 54.35 ± 11.23 μg) and hypromellose capsules (Testes: 62.36 ± 17.10 μg; Ovary: 63.96 ± 7.51 μg), likely due to the inherent antioxidant properties of gelatin. The results obtained in this study demonstrate that the cryotube can be replaced by gelatin capsules for vitrification of both gonadal tissues of zebrafish, being a sustainable and accessible alternative as it is a low-cost and environmentally friendly container., Competing Interests: Declaration of competing interest TSF and DPSJ filed (September/2022) a patent (BR10202201781) application on the methodology described in the study. The remaining authors have no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Non-permeable disaccharides are widely used as cryoprotectant agents due to their low cytotoxicity, but their protective effect is insufficient when the disaccharides are present only extracellularly. On the other hand, cryoprotectant agent (CPA)-free cryopreservation has been recently achieved by instantaneously inkjet-freezing cells as tiny droplets. However, CPA-free cryopreservation requires skilled handling operations due to instability of the vitreous water without the CPA. In this study, the effectiveness of separately adding two types of disaccharides in inkjet freezing of 3T3 cells was evaluated and the following results were obtained. First, trehalose showed the highest effect at 0.57 M, twice the plasma osmolarity, with a maximum cell viability of over 90 % when freezing 70 pL droplets. However, higher concentrations of trehalose decreased cell viability due to damage caused by dehydration. Similarly, sucrose gave cell viability close to 90 % at 0.57 M with 70 pL droplets, and higher concentrations decreased cell viability. Next, the relationship between minimum trehalose concentrations to prevent intracellular and extracellular ice crystal formation and droplet size was analyzed. The results indicated that trehalose of less than 0.57 M was able to inhibit intracellular ice crystal formation even in the largest droplet used in this study, 450 pL, while trehalose of nearly 0.57 M was required to inhibit extracellular ice crystal formation in the smallest droplet, 70 pL. In other words, the suppression of extracellular ice crystals by the addition of CPA was shown to be crucial in improving the viability of inkjet superflash freezing., Competing Interests: Declaration of Competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3'UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Probiotics offer health advantages when consumed in adequate quantities. As ongoing research identifies promising new strains, ensuring their viability and functionality through simple preservation methods is vital for success within the probiotic industry. This study employed a factorial design to investigate the combined effects of four cryoprotectants [C1: MRS broth + 14 % (w/v) glycerol, C2: Aqueous solution containing 4 % (w/v) trehalose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate, C3: Aqueous solution containing 10 % (w/v) skimmed milk and 4 % (w/v) sodium glutamate, C4: Aqueous solution containing 4 % (w/v) sucrose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate] and three methods of preservation (P1: -86 °C freezing, P2: -196 °C liquid nitrogen freezing, and P3: storing at 4 °C after lyophilization) on the cell viability of three potentially probiotic strains over 12 months. Pediococcus sp P15 and Weissella cibaria ml6 had the highest viability under treatments C3 and C2, after 12 months of storage, respectively. Meanwhile, Lactococcus lactis ml3 demonstrated the highest viability in both treatments C2 and C4 (P ≤ 0.05). According to the results freezing, either P1 or P2, is the most effective preservation method for P. sp P15 and W. cibaria ml6. Meanwhile, L. lactis ml3 showed the highest colony count under treatment (P1) after 12 months of storage (P ≤ 0.05). Among the tested conditions, P. sp P15 and L. lactis ml3 exhibited the highest viability and bile salt resistance when stored under P1C1. For W. cibaria ml6, the optimal storage condition was P2C2 (frozen in liquid nitrogen with cryoprotectant C2)., (Copyright © 2024. Published by Elsevier Inc.)

The fundamental interactions between plant cells and cryoprotectants during vitrification are understudied in the field of plant cryopreservation. Within this area of research, real time cryoprotectant permeation into plant cells is even less documented. In this study, we monitor the real time permeation of individual cryoprotectants into rice callus cells when in mixtures with other cryoprotectants. Specifically, we use coherent anti-Stokes Raman scattering (CARS) microscopy to observe the permeation of individually deuterated DMSO, ethylene glycol, and glycerol in plant vitrification solution 2 (PVS2) by probing vibrational frequencies that correspond to C-D stretching modes of the cryoprotectant molecules. Additionally, we measure cell plasma membrane responses to PVS2 exposure using brightfield microscopy. We conclude that the permeation of PVS2 components into plant cells occurs faster than the first cell plasma membrane responses observed and therefore permeation and cell plasma membrane response do not appear to be directly correlated. In addition, we observe that cryoprotectant permeation into plant cells occurs more quickly and more uniformly when cryoprotectants are in PVS2 solution than when they are in single component aqueous solutions., (Copyright © 2024 Society for Cryobiology. All rights reserved.)

