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Summary: In this study, the alternating magnetic field (AMF) with frequency of 50, 100, 150, 200 and 250 Hz at magnetic field intensity of 5 mT was used to assist the freezing of tilapia fillets, with no magnetic field (NMF) as a control group. The evaluation of thawed tilapia fillets encompassed an assessment of freezing curve, texture, microstructure, moisture state and protein structure, followed by a subsequent analysis of their correlation. The findings showed that the application of a magnetic field during the freezing process yielded favourable outcomes in enhancing the overall quality of tilapia fillets. Among them, 200 Hz had the best effect. Under the condition, the frozen tilapia fillets assisted by magnetic field showed the shortest freezing time, the least decrease in hardness and elasticity and the best chewiness after thawing. The size of the ice crystals generated during freezing was small and uniform, and the fractal dimension of the frozen sections was the highest. Muscle tissue was the least damaged, which led to the improvement of its water‐holding capacity. Magnetic field‐assisted freezing was found to maintain the α‐helix and β‐sheet in the secondary structure of proteins. The correlation analysis showed that cohesion, elasticity, fractal dimension, FD, A2, A22 and β‐turn were closely related to the quality changes of tilapia fillets after magnetic field treatment. [ABSTRACT FROM AUTHOR]

3D tissue‐engineered models are poised to facilitate understanding of skeletal muscle pathophysiology and identify novel therapeutic agents to improve muscle health. Adopting these culture models within the broader biology community is a challenge as many models involve complex methodologies and significant investments of time and resources to optimize manufacturing protocols. To alleviate this barrier, a protocol with commercially available reagents is developed to cryopreserve myoblasts in a 96‐well compatible format that allows tissues to be transferred to users without expertise in 2D or 3D skeletal muscle cell culture. This report validates that myoblasts encapsulated in a hydrogel and cryopreserved in paper‐based scaffolds maintain cell viability, differentiation, and function via acetylcholine‐induced transient calcium responses. Furthermore, successful shipping of myoblasts cryopreserved in paper‐based scaffolds to intra‐provincial and international collaborators is demonstrated who successfully thaw, culture, and use the 3D muscle tissues. Finally, the application of this method is confirmed for studying muscle endogenous repair by seeding freshly isolated skeletal muscle stem cells to cryopreserved then differentiated and injured tissues, demonstrating expected responses to a known stimulator of muscle stem cell self‐renewal, p38α/β MAPKi. Altogether, the 3D myoblast cryopreservation protocol offers broadened access of a complex skeletal muscle tissue model to the research community. [ABSTRACT FROM AUTHOR]

Sugars, as cryoprotectants, can be added to NP formulations before lyophilization to ensure that NPs retain their size during long‐term storage. In this study, mannitol, glucose, and sucrose were used at different concentrations to extend the storage time of doxorubicin (DOX)‐loaded PLGA‐NPs prepared using two different copolymer ratios (75 : 25 and 50 : 50) at different storage temperatures (−20, +4, and +25 °C). After lyophilization, a glucose or sucrose concentration of only 5 % for DOX‐PLGA‐75 : 25‐NP and a glucose concentration of 10 % for DOX‐PLGA‐50 : 50‐NP were found to prevent agglomeration for up to 4 months at −20 °C. It is noteworthy that after storage at 4 °C for up to 4 months, agglomeration was prevented using 5 % sucrose and 10 % glucose in DOX‐PLGA‐50 : 50‐NP and 10 % sucrose content in both NP types. On the other hand, as expected, no sugar type or concentration could prevent agglomeration during long‐term storage of NPs at 25 °C. The sizes and encapsulation efficiencies (EE%) before lyophilization were 221.8±2.20 and 45.4 % for DOX‐PLGA‐50 : 50‐NP and 221.5±1.31 and 48.7 % for DOX‐PLGA‐75 : 25‐NP, respectively. Furthermore, the release profiles and encapsulation of DOX in both NPs were investigated by UV‐Vis analyses. DSC analysis revealed that DOX was present in the free form and amorphous in the NPs. Consequently, PLGA copolymer ratios were found to have an impact on the long‐term stability of DOX loaded PLGA lyophilizates, and DOX‐loaded NPs prepared with PLGA 75 : 25 or PLGA 50 : 50 can be stored at −20 and +4 °C for up to 4 months when combined with appropriate sugar contents and concentrations, demonstrating the versatility of these materials. [ABSTRACT FROM AUTHOR]

BACKGROUND: The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long‐term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. RESULTS: The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low‐temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze–thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice‐growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice‐growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. CONCLUSION: The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Cryopreservation of biological samples, including cells, tissues, and organs, has become an essential component in various biomedical research and applications, such as cellular therapy, tissue engineering, organ transplantation, and conservation of endangered species. However, it faces critical challenges throughout the cryopreservation process, such as loading/unloading of cryoprotective agent (CPA), ice inhibition during cooling, and ultrafast and uniform heating during rewarming. Applying nanomaterials in cryopreservation has emerged as a promising solution to address these challenges in each step due to their unique properties. For instance, in order to deliver nonpermeating CPA into cells, some nanomaterials, such as polymeric nanocapsule, can carry nonpermeating CPA to penetrate into the cells, regulating the intracellular ice crystal. During cooling, some nanomaterials, such as graphene oxide, can bind to basal or prism planes of ice crystals, suppressing the ice growth. During rewarming, some nanomaterials, such as magnetic nanoparticles, can improve the heating performance, preventing devitrification and recrystallization during rewarming. However, challenges in nanomaterials‐assisted cryopreservation remain, including the need for comprehensive studies on nanomaterials toxicity and the development of scalable manufacturing processes for industrial applications. This review examines the role of nanomaterials in cryopreservation, focusing on their mechanisms, applications, and associated challenges. [ABSTRACT FROM AUTHOR]

Cryopreservation of bovine oocytes and embryos is essential for long‐term preservation and widespread distribution of genetic material, particularly in bovine in vitro embryo production, which has witnessed substantial growth in the past decade due to advancements in reproductive biotechnologies. Among current cryopreservation methods, vitrification has emerged as the preferred cryopreservation technique over slow freezing for preserving oocytes and in vitro‐produced (IVP) embryos, as it effectively addresses membrane chilling injury and ice crystal formation. Nonetheless, challenges remain and a simple and robust vitrification protocol that guarantees the efficiency and viability after warming has not yet been developed. Furthermore, although slow cooling can easily be adapted for direct transfer, an easier and more practical vitrification protocol for IVP embryos is required to allow the transfer of IVP embryos on farms using in‐straw dilution. In addition, the susceptibility of bovine oocytes and embryos to cryoinjuries highlights the need for novel strategies to improve their cryotolerance. This manuscript examines various methodological approaches for increasing the viability of bovine oocytes and IVP embryos during vitrification. Strategies such as modifying lipid content or mitigating oxidative damage have shown promise in improving cryotolerance. Additionally, mathematical modelling of oocyte and embryo membrane permeability has facilitated the rational design of cryopreservation protocols, optimizing the exposure time and concentration of cryoprotectants to reduce cytotoxicity. [ABSTRACT FROM AUTHOR]