Freezing of artichoke is a promising alternative to storing it in brine and canning. The perishable vegetable was vacuum infused with inulin to improve freezing tolerance. Artichokes with and without inulin were frozen by static, air blast and individual quick freezing (IQF) methods and thawed by microwave, 25 °C and 4 °C temperature levels at each month of 6-months storage. Process conditions were evaluated by multivariate analysis of variance (MANOVA) and were found significant on the quality parameters. Inulin infusion better conserved the a w , color, texture, ascorbic acid and overall integrity of artichokes during frozen storage. Inulin incorporation and IQF showed mutual positive effect on drip loss. Polyphenol oxidase (PPO) activity values fitted to 2nd order kinetic and the highest residuals were determined in static freezing. PPO showed alleviating effect on total phenolic content. Vacuum impregnation caused a color difference prior to freezing, but was found effective for maintaining color during storage. As a result, the use of quick freezing techniques together with the addition of cryoprotectant was effective in the preservation of artichoke quality attributes during frozen storage., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell-cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 μM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 μM naringenin compared to 200 μM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150 μM naringenin compared to all groups except 100 μM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 μM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 μM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 μM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03 % (114/200) vs. 46.50 ± 0.04 % (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 μM concentration., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest regarding the publication of this research article., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Spermatogonia cryopreservation can be a strategy for future conservation actions. The neotropical Siluriformes Pseudopimelodus mangurus was already classified as vulnerable on the Red List of Threatened Species. P. mangurus spermatogonial cells were isolated, assessed, and cryopreserved. Fragments of the testis were enzymatically dissociated, purified using Percoll density gradient, and submitted to differential plating. Fractionated cells were evaluated by microscopy, ddx4 (vasa) relative expression, and alkaline phosphatase activity. Cryopreservation was conducted using ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and propanediol at 1 M, 1.5 M, and 2 M. Cell viability was evaluated and cell concentration was determined. Cell fractions from 20 % and 30 % Percoll gradient bands showed the highest concentrations of spermatogonia. The fraction mix showed 54 % purity and 93 % viability. After differential plating, 60 % purity and 92 % viability were obtained. Spermatogonial cells showed high alkaline phosphatase activity compared to spermatocytes and spermatids. The relative spermatogonial ddx4 expression from the Percoll density gradient was about twice as high as in samples from the testis and the differential plating. The increased ddx4 expression indicated the enrichment of spermatogonial cells by density gradient step and dead cells expressing ddx4 in differential plating, or ddx4 decreasing expression during cell culture. For this reason, cells from the Percoll gradient were chosen for cryopreservation. Propanediol at 1 M demonstrated the best condition for spermatogonial cell cryopreservation, presenting 98 % viability, while dimethylacetamide at 2 M represented the least favorable condition, with approximately 47 % viability. These findings are essential for P. mangurus spermatogonial cell cryopreservation, aiming to generate a spermatogonia cryobank for future conservation efforts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Grapevine (Vitis vinifera L.) crops are continuously exposed to biotic and abiotic stresses, which can cause genetic and epigenetic alterations. To determine the possible effects of grapevine cryopreservation on the regulation of DNA demethylase genes, this work studied the expression of DNA demethylase genes in cryopreserved and post-cryopreserved grapevine tissues. V. vinifera DNA demethylases were characterized by in silico analysis, and gene expression quantification was conducted by RT‒qPCR. Three DNA demethylase sequences were found: VIT&#95;13s0074g00450 (VvDMT), VIT&#95;08s0007g03920 (VvROS1), and VIT&#95;06s0061g01270 (VvDML3). Phylogenetic analysis revealed that the sequences from V. vinifera and A. thaliana had a common ancestry. In the promoters of responsive elements to transcription factors such as AP-2, Myb, bZIP, TBP, and GATA, the conserved domains RRM DME and Perm CXXC were detected. These responsive elements play roles in the response to abiotic stress and the regulation of cell growth. These data helped us characterize the V. vinifera DNA demethylase genes. Gene expression analysis indicated that plant vitrification solution 2 (PVS2) treatment does not alter the expression of DNA demethylase genes. The expression levels of VvDMT and VvROS1 increased in response to cryopreservation by vitrification. Furthermore, in post-cryopreservation, VvROS1 was highly induced, and VvDML3 was repressed in all the treatment groups. Gene expression differences between different treatments and tissues may play roles in controlling methylation patterns during gene regulation in tissues stressed by cryopreservation procedures and in the post-cryopreservation period during plant growth and development., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

When cells are cryopreserved, they go through a freezing process with several distinct phases (i.e., cooling until nucleation, ice nucleation, ice crystal growth and cooling to a final temperature). Conventional cell freezing approaches often employ a single cooling rate to describe and optimize the entire freezing process, which neglects its complexity and does not provide insight into the effects of the different freezing phases. The aim of this work was to elucidate the impact of each freezing phase by varying different process parameters per phase. Hereto, spin freezing was used to freeze Jurkat T cells in either a Me 2 SO-based or Me 2 SO-free formulation. The cooling rates before ice nucleation and after total ice crystallization impacted cell viability, resulting in viability ranging from 26.7% to 52.8% for the Me 2 SO-free formulation, and 22.5%-42.6% for the Me 2 SO-based formulation. Interestingly, the degree of supercooling upon nucleation did not exhibit a significant effect on cell viability in this work. However, the rate of ice crystal formation emerged as a crucial factor, with viability ranging from 2.4% to 53.2% for the Me 2 SO-free formulation, and 0.3%-53.2% for the Me 2 SO-based formulation, depending on the freezing rate. A morphological study of the cells post-cryopreservation was performed using confocal microscopy, and it was found that cytoskeleton integrity and cell volume were impacted, depending on the formulation-process parameter combination. These findings underscore the importance of scrutinizing all cooling and freezing phases, as each phase impacted post-thaw viability in a distinct way, depending of the specific formulation used., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Subcooled liquid nitrogen and nitrogen slush are often considered for high-speed cooling, but their preparation and maintenance are not easy. To address this issue, a unique device was designed to prepare subcooled liquid argon (SLA) using liquid nitrogen (LN). The cooling process was mathematically modeled to predict the preparation time. If the interlayer space between LN and liquid argon is filled with nitrogen gas, liquid argon could be cooled to 3.5 K subcooling within 1 h. If the interlayer is filled with air, 2 h are required to achieve the same subcooled state. An additional 1000 mL of LN was required for the preparation of 600 mL of 3.5 K SLA. The cooling tests of 3 μL microdroplets in 3 mm-6 mm capillary quartz tubes were duplicated to evaluate the potential of SLA. It was found that the cooling rate of microdroplet in the 3.5 K subcooled SLA is very close to that in the 3 K subcooled LN, higher than that in the saturated LN. The convenience of preparation and maintenance of SLA can make it good choice of cryogen for cryopreservation of biomaterials., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

The most widely used method of platelet cryopreservation requires the addition of dimethyl sulfoxide (DMSO; Me2SO) as a cryoprotective agent (CPA) and pre-freeze removal of Me2SO before freezing to mitigate toxicity. However, alternative CPAs such as deep eutectic solvents (DES), which are less toxic could simplify this process. The aim of this study was to determine the effectiveness of a Proline-Glycerol (Prol-Gly 1:3) DES as a platelet CPA. Platelets were cryopreserved at -80 °C using 10 % Prol-Gly 1:3 (DES; n = 6), or in the absence of a cryoprotectant (no CPA; n = 6). Platelets were also cryopreserved according to the gold-standard blood-banking method using 5.5 % Me2SO (n = 6), with centrifugation and pre-freeze removal of the excess Me2SO. Platelet quality was assessed by flow cytometry and thromboelastography (TEG). Post-thaw recovery was similar between the three groups. The abundance of labile platelet glycoproteins GPIbα and GPVI were highest in the DES group, however, markers of activation (CD62P and annexin-V) were also higher in this group. In terms of function, the strength of the clot (maximum amplitude; TEG) and extent of clot retraction was better with DES platelets compared to no CPA, but lower than Me2SO platelets. DES provides a cryoprotective advantage to platelets when compared to no CPA. Importantly, when compared to Me2SO platelets, most quality parameters were similar in DES platelets. The major advantage with using a DES is biocompatibility, therefore it does not need to be removed prior to transfusion. This greatly simplifies the freezing and thawing process, avoiding the toxic effects of Me2SO., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

We studied the impact of modulating cholesterol levels in zebrafish sperm plasma membranes using cholesterol-loaded methyl-β-cyclodextrin (CLC) and unloaded methyl-β-cyclodextrin (MβC). Zebrafish sperm were treated with these substances before cryopreservation, and post-thaw sperm motility and in vitro fertilization (IVF) rates were compared between treated and untreated samples. Our findings indicate that adding cholesterol to sperm membranes increases post-thaw motility, motile cell count, and motile cell survival within a 0.5-4.0 mg per 1.2 × 10 8  cell concentration range. Conversely, depleting cholesterol using MβC at 1.0 and 2.0 mg per 1.2 × 10 8  cells reduced these parameters. On average, all CLC-treated sperm samples produced a 15 % higher IVF rate compared to untreated sperm. Including CLC in the extender before cryopreservation is beneficial for post-thaw sperm quantity and quality in zebrafish., Competing Interests: Declaration of competing interest No competing personal or financial interests exist. We have not used any generative AI or AI assisted technologies during the preparation of this manuscript, except for the MS-Word Editor grammar and spell check., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to assess the effect of lyophilized freezing extenders, which can be stored at room temperature, on stallion post-thaw sperm total motility (TM). Ejaculates of 28 stallions were frozen with four different extenders: two commercial freezing extenders offered worldwide and two novel lyophilized extenders (STAR and MX3), and two different cryopreservation protocols (CP1 with an equilibration period of 20 min. and CP2 with an equilibration period of 60 min.). The TM was assessed after thaw. Mean TM did not show significant differences between cryopreservation protocols within each extender. Mean TM was greater in samples diluted with STAR than in samples diluted with Botucrio (P ˂ 0.05), but no significant differences were observed for this variable between the other studied extenders. From all evaluated samples, twenty ejaculates showed the greatest TM when using the lyophilized extenders and the CP1. Thus, lyophilized extenders are a promising option for stallion sperm cryopreservation and have the advantage of storage and distribution at room temperature for at least one year., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cryoprotective agents play a critical role in minimizing cell damage caused by ice formation during cryopreservation. However, high concentrations of CPAs are toxic to cells and tissues. Required concentrations of CPAs can be reduced by utilizing higher cooling and warming rates, but insight into the thermophysical properties of biological solutions in the vitrification method is necessary for the development of cryopreservation protocols. Most studies on thermophysical properties under ultra-rapid cooling conditions have been qualitatively based on visualization. Differential scanning calorimetry methods are ideal for studying the behavior of biomaterials in various freezing conditions quantitatively and accurately, though previous studies have been predominantly restricted to slower cooling rates. Here, we developed an ultra-rapid cooling method for DSC that can achieve minimal cooling rates exceeding 2000 °C/min. We investigated the thermophysical vitrification behavior of ternary solutions of phosphate buffer saline (1X), dimethyl sulfoxide or glycerol and ice blocking polymers (X-1000 or Z-1000). We quantified the impact of solute concentration on ice crystal formation during rapid cooling. Our findings support the expectation that increasing the solute concentration reduces the amount of ice formation, including devitrification. Devitrification increases from 0 % to 40 % (v/v) Me 2 SO and then reduces significantly. The relative amounts of devitrification to the total ice formation are 0 %, 60 %, 0 % in 20 %, 40 %, 60 % (v/v) Me 2 SO, and 2 %, 48 %, 49 % in 20 %, 40 %, 60 % (v/v) glycerol, respectively. The results suggest that at low concentrations, such as below 20 % (v/v) for Me 2 SO or glycerol, increasing the warming rate after ultra-rapid freezing is not essential to eliminate devitrification. Furthermore, ice blocking polymers do not reduce ice formation substantially and cannot eliminate devitrification under ultra-rapid cooling conditions. In conclusion, our results provide insights into the impact of solute concentration on ice formation and devitrification during rapid cooling, which can be practical for optimizing cryopreservation protocols., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryopreservation is highly desired for long-term maintenance of the viability of living biosamples, while effective cell cryopreservation still relies heavily on the addition of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). However, the intrinsic toxicity of DMSO is still a bottleneck, which could not only cause the clinical side effect but also induce cell genetic variants. In the meantime, the addition of FBS may bring potentially the risk of pathogenic microorganism contamination. The liquid marbles (LMs), a novel biotechnology tool for cell cryopreservation, which not only have a small volume system that facilitated recovery, but the hydrophobic shell also resisted the harm to cells caused by adverse environments. Previous LM-based cell cryopreservation relied heavily on the addition of FBS. In this work, we introduced acidic polyaspartic acid and polyglutamic acid as cryoprotectants to construct LM systems. LMs could burst in an instant to facilitate and achieve ultrarapid recovery process, and the hydrophilic carboxyl groups of the cryoprotectants could form hydrogen bonds with water molecules and further inhibit ice growth/formation to protect cells from cryoinjuries. The L929 cells could be well cryopreserved by acidic polyamino acid-based LMs. This new biotechnology platform is expected to be widely used for cell cryopreservation, which has the potential to propel LMs for the preservation of various functional cells in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ischemia is a major limiting factor in Vascularized Composite Allotransplantation (VCA) as irreversible muscular injury can occur after as early as 4-6 h of static cold storage (SCS). Organ preservation technologies have led to the development of storage protocols extending rat liver ex vivo preservation up to 4 days. Development of such a protocol for VCAs has the added challenge of inherent ice nucleating factors of the graft, therefore, this study focused on developing a robust protocol for VCA supercooling. Rodent partial hindlimbs underwent subnormothermic machine perfusion (SNMP) with several loading solutions, followed by a storage solution with cryoprotective agents (CPA) developed for VCAs. Storage occurred in suspended animation for 24h and VCAs were recovered using SNMP with modified Steen. This study shows a robust VCA supercooling preservation protocol in a rodent model. Further optimization is expected to allow for its application in a transplantation model, which would be a breakthrough in the field of VCA preservation., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