Cryopreservation is a common way for long‐term storage of therapeutical proteins, erythrocytes, and mammalian cells. For cryoprotection of these biosamples to keep their structural integrity and biological activities, it is essential to incorporate highly efficient cryoprotectants. Currently, permeable small molecular cryoprotectants such as glycerol and dimethyl sulfoxide dominate in cryostorage applications, but they are harmful to cells and human health. As acting in the extracellular space, membrane‐impermeable macromolecular cryoprotectants, which exert remarkable membrane stabilization against cryo‐injury and are easily removed post‐thaw, are promising candidates with biocompatibility and feasibility. Water‐soluble hydroxyl‐containing polymers such as poly(vinyl alcohol) and polyol‐based polymers are potent ice recrystallization inhibitors, while polyampholytes, polyzwitterions, and bio‐inspired (glyco)polypeptides can significantly increase post‐thaw recovery with reduced membrane damages. In this review, the synthetic macromolecular cryoprotectants are systematically summarized based on their synthesis routes, practical utilities, and cryoprotective mechanisms. It provides a valuable insight in development of highly efficient macromolecular cryoprotectants with valid ice recrystallization inhibition activity for highly efficient and safe cryopreservation of cells. [ABSTRACT FROM AUTHOR]

BACKGROUND: The present study aimed to investigate the cryoprotective effect of epigallocatechin gallate (EGCG) replacing sucrose on surimi during frozen storage. Substitution or partial substitution of 0.1% EGCG for sucrose (1.5%) was added to surimi, and the surimi samples without and with commercial cryoprotectants (4% sucrose and 4% sorbitol) were used as the control group. RESULTS: The results obtained suggest that, with the increase in frozen storage time, the structural performance of surimi protein gradually weakened (e.g. the decrease in the surface hydrophobicity, the increase in the total sulfhydryl and solubility, and the protein myosin heavy chain bands became shallow) and surimi gel quality gradually deteriorated (e.g. the decrease in water‐holding capacity, gel strength and all texture profile attributes). However, compared with the other three group surimi samples during the frozen period, the surimi proteins with partial replacement of sucrose by EGCG had a higher total sulfhydryl group content and solubility of proteins, as well as lower surface hydrophobicity of protein, suggesting that the addition of EGCG as a partial substitute for sucrose can enhance the antifreeze ability of surimi. Meanwhile, the surimi gel with the partial replacement of sucrose by EGCG had a higher water retention capacity, gel strength and texture attributes (e.g. hardness, springiness, cohesiveness, chewiness, and resilience), indicating that the addition of EGCG as a partial substitute for sucrose can inhibit the deterioration of surimi gel quality. CONCLUSION: Overall, EGCG partially replacing sucrose can play an alternative cryoprotectant with a lower sweetness to prevent the quality of surimi from deteriorating. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Research on the development of innovative cryobanking techniques can aid coral restoration and conservation. This study applied a customized freezing device and "cryojig" along with vitrification and laser warming techniques to create the first cryorepository for pelagic‐phase larvae of the corals Seriatopora caliendrum, Pocillopora verrucosa, Stylophora pistillata, and Pocillopora acuta. Three vitrification solutions (VSs) were prepared, with each composed of two of four cryoprotective agents. These included VS1: 2 M ethylene glycol (EG) and 1 M propylene glycol; VS2: 2 M EG and 1 M glycerol; VS3: 2 M dimethyl sulfoxide and 1 M EG. Larvae were then mixed with the VSs, 40% (w/v) Ficoll, and 10% (v/v) gold nanoparticles (GNPs) at a final concentration of 1.2 × 1018 particles/m3 and using our customized device for vitrification and laser warming. Over a thousand coral larvae (576 S. caliendrum, 376 P. verrucosa, 238 S. pistillata, and 524 P. acuta) were successfully stored in the cryorepository and the proportions of coral larvae that were noted to be actively swimming or settled after vitrification and laser warming. This is the first project to establish a coral larvae cryorepository, and the methods applied could represent a critical tool for reef restoration and maintenance of marine biodiversity. [ABSTRACT FROM AUTHOR]

Devising an approach to deterministically position organisms can impact various fields such as bioimaging, cybernetics, cryopreservation, and organism‐integrated devices. This requires continuously assessing the locations of randomly distributed organisms to collect and transfer them to target spaces without harm. Here, an aspiration‐assisted adaptive printing system is developed that tracks, harvests, and relocates living and moving organisms on target spaces via a pick‐and‐place mechanism that continuously adapts to updated visual and spatial information about the organisms and target spaces. These adaptive printing strategies successfully positioned a single static organism, multiple organisms in droplets, and a single moving organism on target spaces. Their capabilities are exemplified by printing vitrification‐ready organisms in cryoprotectant droplets, sorting live organisms from dead ones, positioning organisms on curved surfaces, organizing organism‐powered displays, and integrating organisms with materials and devices in customizable shapes. These printing strategies can ultimately lead to autonomous biomanufacturing methods to evaluate and assemble organisms for a variety of single and multi‐organism‐based applications. [ABSTRACT FROM AUTHOR]

Cryopreservation of metaphase II (MII) oocytes is crucial to preserve female fertility. However, the existing vitrification technology just provides partial cryogenic protection for oocytes and must use high concentrations of toxic penetrating cryoprotective agents (CPAs, up to 4.8 M). Here, a multi‐scale ice inhibition platform is proposed, which effectively suppresses ice crystal morphology, ice recrystallization (IR), temperature gradient, and devitrification. This platform achieves mouse oocyte survival rate of 98.6% using the low concentration of CPA (3.1 M) while also possessing batch oocyte cryopreservation ability. In contrast, recovered oocytes show a much more modest variation of genes compared with the conventional method (85 vs. 1396 genes), retaining normal fertilization, embryonic development, and birth to healthy offspring. Diverging from the conventional vitrification method with partial cryogenic protection capability, this platform introduces full‐cycle ice inhibition, offering promising prospects for achieving high‐quality and scalable fertility preservation. [ABSTRACT FROM AUTHOR]

The availability of microbial biobanks for the storage of individual gut microbiota members or their derived and artificially assembled consortia has become fundamental for in vitro investigation of the molecular mechanisms behind microbe–microbe and/or microbe–host interactions. However, to preserve bacterial viability, adequate storage and processing technologies are required. In this study, the effects on cell viability of seven different combinations of cryoprotective agents were evaluated by flow cytometry for 53 bacterial species representing key members of the human gut microbiota after one and 3 months of cryopreservation at −80°C. The obtained results highlighted that no universal cryoprotectant was identified capable of guaranteeing effective recovery of intact cells after cryopreservation for all tested bacteria. However, the presence of inulin or skimmed milk provided high levels of viability protection during cryoexposure. These results were further corroborated by cryopreserving 10 artificial gut microbiota produced through in vitro continuous fermentation system technology. Indeed, in this case, the inclusion of inulin or skimmed milk resulted in a high recovery of viable cells, while also allowing consistent and reliable preservation of the artificial gut microbiota biodiversity. Overall, these results suggest that, although the efficacy of various cryoprotective agents is species‐specific, some cryoprotectants based on glycerol and the addition of inulin or skimmed milk are preferable to retain viability and biodiversity for both single bacterial species and artificial gut microbiota. [ABSTRACT FROM AUTHOR]

Our previous research has shown that melatonin (MLT) can reduce cryopreserved ovarian damage in mice. Yet, the molecular mechanism of MLT protection is still unclear. Some studies have shown that melatonin receptor 1 (MT1) is very important for animal reproductive system. To evaluate whether MLT exerts its protective effect on cryopreserved mice ovarian tissue via MT1, we added antagonist of MT1/MT2 (Luzindor) or antagonist of MT2 (4P‐PDOT) to the freezing solution, followed by cryopreservation and thawing of ovarian tissue. The levels of total superoxide dismutase (T‐SOD), catalase (CAT), nitric oxide (NO) and malondialdehyde (MDA) were detected. Besides, by using RT‐PCR and Western blotting, the expression of Bcl‐2, Bax and Nrf2/HO‐1 signalling pathway‐related proteins was detected. These findings demonstrated that compared with the melatonin group, the addition of Luzindor increased apoptosis, NO and MDA activities, decreased CAT and T‐SOD activities and inhibited Nrf2/HO‐1 signalling pathway. In conclusion, melatonin can play a protective role in cryopreserved ovarian tissue of mice through MT1 receptor. [ABSTRACT FROM AUTHOR]

Background: The effects of novel non‐cytotoxic and immunotherapy drugs for cancer treatment on human testicular function have not been studied systematically. Objectives: The present study aimed to characterize effects of non‐cytotoxic and immunotherapy drugs in patients with cancers who had not been previously treated with gonadotoxic chemo‐ or radiotherapy. Materials and methods: This study involved 34 men, not previously treated with gonadotoxic regimens, in a mixed longitudinal (Cohort 1: 19 men about to start and approximately 1 year on non‐cytotoxic and immunotherapy treatment) and cross‐sectional (Cohort 2: 15 men already on non‐cytotoxic and immunotherapy treatment) study using data modeling to estimate within‐person time‐course changes in testicular exocrine and endocrine functions. Cohort 1 provided 45 paired semen and blood samples (34 prior to and nine during treatment) and Cohort 2 provided 45 sets of samples (15 pre‐treatment, 30 on treatment), including six men in Cohort 2 who had pre‐treatment spermatozoa cryostorage prior to the study. Men on non‐cytotoxic and immunotherapy treatment had undergone a median of 33.5 months long‐term treatment. Results: Spermatozoa output and concentration were reduced by about 50%, with corresponding increases in serum follicle‐stimulating hormone and decreases in serum inhibin B. Serum testosterone, luteinizing hormone, and sex hormone‐binding globulin were unaffected by non‐cytotoxic and immunotherapy treatment. Conclusion: Within limits of the present study of sample size and duration of on‐non‐cytotoxic and immunotherapy treatment, non‐cytotoxic and immunotherapy drugs have a modest effects on testicular exocrine function (sperm production) or its hormonal correlates (follicle‐stimulating hormone, inhibin B), with minimal impact on testicular endocrine (testosterone, luteinizing hormone) function. [ABSTRACT FROM AUTHOR]

Well‐designed cryoprotectants are crucial for successful probiotic application. In this study, based on literature analysis, characteristics and economic analysis of cryoprotectants, the sucrose (8.2%)–skim milk (8.0%)–inulin (8.2%) combination was proposed and optimised to enhance freeze‐drying resistance of probiotics. Survival, viable cell counts and inactivation rate during storage at 4°C of Lactiplantibacillus plantarum grx16 protected by the combination was 94.9%, 1.89 × 1011 cfu/g and 0.0116. The optimised sucrose–skim milk–polymer prebiotics combination could also effectively protect several other probiotics, with the survival rate being 89.2–95.6%. These results will aid in the rational development of synergistic cryoprotectant formulations for probiotics. [ABSTRACT FROM AUTHOR]

Meat and eggs from chicken are the major source of animal protein for the human population. The cryopreservation of poultry species is needed to guarantee sustainable production. Here, we describe the existing cryopreservation technologies for avian reproductive cells using embryonic germ cells, spermatozoa and ovarian tissues. We outline strategies to reconstitute chicken breeds from their cryopreserved embryonic germ cells using surrogate hosts and discuss the perspectives for genetic conservation and reconstitution of chicken and wild avian species using surrogate host animals. [ABSTRACT FROM AUTHOR]

Cryopreservation is a valuable technique used to assist in the genetic improvement of cultured stocks and provide a continuous supply of good‐quality semen for artificial insemination. Conserving semen by cryopreservation serves several purposes (e.g. artificial reproductive technologies and species conservation) and is also used in the clinical treatment of human infertility. However, the lifespan of cryopreserved semen is influenced by a range of factors, including storage temperature, cooling rate, chemical composition of the extender, the concentration of cryoprotectant, reactive oxygen species, seminal plasma composition and hygienic control. The choice of cryoprotectant is a vital factor underlying the success of animal semen cryopreservation. In this regard, extensive research has been carried out on various cryoprotectants, such as egg yolk, dimethyl sulfoxide, methanol, ethylene glycol and dimethylacetamide. Recent studies have also described the use of a range of new cryoprotectants for cryopreservation, including compounds of plant origin (soy), amino acids, antifreeze proteins, carbohydrates and cyclodextrins. Moreover, semen cryopreservation and storage require the use of liquid nitrogen or ultralow refrigeration methods for both long‐ and short‐term storage. This review summarizes the general methods used for freezing semen and discusses the use of traditional and newly emerging cryoprotectants (permeable and non‐permeable) for the cryopreservation of semen in selected fish and mammalian species. [ABSTRACT FROM AUTHOR]

The possibility of embryo cryopreservation is important for applying the genome resource banking (GRB) concept to those mammalian species that exhibit embryonal diapause in their early development. Odc1 encodes ODC1, which is a key enzyme in polyamine synthesis. RhoA is an essential part of Rho/ROCK system. Both Odc1 and RhoA play an important role in preimplantation embryo development. Studying these systems in mammalian species with obligate or experimentally designed embryonic diapause may provide insight into the molecular machinery underlying embryo dormancy and re‐activation. The effect of cryopreservation procedures on the expression of the Odc1 and RhoA in diapausing embryos has not been properly studied yet. The purpose of this work is to address the possibility of cryopreservation diapausing embryos and to estimate the expression of the Odc1 and RhoA genes in diapausing and non‐diapausing embryos before and after freeze–thaw procedures using ovariectomized progesterone treated mice as a model. Both diapausing and non‐diapausing in vivo‐derived embryos continued their development in vitro after freezing–thawing as evidenced by blastocoel re‐expansion. Although cryopreservation dramatically decreased the expression of the Odc1 and RhoA genes in non‐diapausing embryos, no such effects have been observed in diapausing embryos where these genes were already at the low level before freeze–thaw procedures. Future studies may attempt to facilitate the re‐activation of diapausing embryos, for example frozen–thawed ones, specifically targeting Odc1 or Rho/ROCK system. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation is a critical tool for safeguarding and managing valuable genetic resources. Protocols for cryopreservation of Xenopus laevis sperm were available but lacking sperm quality evaluation and scalability and the outcomes were inconsistent. The goal of this study was to begin developing a center‐level cryopreservation pathway for this species by integrating French straws as containers that would facilitate germplasm repository development. The objectives were to analyze the effect of: (1) three sperm concentrations (33, 50, and 100 × 106 sperm/mL) on post‐thaw fertilization, (2) three final concentrations (2.5%, 5%, and 10%) of dimethyl sulfoxide, methanol, and dimethylformamide (DMFA) on sperm membrane integrity of fresh and frozen samples, (3) two concentrations (5% and 10%) of DMFA with and without 5% sucrose at four cooling rates (5, 10, 20, and 40°C/min) on sperm membrane integrity and motility, and (4) egg exposure to different concentrations of DMFA on fertilization. Few differences in sperm viability were found among fresh samples incubated in cryoprotectants, but thawed samples frozen in methanol or DMFA presented higher membrane integrity. Samples frozen in 10% DMFA at 20°C/min showed higher membrane integrity (60 ± 7%) than other DMFA concentrations and cooling rates, and the same total motility (30 ± 7%) as at 10°C/min. Higher DMFA concentrations (10%–13%) were detrimental for embryo development compared to lower concentrations (<6%). This study provided a reliable protocol for sperm cryopreservation in Xenopus laevis to yield an application pathway with potential for high throughput that can be used as a roadmap for work with other species. Research Highlight: A reliable and scalable cryopreservation pathway is needed to protect the valuable Xenopus laevis genetic resources.Sperm quality was higher when frozen in 10% DMFA at 20°C/min.Embryo survival was lower in 10% DMFA than in ≤ 5% DMFA. [ABSTRACT FROM AUTHOR]