In medicine, ovarian tissue cryopreservation exists for fertility preservation of cancer patients. In fact, ovarian tissue frozen for subsequent thawing and re-transplantation can be contaminated with cancer cells. Therefore, investigations on the effect of cryopreservation on the post-thawed viability of such cells are relevant. Speed of warming is a key parameter of cell cryopreservation. However, the data about comparative viability of cancer cells cryopreserved with different parameters of warming are limited. The aim of our investigations was to assess the malignancy of cryopreserved cancer cells after conventional cooling followed by relatively slow and quick speed of warming. In vitro cultured breast cancer cells of lines ZR-75-1 and MD0MD-231 in form of compacted fragments (as a model of solid tumors) were frozen following a protocol usually used for freezing of ovarian tissue (6 % ethylene glycol+6 % glycerol+0.15 M sucrose, -0.3 °C/min). Cells were warmed by two routine regimes of warming: at 37 °C ("slow" warming) and 100 °C ("quick" warming). Biological properties of cells were investigated: viability, proliferation rate, 2D- and 3D-migration, transmembrane movement and invasion. Quick warming at 100 °C in comparison with slow warming at 37 °C exhibited significantly higher cell survival for MDA-MB-231 cells: 70.1 % vs. 63.2 % and for ZR-75-1 86.8 % vs. 82.9 %, respectively. The cell motility including 2D movement and 3D transmembrane migration were higher after quick thawing at 100 °C. Invasive abilities of cells after cryopreservation were higher than that of fresh (non-treated cells). Both thawing regimes showed a similar rate of cell proliferation. Cryopreservation procedures, and especially this one with quick thawing, increase malignancy of ZR-75-1 and MDA-MB-231 breast cancer cells and risk of metastasis., (Copyright © 2024. Published by Elsevier Inc.)

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Some synthetic polymers can be used at low concentrations to reduce the toxicity of conventional cryoprotectant agents. In this study we investigated whether the addition of synthetic polymers to a conventional cryoprotectant solution would improve the cryopreservation of bovine ovarian tissue. Freshly collected ovaries from ten adult crossbred cows were incised using a scalpel in the frontal section. From each cow, ovarian cortical slices of 1 mm thickness were divided into 30 fragments of 3 × 3 mm, of which 10 served as fresh controls, 10 were vitrified with conventional cryoprotectant agents (2.93 M glycerol, 27 % w/v; 4.35 M ethylene glycol, 27 % w/v), and 10 were vitrified using the same cryoprotectant agents in addition to synthetic polymers (0.2 % PVP K-12, 0.2 % SuperCool X-1000 ™ w/v and 0.4 % SuperCool Z-1000 ™ w/v). After warming, histology was used to assess follicular quantity and integrity, while in vitro culture of mechanically isolated follicles encapsulated in an alginate matrix was performed for 15 days to assess their growth and hormonal production. Vitrified ovarian tissues presented abnormal morphology, a higher percentage of atretic follicles, and their isolated follicles had lower survival rates and lower frequency of antrum formation during in vitro culture compared to those from fresh tissue. At the end of culture, the follicles that had been cryopreserved produced less estradiol and progesterone than the fresh ones. The addition of synthetic polymers during tissue vitrification did not modify any of these parameters. We conclude that, under the conditions of this study, the use of this combination of synthetic polymers for tissue vitrification did not enhance the preservation of the morphological or functional integrity of bovine ovarian follicles., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Glycans are carbohydrates present in every organism that bind to specific molecules such as lectins, a diverse group of proteins. Glycans are vital to cell proliferation and protein trafficking. In addition, embryogenesis is a critical phase in the development of marine organisms. This study investigated the effects of chilling and cryoprotective agents (CPAs) on glycans in the embryos of Stenopus hispidus. The glycan profiles of embryos of S. hispidus at the heartbeat stage were analyzed using lectin arrays. The results of analyses revealed that mannose was the most abundant glycan in the S. hispidus embryos; mannose is crucial to cell proliferation, providing the energy required for embryonic growth. Additionally, the results reveled that chilling altered the content of several glycans, including fucose and Gla-GlcNAc. Chilling may promote monosaccharide accumulation, facilitating osmotic regulation of cells and signal molecules to aid S. hispidus embryos in adapting to cold conditions. Changes were also observed in the lectins NPA, orysata, PALa, ASA, discoidin II, discoidin I, UDA, PA-IIL, and PHA-P after the samples were treated with different CPAs. DMSO may minimize cell damage during exposure to chilling by preserving cell structures, membrane properties, and functions. The present study is the first to investigate the profiles and functions of glycans in shrimp embryos subjected to low-temperature injuries. This study enhances the understanding of cell reproduction during embryogenesis and provides valuable information for the study of glycans in embryos., Competing Interests: Declaration of competing interest I declare that the authors have no competing interests as defined by the journal, or other interests that might be perceived to influence the results and/or discussion reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Cryopreservation of goat spermatozoa is challenging due to several factors, including one of the most essential, i.e., oxidative stress. It is particularly essential in goat semen due to its scanty ejaculate volume and high sperm concentration. This leaves a narrow sperm-to-seminal plasma ratio owing to marginal antioxidant support; moreover, semen extension further dilutes the antioxidant level, leading to an imbalance of oxidant-antioxidant equilibrium. The present study aimed to evaluate the effect of quercetin on curtailing oxidative stress and its reflection on the post-thaw survivability and membrane integrity of goat spermatozoa. For this study, six bucks were selected. Six ejaculates from each buck totaling 36 ejaculates were collected, which were then split into five parts; furthermore, each part was added with a semen extender having a particular concentration of additive. Group C without quercetin and T 1 containing Vitamin E at 3 mmol/mL were considered the control and positive control respectively, whereas T 2 , T 3 , and T 4 contain 10, 20, and 30 μmol/mL of Quercetin respectively. The final sperm concentration of each group was kept at 200 × 10 6 spermatozoa/mL. All groups were subjected to equilibration at 4 °C for 4 h, then filled in French mini (0.25 mL) straws, followed by sealing and cryopreservation. Samples after 72 h of cryopreservation were subjected to evaluation of plasma membrane integrity and viability through staining, acrosomal integrity, and mitochondrial membrane activity through flow cytometry. Evaluation of sperm kinematics as well as the oxidant-antioxidant status of sperm (ROS and nitric oxide) and seminal plasma (SOD, CAT, GPx, FRAP, and lipid peroxidation through MDA estimation) were also carried out. Quercetin, when supplemented at 20 μmol/mL in buck semen extender, significantly (p < 0.01) improved cryopreserved sperm functions in terms of plasma membrane integrity, viability, acrosomal integrity, mitochondrial membrane activity, and sperm kinematics of buck semen. Similarly, Quercetin supplementation at 20 μmol/mL significantly reduced reactive oxygen and nitrogen species (RONS) in sperm and improved the antioxidant status of seminal plasma, which was indicated by reduced oxidative damage and improved the antioxidant status of buck semen. In conclusion, Quercetin at 20 μmol/mL reduced oxidative stress, improved semen antioxidant status, and improved sperm membranes integrity and kinematics., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead 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., Competing Interests: Declaration of competing interest Some authors declare competing interests. Drs. B.E. Uygun, K. Uygun, S.N. Tessier, and M. Toner have patent applications relevant to this study. Drs. B.E. Uygun, K. Uygun, S.N. Tessier, and M. Toner have a financial interest in and serve on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing high subzero organ preservation technology. Competing interests for MGH investigators are managed by the MGH and MGB in accordance with their conflict-of-interest policies., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

A cryopreservation protocol has been developed for embryogenic cultures (ECs) of Castanea mollissima, an important economic species of the Castanea genus in China. We achieved 100 % regrowth when ECs were treated with Plant Vitrification Solution 2 (PVS2) for 30, 60 and 90 min on ice. Optimal PVS2 treatment for cryopreservation was determined to be 30 min on ice based on the highest biomass regrowth after thawing. Fluorescein diacetate (FDA) staining could rapidly and reliably determine post-thaw cell viability and its use facilitated the optimization of the cryopreservation protocols. Although the proliferation rate of the re-established ECs remained largely unchanged compared to non-cryopreserved ECs, the capacity of the re-established ECs to differentiate (on two media) into somatic embryos nearly doubled to approximately 2200-2300 globular somatic embryos per 1 g of re-established ECs. Based on cell cluster size analysis, this enhanced growth is primarily attributed to the presence of significantly greater cell clusters with a diameter of 100-200 μm, which have the highest level of differentiation ability. In order to understand the increased embryogenic potential following cryopreservation, we analyzed the expression of key genes related to somatic embryogenesis. Genes CmWUS and CmABP1 were downregulated while CmLEC1, CmAGL15, CmGRF2, and CmFUS3 were upregulated in re-established ECs when compared to non-cryopreserved ECs., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author declares that there are no conflicts of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

The freezability of chicken spermatozoa is low, therefore, effective cryoprotectants is desiderated. Antifreeze proteins (AFPs) are widely found in cold-tolerant species and help them to survive in freezing environments. This study was the first to evaluate the effects of different concentrations of plant-originated antifreeze glycoprotein (AFGP) (0, 0.1, 1, and 5 μg/mL) on post-thawed sperm motion characteristics, morphology, mitochondrial function, antioxidant activity, and fertilizing potential in chickens. Results showed that the total motility of 0.1 to 1 μg/mL AFGP groups were significantly higher than those of the 5 μg/mL AFGP group (P < 0.05). The post-thawed sperm viability of 0.1 μg/mL AFGP group was significantly higher than any of test groups (P < 0.05). Higher abnormal morphology rate of post-thawed sperm was observed in the control group (0 μg/mL AFGP) than in the 0.1, 1, and 5 μg/mL AFGP groups (P < 0.05). The concentrations of malondialdehyde (MDA) decreased gradually with the increase of AFGP concentration. ATP was significantly higher in the 0.1 and 1 μg/mL AFGP groups than those of control and any of test groups (P < 0.05). The 0.1 to 1 μg/mL AFGP groups had increased mitochondrial membrane potential (MMP) level (P > 0.05). The 0.1 μg/mL AFGP group had the highest average fertility (61.36%) compared with control group (57.02%) and any of test groups of chickens at 31 wk of age, and the 1 μg/mL AFGP group had the highest average fertility (37.72%) compared with control group (21.73%) and any of test groups of chickens at 65 wk of age. In conclusion, the results from this study suggest lower concentration of AFGP (0.1-1 μg/mL) showed positive effect for sperm function. This study inspires the continuous evaluation and seeking right way of adopting different kinds of AFPs in rooster semen cryopreservation., Competing Interests: DISCLOSURES The authors declare that they have no conflicts of interest to this work., (Copyright © 2024. Published by Elsevier Inc.)

Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 μg/mL (G0, 0 μM); 0.5 μg/mL (G0.5, 0.15 μM), or 1 μg/mL (G1, 0.3 μM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 μg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest. Parts of the figures were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen., Competing Interests: Declaration of competing interest A number of products used in this study are proprietary (Botupharma) and Drs. Marco A. Alvarenga and Frederico O. Papa are co-owners of Botupharma., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cryopreserved testicular tissue offers a promising method to restore fertility in male infertility patients. Current protocols rely on high concentrations of penetrating cryoprotectants (pCPAs), such as dimethyl sulfoxide (DMSO), which necessitating complex washing procedures and posing risks of toxicity. Hydrogel encapsulation presents a non-toxic alternative for cellular cryopreservation. This study investigates the effects of various types, concentrations, and thicknesses of hydrogel encapsulation on the cryopreservation of mouse testicular tissue. Testicular tissues loaded with varying concentrations of DMSO were encapsulated in alginate or gelatin-methacryloyl (GelMA) hydrogels. We evaluated hydrogels as potential CPAs to reduce pCPA concentrations and determine optimal combinations for cryopreservation. Post-cryopreservation, tissues were cultured using organ culture methods to assess spermatogenesis progression. Cryomicroscopy and differential scanning calorimetry (DSC) were used to examine ice crystal formation, melting enthalpy, and non-freezing water content in different hydrogels during cooling. Results indicate that 3 % alginate or 5 % GelMA hydrogel with thin encapsulation optimally preserves mouse testicular tissue. Using 20 % DMSO in 5 % GelMA thin encapsulation showed comparable apoptosis rates, improved morphology, higher mitochondrial activity, and enhanced antioxidant capacity compared to conventional 30 % DMSO without encapsulation. This suggests that hydrogel encapsulation reduces pCPA concentration by 10 %, thereby mitigating toxic damage. Hydrogel encapsulation can reduce basement membrane shrinkage of testicular tissue during cryopreservation. Moreover, frozen tissues remained viable with preserved germ cells after being cultured for one week on alginate methacryloyl (AlgMA) hydrogel using the gas-liquid interphase method. Cryomicroscopy and DSC studies confirmed the hydrogel's ability to inhibit ice crystal growth. In conclusion, this study introduces novel strategies for male fertility preservation and advances cryopreservation technology for clinical applications in assisted reproduction., Competing Interests: Declaration of Competing Interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Semen freezing and storing has been widely used in reproductive biotechnology, being applied to certain males of livestock breeds or animal species with economic value such as the Angora goat. The development of a semen extender with the cryoprotective agents can prevent the deterioration of sperm parameters after thawing. This study aimed to investigate lipid mixtures (from a liposome kit, Lps) and melatonin (Mel) at different doses to prevent the deterioration of sperm parameters and to provide the cryoprotective effects on sperm DNA. The Angora goat ejaculates were collected and pooled. They were divided into seven equal volumes, and each of them was diluted with the extenders of the experimental groups with additives (Lps 321.99 μg/mL, Lps 841.33 μg/mL, Mel 0.25 mM, Mel 1 mM, Lps 321.99 μg/mL + Mel 1 mM, Lps 841.33 μg/mL + Mel 0.25 mM) and no additives (control group). After the freeze-thawing process, motility, viability, acrosome integrity, DNA double-strand breaks, and abnormal DNA integrity were assessed for different extender groups. It was determined that the use of Lps alone at low dose or the combination of Lps and Mel had significant cryoprotective effects on motility, viability, acrosome integrity, and DNA damage in Angora goat sperm. This study will help us to understand the effects of Lps and Mel used alone or in combination at different doses and which doses give the optimum spermatological parameter rates following the freeze-thawing process, and hence it will shed light on further studies., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Nowadays the significant role of biobanks in medical, diagnostic, industrial, and environmental research is well known. Bacterial biobanks could be used as a good resource for designing new treatments, biomedical and industrial researches, and laboratory diagnostics. To have a collection of bacteria from clinical samples and maintain their long-term viability, their preservation needs appropriate protective agents, like cryoprotectants and lyoprotectants. In this study, we collected and characterized Gram-negative and Gram-positive bacteria carrying important antibiotic resistance markers from different clinical samples of hospitalized children. Sucrose (10%), skimmed milk (10%), skimmed milk plus sodium glutamate (10% + 1%), and bovine serum albumin (BSA, 10%) were used as lyoprotectants during the freeze-drying procedure. The survival rate of the lyophilized samples was calculated by dilution plating and measuring the colony forming unit (CFU) after 3 months of storage. The culture analysis results indicated that 25 of the 27 studied bacterial genera (Dilutions 10 -3 to 10 -6 ), including Shigella, Methicillin-resistant S. aureus, Acinetobacter spp., Escherichia spp., Pseudomonas spp., Klebsiella spp., Enterococcus spp., were recovered in cultured fractions from all preservation conditions, while 2 genera were only detected in a single preservation condition (2/27, 7.4%). Based on the results, sucrose (10%) and skimmed milk (10%) presented the most protective features. The survival rates varied significantly according to types of the bacteria. Collectively, our results showed a diversity in the recovery of different bacterial genera after lyophilization. While statistically no significant difference was detected among the studied protective agents, sucrose (10%) and skimmed milk (10%) exhibited more effective lyoprotective properties for both Gram-positive and Gram-negative bacteria among the clinical isolates in our study., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Oocyte cryopreservation is useful for human fertility treatment and strain preservation in both experimental and domestic animals. However, the embryonic development of vitrified rat oocytes was lower than that of vitrified embryos. To increase the viability of vitrified oocytes, intracellular ice formation during cooling and warming must be prevented. Rapid warming is important to prevent ice formation. Furthermore, suppressing the spontaneous activation of oocytes is also important because vitrification promotes the spontaneous activation of rat oocytes, and thus compromise developmental competence of the gametes. MG132, a proteasome inhibitor, suppresses the spontaneous activation of rat oocytes. Here, we examined the effects of rapid warming and MG132 treatment on the survival and embryonic development of vitrified rat oocytes. The warming rate was adjusted by changing the vitrification solution volume and warming solution temperature. The survival rate of oocytes vitrified in 10 μL solution and warmed at 50 °C (94%) was significantly higher than that of oocytes vitrified in 100 μL and 10 μL solution and warmed at 37 °C (49% and 81%, respectively). Furthermore, the rate of embryonic development of vitrified oocytes treated with MG132 during vitrification, warming, and intracytoplasmic sperm injection (ICSI) (44%) was significantly higher than that of untreated gametes (10%). Offspring were obtained after transferring embryos derived from MG132-treated vitrified oocytes (14%). Altogether, the survivability of vitrified rat oocytes increased by rapid warming, and MG132 improved embryonic development after ICSI., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

In this clinical study, we investigated the potential of melatonin (MT) supplementation in the freeze-thaw medium used for cryopreserved human oocytes. In total, 152 patients who underwent in vitro fertilization between January 2020 and December 2022 were included and categorized into different groups as follows: the donor group, comprising 108 patients who donated their oocytes, with 34 patients using a vitrification and warming medium supplemented with MT (D-MT subgroup) and 74 patients using conventional medium without MT (D-0 subgroup); and the autologous group, comprising 38 patients who used their own oocytes, with 19 patients using medium supplemented with MT (A-MT subgroup) and 19 patients using medium without MT (A-0 subgroup). After thawing, the surviving oocytes in the D-MT and A-MT subgroups and D-0 and A-0 subgroups were cultured in a fertilization media with and without 10 -9  MMT for 2.5 h, respectively, followed by intracytoplasmic sperm injection insemination, embryo culture, and transfer. The survival, cleavage, high-quality embryo, clinical pregnancy, ongoing pregnancy, and implantation rates were significantly higher in the D-MT subgroup than in the D-0 subgroup (all P < 0.05). Similarly, the survival, fertilization, high-quality embryo, and high-quality blastocyst rates were significantly higher in the A-MT subgroup than in the A-0 subgroup (all P < 0.05). These findings indicate that MT addition during cryopreservation can enhance the development of vitrified-warmed human oocytes and improve clinical outcomes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