Cryopreservation is one of the reliable techniques for long‐term storage of sperm. The success of this technique depends on the choice of cryoprotectant; therefore, a plethora of literature has reported the effects of different cryoprotective agents so far. Kappa‐carrageenan (κ‐carrageenan) is a hydrocolloid polysaccharide extracted from red marine seaweed. Its unique property makes it a promising option as a non‐colligative cryoprotectant. The current study aims to evaluate the cryoprotective effect of k‐carrageenan along with glycerol on ram sperm quality both after equilibration and freezing. Nine Kajli rams were utilized in this experiment for semen collection through an artificial vagina maintained at 42°C. Qualified samples were diluted in tris egg yolk glycerol (TEYG) extender containing different concentrations of k‐carrageenan as 0 mg/mL (control), 0.2, 0.5, 0.8 and 1 mg/mL. Post‐thaw assessment was done at 37°C after 24 h of storage, which showed a significant improvement (p <.05) in sperm viability, motility, membrane and acrosome integrity in an extender containing k‐carrageenan at a concentration of 0.5 mg/mL compared to control. It is concluded from the current study that the combination of glycerol and 0.5 mg/mL concentration of k‐carrageenan improved the sperm post‐thaw quality. [ABSTRACT FROM AUTHOR]

This study aims to reduce the production of flocculent material and stratification of lemon juice during freezing and thawing, using electric or magnetic fields to assist with the freezing of lemon juice. This study investigated the effect of 10–30 kV/cm electric field (EF) and 2–10 mT magnetic field (MF) on the quality of frozen lemon juice. Electromagnetic field treatment can shorten the freezing time and reduce its particle size, resulting in a lower polymer dispersion index and lower enzyme activity, thus improving its cloud stability. When the EF strength was 20 kV/cm and the MF strength was 6 mT, it resulted in the highest cloud stability, which increased by 15.50 and 16.95%, respectively. In addition, we studied the effect of electromagnetic fields on the physico‐chemical properties and bioactive components of lemon juice. The results showed that it did not affect its bioactive compounds activity and antioxidant activity, and did not cause loss of lemon juice flavor. The electromagnetic field treatment improved the quality of the juice and can be used as a potential novel processing technology for fruit juices. Practical Applications: This study provides a simple and novel method to improve the stability of frozen lemon juice after thawing. The use of physical fields such as electric and magnetic field treatments to assist in freezing lemon juice improves the stability of lemon juice after thawing and provides a new method to improve the quality of fruit juice products. [ABSTRACT FROM AUTHOR]

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open‐source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra‐ and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum‐/xeno‐free medium that included 0.7 M Me2SO and 100 mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2SO compared to serum‐containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum‐free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics. LAY DESCRIPTION: Research in the field of cryobiology involves many technics linked with the assessment of cell recovery after cooling/warming as well as quantification, identification and description of physical processes occurring in and out of the cells. These processes can affect the overall outcome of cryopreservation. Thus, the investigation of the mechanisms of cryoprotection and cryodamage is of high importance. In this work, we have developed a system for cryomicroscopy utilising open‐source platform for prototyping electronic devices and systems Arduino. The platform includes hardware and software designed to develop various projects. It also supports a programming language. The language was used to create a code to control cooling rate of the sample undergoing microscopy. The system was able to visualise the structure of the forming ice crystals, recrystallisation, salt eutectic crystallisation/melting and intracellular phase transitions. It can be utilised in combination with other approaches and techniques. The system helped us to solve specific tasks of cryobiological experimentation connected with the establishment of mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs). The microscopy data allowed us to compare different media developed for cryopreservation of testicular cells. The serum‐containing cryoprotective medium (1.4 M Me2SO and 10% foetal bowine serum) was able to suppress darkening of cell, known as 'flashing' associated with intracellular phase transitions by moderately dehydrating cells, and salt eutectic transitions during cooling/warming. However, the media contains relatively high Me2SO concentration and may be toxic. Additionally, the use of serum in the media can result in transferring infection when cryopreserved cells are used in medicine or veterinary, or lead to instability of the composition of serum‐containing media from batch to batch. In the course of the research, we have shown that using the serum‐/xeno‐free medium supplemented with 0.7 M Me2SO and 100 mg/mL Dex (0.7 Me2SO+Dex) can also reduce cell 'flashing'. The medium influenced the size and shape of extracellular ice crystals in a way that can potentially reduce the risk of mechanical cell damage. This medium promoted amorphous solidification in the space between the extracellular ice crystals, thus, lowering the salt eutectic transitions and recrystallisation that could otherwise cause additional cell damage. Therefore, application of the developed system for cryomicroscopy, which allowed real‐time visualisation of cryobiological processes, made it possible to draw a conclusion about cryoprotecitive properties of medium for ICs. 0.7 Me2SO+Dex can be the basis for the creation of commercial serum‐free media with a defined composition that does not require removal before use. [ABSTRACT FROM AUTHOR]

This review describes the freeze‐dried mesenchymal stem cells (MSCs) and their ability to restore damaged tissues and organs. An analysis of the literature shows that after the lyophilization MSCs retain >80% of paracrine factors and that the mechanism of their action on the restoration of damaged tissues and organs is similar to the mechanism of action of paracrine factors in fresh and cryopreserved mesenchymal stem cells. Based on the own materials, the use of paracrine factors of freeze‐dried MSCs in vivo and in vitro for the treatment of various diseases of organs and tissues has shown to be effective. The study also discusses about the advantages and disadvantages of freeze‐dried MSCs versus cryopreserved MSCs. However, for the effective use of freeze‐dried MSCs in clinical practice, a more detailed study of the mechanism of interaction of paracrine factors of freeze‐dried MSCs with target cells and tissues is required. It is also necessary to identify possible other specific paracrine factors of freeze‐dried MSCs. In addition, develop new therapeutic strategies for the use of freeze‐dried MSCs in regenerative medicine and tissue bioengineering. [ABSTRACT FROM AUTHOR]