The cryopreservation of endothelial cell monolayers is an important step that bridges the cryopreservation of cells in suspension to that of tissues. Previous studies have identified clear distinctions in freezing mechanisms between cells in suspension and in monolayers, as well as developed novel protocols for monolayer cryopreservation. Recently, our group has shown that human umbilical vein endothelial cell (HUVEC) and porcine corneal endothelial cell (PCEC) monolayers grown on Rinzl plastic substrate can be cryopreserved in 5% dimethyl sulfoxide, 6% hydroxyethyl starch, and 2% chondroitin sulfate, following a slow-cooling protocol (-1 °C/min) with rapid plunge into liquid nitrogen from -40 °C. However, membrane integrity assessments were done immediately post thaw, which may result in an overestimation of cell viability due to possible delayed injury responses. Here, we show that for the optimal protocol condition of plunge at the -40 °C interrupt temperature, HUVEC and PCEC monolayers exhibited no significant immediate post-thaw injuries nor delayed injury responses during the 24-h post-thaw overnight culture period. HUVEC monolayers experienced no significant impact to their natural growth rate during the post-thaw culture, while PCEC monolayers experienced significantly higher growth than the unfrozen controls. The difference in the low-temperature responses between HUVEC and PCEC monolayers was further shown under high temperature plunge conditions. At these suboptimal plunge temperatures, HUVEC monolayers exhibited moderate immediate membrane injury but a pronounced delayed injury response during the 24-h post-thaw culture, while PCEC monolayers showed significant immediate membrane injury but no additional delayed injury response during the same period. Therefore, we provide further validation of our group's previously designed endothelial monolayer cryopreservation protocol for HUVEC and PCEC monolayers, and we identify several cell-type-specific responses to the freezing process., Competing Interests: Declaration of competing interest J.A.W. Elliott and L.A. Marquez-Curtis are co-inventors on a filed patent: N. Eskandari, J.A.W. Elliott, L.E. McGann, J.A. Nychka, L.A. Marquez-Curtis, “Cryopreservation of cell monolayers”., (Copyright © 2024. Published by Elsevier Inc.)

Over half of the world's buffalo (Bubalus bubalis) inhabit India, and buffaloes frequently encounter health challenges that resist conventional treatments, prompting the exploration of alternative therapeutic strategies. One promising approach is stem cell therapy, particularly multipotent mesenchymal/stromal stem cells (MSCs). These cells have shown significant efficacy in addressing various diseases in livestock that exhibit resistance to conventional therapies. Adipose tissue-derived MSCs (ADSCs) have garnered attention due to their accessibility and robust expansion potential. The current study comprehensively characterises buffalo ADSCs (bADSCs), confirming their identity as MSCs capable of differentiating into diverse cell lineages-the identified characteristics position bADSCs as promising candidates for applications in regenerative medicine, applicable in veterinary contexts. Notably, the study established that a cryoprotective solution comprising 10 % dimethyl sulfoxide and 90 % fetal bovine serum is optimal for preserving bADSCs. This cryoprotective solution maintains vital parameters, including viability, apoptosis, senescence, cell adherence, adherent cell viability, metabolic and clonogenic efficiency, and the activity of reactive oxygen species and trilineage differentiation potential following thawing. These findings lay the foundation for developing a cryo-banking system for bADSCs. Subsequent research efforts are focused on exploring the therapeutic potential of bADSCs in specific disease models and clinical settings. The outcomes of such investigations may pave the way for innovative and effective treatments, further enhancing our understanding of the regenerative capabilities of bADSCs., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Vitrification under isochoric (constant-volume or volumetrically confined) conditions has emerged as an intriguing new cryopreservation modality, but the physical complexities of the process confound straight-forward interpretation of experimental results. In particular, the signature pressure-based ice detection used in many isochoric techniques becomes paradoxical during vitrification, wherein the emergence of a sharp increase in pressure reliably indicates the presence of ice, but avoidance of this increase does not necessarily indicate its absence. This phenomenon arises from the rich interplay between thermochemical and thermovolumetric effects in isochoric systems, and muddies efforts to confirm the degree to which a sample has vitrified. In this work, we seek to aid interpretation of isochoric vitrification experiments by calculating thermodynamic limits on the maximum amount of ice that may form without being detected by pressure, and by clarifying the myriad physical processes at play. Neglecting kinetic effects, we develop a simplified thermodynamic model accounting for thermal contraction, cavity formation, ice growth, solute ripening, and glass formation, we evaluate it for a range of chamber materials and solution compositions, and we validate against the acutely limited data available. Our results provide both counter-intuitive insights- lower-concentration solutions may contract less while producing more pressure-undetectable ice growth for example- and a general phenomenological framework by which to evaluate the process of vitrification in isochoric systems. We anticipate that the model herein will enable design of future isochoric protocols with minimized risk of pressure-undetectable ice formation, and provide a thermodynamic foundation from which to build an increasingly rigorous multi-physics understanding of isochoric vitrification., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cryopreservation involves exposing sperm to stressful conditions that affect cell viability. The high quality of the Azerbaijani water buffalo's by-products, such as buffalo milk, makes it a species of significant importance. Our focus is on protecting its genetic resources by preserving its sperm, as their numbers will decrease in the coming years and they are at risk of extinction. This study's goal was to ascertain how apigenin (A) and trans-ferulic acid (t-FA) affected the semen quality of Azari water buffalo bulls under cryopreservation. Pooled buffalo sperm (n = 35 ejaculations) were diluted in a Tris-based diluent also containing varying amounts of apigenin (0.2, 0.4, 0.6, and 0.8 mM) and trans-ferulic acid (2.5, 5, 10 and 20 mM). Following a freeze-thaw procedure, samples were assayed for total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione activity (GSH), glutathione peroxidase (GPx), progressive motility and total motility, motility properties, plasma membrane functionality, and viability. Sixty days after insemination, the rectal examination was performed on 38 buffaloes that had undergone sexual breeding to confirm pregnancy. The results of the study show that the addition of A-0.2, A-0.4, and t-FA-10 to buffalo semen increases the percentage of intact plasma membrane, motility, and sperm viability, as well as the levels of GSH, GPx, CAT. and TAC. In addition, there is a decrease in MDA and DNA damage after cryopreservation. Furthermore, the results show that 0.4 mM apigenin significantly increases conception rates compared to the control group. The base extender of Tris supplemented with A (0.4 and 0.2 mM) and t-FA (10 mM) improves the antioxidant indices of both frozen and thawed buffalo sperm, which in turn improves post-thawing sperm quality and in vivo fertility improves buffalo sperm., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