Vitrification‐based cryopreservation is a promising approach to achieving long‐term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction‐dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105 °C min−1) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction‐dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated. [ABSTRACT FROM AUTHOR]

Globalization and habitat destruction pose a significant threat to wildlife felids. Even though conservation banks for genetic materials have been created, the sperm cryopreservation with minimal cell damage is still a great challenge. Thus, this study aimed to compare the effects of two commercial extenders with different concentrations of alternative cryoprotectants on thawed sperm quality of domestic cats. Five adult cats were anaesthetized (using a combination of 40 μg/kg medetomidine associated to 5 mg/kg ketamine), and the semen was collected by electroejaculation (electrical stimulation of 2–3 V). Semen samples were evaluated for sperm characteristics (kinetics, morphology, membrane integrity and morphometry). Subsequently, they were sorted into two aliquots and centrifuged. The aliquots were added to a commercial extender containing 3% glycerol and 2% methylformamide (extender I) or 2% glycerol and 3% methylformamide (extender II), frozen, thawed (37°C/30 s) and reevaluated. Comparatively, the sperm kinetics and membrane integrity of fresh semen were higher (p <.002) than frozen samples in extender I and II. Total and progressive motility were lowest in the thawed samples. However, the subjective analysis indicated high sperm motility, since the kinetics evaluation was impaired by the low cell number in the thawed samples. There were no differences in sperm morphology between the groups. In the sperm morphometric analysis, a significant difference (p =.04) was identified in the length of the intermediate piece in extender II samples compared with fresh and extender I. Thus, it can be concluded that although the concentrations tested did not maintain the kinetic parameters and membrane integrity of spermatozoa after thawing, the extender with a lower concentration of glycerol was less toxic for maintaining the midpiece length. [ABSTRACT FROM AUTHOR]

BACKGROUND: Frozen storage often leads to quality deterioration of surimi‐based products. At present, most of the research focuses on improving the quality of surimi products by adding cryoprotectants, and there are few studies available on preparation technology. Therefore, the effects of different gelation‐freezing treatments, high temperature heating‐freezing treatment (HF), low temperature heating‐high temperature heating‐freezing treatment (LHF) and low temperature heating‐freezing‐high temperature heating treatment (LFH) on the quality changes of surimi gels containing hydroxypropyl distarch phosphate (HPDSP) during frozen storage were investigated. RESULTS: With the extension of frozen storage time, the quality of surimi gel in all groups decreased, but the quality of surimi gel with HPDSP was better than that of surimi gel without HPDSP. Compared with HF and LHF, the change range of breaking force, hardness, gumminess, whiteness and disulfide bond content of HPDSP‐surimi gel treated with LFH was the least during the frozen storage. In the reheating process of LFH, HPDSP could absorb the water lost during freezing. Therefore, the change in the transverse relaxation time of HPDSP‐surimi gels treated with LFH was smaller, with more immobile water and less free water and P22 of 96.81% and P23 of 0% at 16 weeks. In addition, the breaking deformation, cohesiveness, resilience, springiness and protein composition of surimi gels with and without HPDSP treated with HF, LHF and LFH did not change significantly during frozen storage. CONCLUSION: The combination of LFH and HPDSP could effectively reduce the quality change of surimi gel during frozen storage. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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

Summary: Alginate‐based encapsulation is an effective probiotics delivery system in the intestine. However, due to calcium ions and gastric juice damaging cell wall/membrane integrity during freeze‐drying and gastrointestinal ingestion, lots of cell lose their viability. This study, taking Lactiplantibacillus plantarum grx16 as an example, indicated that co‐encapsulation of cryoprotectants and porous starch within calcium alginate capsules (CAC) could combinedly improve the viability of probiotics encapsulated. Compared with calcium alginate‐protectant capsule (CAC‐Pro) (50.67%), the combination of porous starch increased the encapsulation rate of PS0.8‐CAC‐Pro to 82.68%. Meanwhile, the death rate constants of PS0.8‐CAC‐Pro decreased by 31.3% (−20 °C), 13.5% (4 °C) and 7.5% (25 °C), respectively. The survival rate of PS0.8‐CAC‐Pro was 81.94% after continuous simulated gastrointestinal digestion, which was significantly higher than that of CACs (69.46%) and CAC‐Pro (76.92%). This study provided an effective approach for modifying alginate capsule to reduce the loss of probiotics viability. [ABSTRACT FROM AUTHOR]

Cryopreservation strives to maximize the viability and biofunctionality of cells and tissues by cooling them to a subzero temperature to facilitate storage and delivery. This technology has enabled clinics and labs to preserve rare and crucial samples and is poised to become more important with rising interest in cell therapy. Here, the biological impact of cooling rates on different cellular components is first described, paying special emphasis on the differences between slow cooling and vitrification with a heat transfer perspective based on the Biot number. This is followed by an overview of various classes of chemical‐based cryoprotective agents including small molecules, antifreeze proteins, hydrogels, and cryoprotective nanomaterials. Most importantly, fundamental concepts of cryopreservation including Mazur's "two‐factor hypothesis" are revisited, gaps in them are highlighted, and experiments to validate reported claims to deepen mechanistic understanding of cryoprotection are proposed. A matric is also introduced to assess the suitability of biomaterials for use in cell therapy to support manufacturers in making strategic choices for storing clinical samples. It is believed that this review would inspire readers to scrutinize fundamental concepts in cryopreservation to facilitate the development of new cryoprotective materials and technologies to support the emerging cell manufacturing and therapy industry. [ABSTRACT FROM AUTHOR]

Coral reefs are threatened by anthropogenic climate change, which causes ocean acidification and warming that can result in coral death and the loss of genetic diversity on reefs around the world. Global efforts to secure the genetics of threatened populations using cryopreservation and biobanking are underway but are limited to coral sperm and larvae, available only during brief annual spawning events. Methods to cryopreserve adult coral tissues to enable biobanking activities year‐round are urgently needed, but are challenging due to the presence of a calcium carbonate skeleton and algal symbionts within the tissues, and chill sensitivity. In this study, vitrification and laser nanowarming permitted successful recovery of adult coral tissues in a novel sample type, the single‐polyp microfragment. Fluorescence and confocal microscopy shows clearly defined green fluorescent protein auto‐fluorescence around the polyp mouth post‐warming, with an overall survival rate of 39.7 ± 7.4% at 24 h post‐warming and 23.3 ± 9.7% at 1 month, but relatively few algal symbionts are present in the tissues, indicating poor survival of these cells. These proof‐of‐concept results provide a basis for continued research and development of a field‐ready protocol for cryopreservation of adult coral tissues, which will be essential to prevent extinctions and support reef restoration. [ABSTRACT FROM AUTHOR]

Rabbit production is well developed in the Mekong Delta and Vietnam, with increased consumption of rabbit meat in both urban and rural areas. Consequently, meat rabbit breeds have become popular in almost all provinces in Vietnam, resulting in a decline in the population of Local Black rabbits over the years. To support breeding programs, there is a crucial need to develop a cryopreservation method. The objective of this study was to develop sperm cryopreservation protocols and determine the optimal cryoprotectant for Local Black rabbit spermatozoa. Rabbit spermatozoa were obtained and diluted with TCG‐EggYolk freezing medium containing 5%, 8%, and 10% glycerol or DMSO in a 1:1 ratio. Glycerol and DMSO‐free mediums served as the controls. Samples were put into 0.5‐mL straws, cooled to 15°C and 5°C, then placed on nitrogen vapour for 15 min and directly immersed in liquid nitrogen at −196°C. Samples were thawed at 37°C for 60 s after being frozen for 72 h Sperm quality measures, such as viability, motility, and membrane integrity, were assessed both before and after freezing. The results showed that the average viability, average motility, and average membrane integrity were 61.9%, 53.9%, and 42.9%, respectively, after thawing with 5% glycerol, which was higher than the two higher concentrations of glycerol (8% and 10%) (p <.05). Similarly, with 8% DMSO, the average viability, average motility, and membrane integrity were 59.2%, 51.0%, and 41.8%, respectively, which were higher than the two concentrations of DMSO (5% and 10%) (p < 0.05). Therefore, the cryopreservation of local black rabbit sperm with 5% glycerol or DMSO 8% resulted in high sperm quality, but considering the practical aspect of minimizing the volume of the preservative, a concentration of 5% glycerol may be the best option. [ABSTRACT FROM AUTHOR]

The role of ex situ conservation facilities or captivity through captive breeding programs is essential in the conservation of the endangered Javan banteng. The development of semen cryopreservation may assist on one side of the conservation plan. However, the male Javan banteng reproductive capability must be considered as it influences the targeted outputs. Studying the potential biomarker for fertility such as osteopontin gene expression is also expected to help predict male fertility. Therefore, this study aimed to analyze the quality of spermatozoa after thawing to help predict the male reproductive capability of Javan banteng. Furthermore, this study investigated the potential role of osteopontin gene expression in male Javan banteng fertility. A positive reinforcement approach was used to accustom the male and female animals as we focused on establishing a collection procedure using neither sedation nor anaesthesia. Semen samples were collected at Taman Safari Indonesia, Bogor, in accordance with the female banteng receptivity. Semen samples were then evaluated and then cryopreserved under field conditions. Our study showed the different predicted reproductive capability of the Javan banteng based on the post-thaw spermatozoa quality, which showed significant differences. The OPN gene showed positive correlations with the progressive motility (r = 0.711, p = 0.048), viability (r = 0.822, p = 0.012), and acrosomal integrity (r = 0.665, p = 0.072) of Javan banteng spermatozoa after thawing. Our study demonstrated the predicted Javan banteng reproductive capability based on various post-thaw spermatozoa variables. This finding is also the first report on the OPN gene potential to be developed as the assessment tool of post-thaw spermatozoa quality of the male Javan banteng. The findings in our study may help give recommendations for future breeding programs, especially in the ex situ conservation sites. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that protecting human amnion and chorion matrices (HACM) during processing enhances the performance of HACM for wound repair and tissue regeneration. We utilised a diabetic (db/db) delayed wound healing mouse model. Treatment of db/db full‐thickness excisional wounds with HACM, processed with a polyampholyte preservative accentuated the proliferative phase of wound healing that decreased the time necessary to heal wounds. Polyampholyte protection improved the preservation of growth factors and cytokines during room temperature storage following E‐beam sterilisation and improved its function in wound healing applications. Our findings indicate protected HACM tissue up‐regulated MIP2, NF‐kB, TNF‐α, KI‐67, and Arg1 (0.6‐fold to 1.5‐fold) but those changes were not statistically significant. Immunofluorescent assessment identifying cell activity illustrated an induction of the proliferative phase of wound healing and a switch from an inflammatory macrophage phenotype (M1) to a pro‐regenerative macrophage phenotype (M2a). Genomic profiling of 282 genes was performed using Nanostring from co‐cultures of human macrophages and fibroblasts. The polyampholyte + HACM‐treated group, compared with the HACM or polyampholyte alone groups, had a statistically significant up‐regulation (32–368 fold) of 12 genes primarily involved in macrophage plasticity including CLC7, CD209, CD36, HSD11B1, ICAM1, IL1RN, IL3RA, ITGAX, LSP1, and PLXDC2 (adj. p‐value < 0.05). The polyampholyte alone group demonstrated statistically significant down‐regulation of four genes ADRA2, COL7A1, CSF3, and PTGS2 (adj. p < 0.05). The HACM alone group up‐regulated four genes ATG14, CXCL11, DNMT3A, and THBD, but the results were not statistically significant. Biomechanical measurements indicated that wounds treated with polyampholyte‐protected HACM had more tensile integrity compared with wounds treated with HACM alone. These findings indicate that better protection of HACM during processing stabilises the HACM matrix, which may lead to improved wound healing outcomes. [ABSTRACT FROM AUTHOR]

Background: Magnetic nanoparticles (MNPs) are used as tracers without ionizing radiation in vascular imaging, molecular imaging, and neuroimaging. The relaxation mechanisms of magnetization in response to excitation magnetic fields are important features of MNPs. The basic relaxation mechanisms include internal rotation (Néel relaxation) and external physical rotation (Brownian relaxation). Accurate measurement of these relaxation times may provide high sensitivity for predicting MNP types and viscosity‐based hydrodynamic states. It is challenging to separately measure the Néel and Brownian relaxation components using sinusoidal excitation in conventional MPI. Purpose: We developed a multi‐exponential relaxation spectral analysis method to separately measure the Néel and Brownian relaxation times in the magnetization recovery process in pulsed vascular MPI. Methods: Synomag‐D samples with different viscosities were excited using pulsed excitation in a trapezoidal‐waveform relaxometer. The samples were excited at different field amplitudes ranging from 0.5 to 10 mT at intervals of 0.5 mT. The inverse Laplace transform‐based spectral analysis of the relaxation‐induced decay signal in the field‐flat phase was performed by using PDCO, a primal‐dual interior method for convex objectives. Néel and Brownian relaxation peaks were elucidated and measured on samples with various glycerol and gelatin concentrations. The sensitivity of viscosity prediction of the decoupled relaxation times was evaluated. A digital vascular phantom was designed to mimic a plaque with viscous MNPs and a catheter with immobilized MNPs. Spectral imaging of the digital vascular phantom was simulated by combining a field‐free point with homogeneous pulsed excitation. The relationship between the Brownian relaxation time from different tissues and the number of periods for signal averages was evaluated for a scan time estimation in the simulation. Results: The relaxation spectra of synomag‐D samples with different viscosity levels exhibited two relaxation time peaks. The Brownian relaxation time had a positive linear relationship with the viscosity in the range 0.9 to 3.2 mPa · s. When the viscosity was >3.2 mPa · s, the Brownian relaxation time saturated and did not change with increasing viscosity. The Néel relaxation time decreased slightly with an increase in the viscosity. The Néel relaxation time exhibited a similar saturation effect when the viscosity level was >3.2 mPa · s for all field amplitudes. The sensitivity of the Brownian relaxation time increased with the field amplitude and was maximized at approximately 4.5 mT. The plaque and catheter regions were differentiated from the vessel region in the simulated Brownian relaxation time map. The simulation results show that the Néel relaxation time was 8.33±0.09 μs in the plaque region, 8.30±0.08 μs in the catheter region, and 8.46±0.11 μs in the vessel region. The Brownian relaxation time was 36.60±2.31 μs in the plaque region, 30.17±1.24 μs in the catheter region, and 31.21±1.53 μs in the vessel region. If we used 20 excitation periods for image acquisition in the simulation, the total scan time of the digital phantom was approximately 100 s. Conclusion: Quantitative assessment of the Néel and Brownian relaxation times through inverse Laplace transform‐based spectral analysis in pulsed excitation, highlighting their potential for use in multi‐contrast vascular MPI. [ABSTRACT FROM AUTHOR]