The collector urchin, Tripneustes gratilla, is an ecologically important member of the grazing community of Hawai'i's coral reefs. Beyond its ability to maintain balance between native seaweeds and corals, T. gratilla has also been used as a food source and a biocontrol agent against alien invasive algae species. Due to overexploitation, habitat degradation, and other stressors, their populations face local extirpation. However, artificial reproductive techniques, such as cryopreservation, could provide more consistent seedstock throughout the year to supplement aquaculture efforts. Although the sperm and larvae of temperate urchins have been successfully cryopreserved, tropical urchins living on coral reefs have not. Here, we investigated the urchin embryos' tolerance to various cryoprotectants and cooling rates to develop a cryopreservation protocol for T. gratilla. We found that using 1 M Me2SO with a cooling rate of 9.7 °C/min on gastrula stage embryos produced the best results with survival rates of up to 85.5% and up to 50.8% maturation to the 4-arm echinopluteus stage, assessed three days after thawing. Continued research could see cryopreservation added to the repertoire of artificial reproductive techniques for T. gratilla, thereby assisting in the preservation of this ecologically important urchin, all while augmenting aquaculture efforts that contribute to coral reef restoration., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author declares that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE., Competing Interests: Declaration of competing interest The authors affirm that they have no competing or conflicts of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

We investigated methods for cryopreserving sperm from the endangered gudgeon, Microphysogobio rapidus, by examining the effects of cryoprotective agent (CPA) concentration, diluent, and dilution ratio on post-thaw sperm quality. The quality of frozen sperm was evaluated in terms of motility and kinematic parameters, viability, DNA damage, and fertilization rate. We evaluated methanol, glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol as CPAs. Sperm motility, velocity, and viability were significantly higher when methanol was used as the CPA (p < 0.05). The diluents tested were Ringer's solution, Kurokura's Extender, Common Carp Sperm Extender (CCSE), and buffered sperm motility-inhibiting saline solution (BSMIS); post-thaw motility was highest when Ringer's solution was used as the diluent. Next, various quantities of methanol were combined with Ringer's solution to identify the optimal dose of methanol. The dilution ratios tested ranged from 1:1 to 1:7. Cryopreserved sperm was thawed at 20 °C for 15 s. The use of 10% methanol with Ringer's solution at a dilution ratio of 1:5 resulted in the highest post-thaw sperm motility, viability, and velocity including VAP, VCL, and VSL. Post-thaw sperm showed significantly greater DNA damage than the control (fresh sperm) (p < 0.05). The fertilization rate was highest with fresh sperm (p < 0.05), followed by sperm frozen with 10% methanol + Ringer's solution. We recommend that the best way to preserve sperm in the studied species is to use a combination of Ringer's solution and 10% methanol at a 1:5 dilution ratio. Our findings will facilitate the artificial fertilization of M. rapidus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Mesenchymal stromal cells (MSCs) have become one of the most investigated and applied cells for cellular therapy and regenerative medicine. In this update of our review published in 2015, we show that studies continue to abound regarding the characterization of MSCs to distinguish them from other similar cell types, the discovery of new tissue sources of MSCs, and the confirmation of their properties and functions that render them suitable as a therapeutic. Because cryopreservation is widely recognized as the only technology that would enable the on-demand availability of MSCs, here we show that although the traditional method of cryopreserving cells by slow cooling in the presence of 10% dimethyl sulfoxide (Me 2 SO) continues to be used by many, several novel MSC cryopreservation approaches have emerged. As in our previous review, we conclude from these recent reports that viable and functional MSCs from diverse tissues can be recovered after cryopreservation using a variety of cryoprotectants, freezing protocols, storage temperatures, and periods of storage. We also show that for logistical reasons there are now more studies devoted to the cryopreservation of tissues from which MSCs are derived. A new topic included in this review covers the application in COVID-19 of MSCs arising from their immunomodulatory and antiviral properties. Due to the inherent heterogeneity in MSC populations from different sources there is still no standardized procedure for their isolation, identification, functional characterization, cryopreservation, and route of administration, and not likely to be a "one-size-fits-all" approach in their applications in cell-based therapy and regenerative medicine., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Patricia Goldstein reports financial support was provided by National Autonomous University of Mexico Directorate General of Academic Staff Affairs. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Pollen, as the male gametophyte, carries half of plant genetic information and is an important source of germplasm. The cryopreservation of pollen can not only preserve germplasm, but also solve the problem of time and space barrier in crossbreeding. So it is of great significance to explore the mechanism of pollen viability maintenance after cryopreservation. In this paper, 10 cultivars of Paeonia lactiflora with different fresh pollen viability that did not change after cryopreservation were taken as objects and the effects of pollen inclusions such as soluble sugar, starch, soluble protein, free amino acids, and proline were explored. The results showed that: (1) The contents of pollen inclusions in the fresh pollen of 10 cultivars were different. After cryopreservation, the contents of starch and free amino acids significantly decreased in 10 cultivars, and the soluble sugar, soluble protein, and proline varied with cultivars. (2) Correlation analysis showed that fresh pollen viability was significantly positively correlated with the soluble sugar (R-values of 0.630) and starch content (R-values of 0.694) in fresh pollen. But after cryopreservation pollen viability was only significantly positively correlated with the starch content (R-values of 0.725). These results suggest that the effects of pollen inclusions on pollen vitality are different before and after cryopreservation. The fresh pollen with higher soluble sugar and starch is more vital. But after cryopreservation, the pollen with high starch content has higher viability. The maintenance of stable pollen viability after cryopreservation appears to be related to starch content or starch metabolism, which requires further to study for a final determination., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)