The current study evaluated the physiochemical quality and gene expression profile of post‐thawed buck semen after supplementation with antioxidants [melatonin (M), L‐carnitine (LC), cysteine (Cys), LC + M, M + Cys, LC + Cys, LC + Cys + M] in comparison with the non‐treated control group. Physical and biochemical characteristics of semen were evaluated following freezing and thawing. Transcript abundance of six selected candidate genes was profile using quantitative real‐time PCR. The data demonstrated significant enhancement of post‐freezing total motility, progressive motility, percentage of live sperm, CASA parameters, plasma membrane and acrosome integrity in all groups supplemented with Cys, LC, M + Cys and LC + Cys compared with the control group. The biochemical analysis of semen indicated that semen groups supplemented with LC and LC + Cys recorded increased levels of GPX and SOD that were coupled with up‐regulation of antioxidant genes (SOD1, GPX1 and NRF2) and mitochondrial transcripts (CPT2 and ATP5F1A). Moreover, H2O2 level and DNA fragmentation percentage were reduced compared with other groups. In conclusion, supplementation of Cys alone or in combination with LC positively improved the post‐thaw physiochemical properties of rabbit semen through activation of bioenergetics‐related mitochondrial genes and cellular antioxidant defence mechanism. [ABSTRACT FROM AUTHOR]

Cryopreservation is the most prevalent method of long‐term cell preservation. Effective cell cryopreservation depends on freezing, adequate storage, and correct thawing techniques. Recent advances in cryopreservation techniques minimize the cellular damage which occurs while processing samples. This article focuses on the fundamentals of cryopreservation techniques and how they can be implemented in a variety of clinical settings. The article presents a brief description of each of the standard cryopreservation procedures, such as slow freezing and vitrification. Alongside that, the membrane permeating and nonpermeating cryoprotectants are briefly discussed, along with current advancements in the field of cryopreservation and variables influencing the cryopreservation process. The diminution of cryoinjury incurred by the cell via the resuscitation process will also be highlighted. In the end application of cryopreservation techniques in many fields, with a special emphasis on stem cell preservation techniques and current advancements presented. Furthermore, the challenges while implementing cryopreservation and the futuristic scope of the fields are illustrated herein. The content of this review sheds light on various ways to enhance the output of the cell preservation process and minimize cryoinjury while improving cell revival. [ABSTRACT FROM AUTHOR]

To increase the red blood cell (RBC) cryopreservation efficiency by metal–organic frameworks (MOFs), a dimensional reduction approach has been proposed. Namely, 3D MOF nanoparticles are progressively reduced to 2D ultra‐thin metal–organic layers (MOLs). We found that 2D MOLs are beneficial for enhanced interactions of the interfacial hydrogen‐bonded water network and increased utilization of inner ordered structures, due to the higher surface‐to‐volume ratio. Specifically, a series of hafnium (Hf)‐based 2D MOLs with different thicknesses (monolayer to stacked multilayers) and densities of hydrogen bonding sites have been synthesized. Both ice recrystallization inhibition activity (IRI) and RBCs cryopreservation assay confirm the pronounced better IRI activity and excellent cell recovery efficiency (up to ≈63 % at a very low concentration of 0.7 mg mL−1) of thin‐layered Hf‐MOLs compared to their 3D counterparts, thereby verifying the dimensional reduction strategy to improved cryoprotectant behaviors. [ABSTRACT FROM AUTHOR]

Developing efficient and eco‐friendly catalysts for selective degradation of waste polyethylene terephthalate (PET) is critical to the circular economy of plastics. Herein, we report the first monatomic oxygen anion (O−)‐rich MgO−Ni catalyst based on a combined theoretical and experimental approach, which achieves a bis(hydroxyethyl) terephthalate yield of 93.7 % with no heavy metal residues detected. DFT calculations and electron paramagnetic resonance characterization indicate that Ni2+ doping not only reduces the formation energy of oxygen vacancies, but also enhances local electron density to facilitate the conversion of adsorbed oxygen into O−. O− plays a crucial role in the deprotonation of ethylene glycol (EG) to EG− (exothermic by −0.6 eV with an activation barrier of 0.4 eV), which is proved effective to break the PET chain via nucleophilic attack on carbonyl carbon. This work reveals the potential of alkaline earth metal‐based catalysts in efficient PET glycolysis. [ABSTRACT FROM AUTHOR]

The combination of electric and magnetic field assisted freezing has potential as a new means of improving the freeze–thaw quality of green peppers. In this work, the quality of the freeze–thawed product was assessed in terms of thawing juice loss, moisture profile, ascorbic acid content, antioxidant activity, flavor, and microstructure. Juice loss was reduced by 16%–68%, freezing time was shortened by 15%–26%, and the nutrient retention rate was higher in the physical field‐assisted case compared to the no‐physical field case. Interestingly, the combined freezing of the two physical fields showed better freezing results compared to a single electric or magnetic field, with juice loss reduced to 3.04%, retention of 82% of calcium ions, retention of ascorbic acid increased by 6%–15%. In addition, the content of hexenal and methyl salicylate and other aromatic substances increased, showing a good flavor quality such as increased umami. The results suggest that combined electric and magnetic field assisted freezing is better in improving the quality of frozen products and may be a potential alternative to freezing and thawing of fruits and vegetables. Practical application: This research provides a simple and novel method for improving the speed and quality of frozen products. These steps combine electric and magnetic fields to explore, improve the quality of frozen products, but also provide a new idea for freezing research. [ABSTRACT FROM AUTHOR]

Eighty‐five sperm samples were cryopreserved and SYBR14/PI, MitoTracker Deep Red FM, FITC‐PSA/PI and chlortetracycline were used for imaging flow cytometry evaluation of sperm viability, mitochondrial membrane potential (MMP), acrosome integrity and sperm capacitation, respectively. Sperm motility was also registered. Sperm motility (46.1 ± 7.7 vs. 24.1% ± 6.5%), sperm viability (49.8 ± 11.5 vs. 32.3% ± 9.6%) and high MMP (49.8% ± 12.4% vs. 34.9% ± 9.9%) decreased significantly (p <.05) during cryopreservation process, in contrast to acrosome‐reacted in viable spermatozoa (1.0% ± 1.6% vs. 1.0% ± 1.0%) and sperm capacitation (10.0 ± 9.8 vs. 8.2% ± 12.4%) that were similar (p >.05) before and after cryopreservation. Positive correlations were found between sperm motility versus high MMP (r =.63), sperm motility versus sperm viability (r =.67) and sperm viability versus high MMP (r =.88). In conclusion, cryopreservation of alpaca spermatozoa is related to a decrease in sperm motility, sperm viability and high MMP, meanwhile acrosome integrity and sperm capacitation are not affected. [ABSTRACT FROM AUTHOR]

Cryopreservation of semen is an important technique to preserve genetic material. Yet, pregnancy rates in jennies after artificial insemination with frozen–thawed donkey semen are poor. This condition has been attributed to the impact of permeable cryoprotectants, that could cause high post‐breeding endometritis. Removal of seminal plasma (SP) prior to semen freezing process is another contributing factor. SP is involved in a multitude of sperm functions and events preceding fertilization and has a mediating effect of sperm capacitation and postcoital uterine inflammatory response. The aim of this study was to evaluate different alternatives in donkey semen cryopreservation with permeable, non‐permeable cryoprotectants, BSA and SP. Thirty ejaculates from 10 donkeys were cryopreserved with different combinations of dimethylformamide (DMF, 5%), sucrose (SUC, 200 mM) and homologous SP (10%): DMF (T1), DMF/SP (T2), SUC/BSA (T3), SUC/BSA/SP (T4), DMF/SUC/BSA (T5), DMF/SUC/BSA/SP (T6), DMF/BSA (T7) and DMF/BSA/SP (T8). After thawing, sperm motility and kinetics were assessed by computerized semen analysis. Sperm vitality (SV) was evaluated by fluorescence microscopy, functional membrane integrity (FMI) by the HOST test, abnormal morphology by eosin‐nigrosin staining and sperm membrane stability by flow cytometry. For statistical analysis, sperm quality indexes (SQi) were obtained, general linear models were carried out and mean comparisons were made by the Tukey test. T1, T2, T5, T6, and T7 had higher and equivalent results for motility, most kinetic parameters and function membrane integrity. Cryopreservation of donkey semen without permeable cryoprotectant (T3 and T4) showed a reduction in motility, kinetics, SV, FMI and SQi. T5 showed a reduction in progressive motility, sperm velocities, IMF and SQi compared to other DMF treatments. T6 and T8 achieved higher SQi values compared to T1, but they were not different compared to T2 and T7. T1 had a smaller sperm population with low‐M540 compared to T3. It is concluded that the use of permeable cryoprotectant is essential to achieve higher post‐thaw quality of donkey semen. In addition, the combined use of BSA, SUC and/or PS may provide additional sperm protection compared to the individual use of DMF. [ABSTRACT FROM AUTHOR]

With the growing sophistication of cloning technology, rescuing cell resources is of great significance for the protection of endangered animals. The Yangtze sturgeon (Acipenser dabryanus), one of the three Acipenseriformes species in the Yangtze River, is critically endangered. Natural reproduction of the Yangtze sturgeon has not been detected since 2000. Less than 20 wild individuals are kept in husbandry, and all are too old to breed. Therefore, it is urgent to rescue the genetic resources of every wild Yangtze sturgeon. Here, we isolated and preserved viable cells from critically endangered postmortem Yangtze sturgeon for the first time. Attempts to rescue and preserve cell resources were carried out from 8 tissues, brain, kidney, gonad, fin, liver, skin, spleen, and muscle of an over 35-year-old female wild Yangtze sturgeon between 11 and 14 hours after death at 19.8°C in an outdoor concrete pond, and only muscle tissue cells could be successfully subcultured and preserved. Furthermore, the cultured cells showed high post-thaw cell viability and normal growth with a population doubling time of 52.98 h. Moreover, they were fibronectin- and desmin-positive, characterizing them as fibroblastic confirmed muscle cells with fibroblastic properties and myogenic origin. Tests for microbial contamination of the cell lines were negative. Chromosome analysis demonstrated that muscle cells possess a modal polyploid chromosome number of 264. The mitochondrial sequence data of COI genes and 12S rRNA confirmed that the developed cell line originated from A. dabryanus. Furthermore, transfection results indicate that muscle cells could be used for gene manipulation and functional studies. These results suggest that viable muscle cells could also be successfully isolated and cryopreserved from the wild Yangtze sturgeon in a short time after death. This report is not only of great significance for the germplasm rescue of critically endangered Yangtze sturgeon but also provides some scientific reference for the germplasm preservation of other endangered fish. [ABSTRACT FROM AUTHOR]

The use of primary material from patients with haematological malignancies as an ex vivo model system for basic and translational research is becoming increasingly popular as researchers continue to understand translational limitations associated with tools like cell lines or organoids.[[1], [3], [5], [7]] Importantly, primary samples contain cancer as well as stroma and immune cells allowing for the interrogation of multi-cellular processes and interactions.[9] Previously, our team and colleagues developed a high-content imaging platform to study single cell anticancer drug action in primary patient samples.[10] Specifically, bone marrow or peripheral blood mononuclear cells (BMMCs or PBMCs) were incubated in non-adherent monolayers in the presence of small molecule drugs. Measuring the drug-induced on-target cytotoxicity of cancer cells versus the off-target cytotoxicity of healthy cells in the same image provided information on a molecule's differential cytotoxicity (Figure S1). A further advantage is the ability to leverage robust laboratory automation to measure drug effects of all lineages of cells simultaneously, independent of a cell being adherent or non-adherent. [Extracted from the article]

In this study, we determined the efficacy of 3,3‐dimethylglutaric anhydride poly‐L‐lysine (DMGA‐PLL) as a cryoprotectant for porcine spermatozoa. Porcine spermatozoa were cryopreserved in a freezing extender containing 3% (v/v) glycerol and various concentrations of DMGA‐PLL. At 12 h after thawing, the motility index of spermatozoa cryopreserved with 0.25% (v/v) DMGA‐PLL (25.9) was significantly (P < 0.01) higher than that of spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA‐PLL (10.0–16.3). In addition, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% DMGA‐PLL (22.8%) was significantly (P < 0.01) higher than that of embryos derived from spermatozoa cryopreserved with 0%, 0.125%, or 0.5% DMGA‐PLL (7.9%–10.9%). The mean number of total piglets born to sows inseminated with spermatozoa cryopreserved without DMGA‐PLL (9.0) was significantly (P < 0.05) lower than that of total piglets born to sows inseminated with spermatozoa stored at 17°C (13.8). However, when spermatozoa cryopreserved with 0.25% DMGA‐PLL were used for artificial insemination, the mean number of total piglets (11.7) was not significantly different from that obtained following artificial insemination using spermatozoa stored at 17°C. The results showed the usefulness of DMGA‐PLL as a cryoprotectant in the cryopreservation of porcine spermatozoa. [ABSTRACT FROM AUTHOR]

Background: Cell therapy is considered a promising strategy for intervertebral disc (IVD) regeneration. However, cell products often require long‐term cryopreservation, which compromises cell viability and potency, thus potentially hindering commercialization and off‐the‐shelf availability. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant, however, DMSO is associated with cytotoxicity and cell viability loss. This study aimed to investigate the effects of DMSO on human nucleus pulposus cells (NPC) and the role of oxidative stress in DMSO‐induced cytotoxicity. Furthermore, we examined the potential of antioxidant N‐acetylcysteine (NAC) supplementation to mitigate the negative effects of DMSO. Methods: NPC were exposed to various concentrations of DMSO with or without a freezing cycle. Cell viability, cell apoptosis and necrosis rates, intracellular reactive oxygen species (ROS) levels, and gene expression of major antioxidant enzymes were evaluated. In addition, NAC was added to cryopreservation medium containing 10% DMSO and its effects on ROS levels and cell viability were assessed. Results: DMSO concentrations ≤1% for 24 h did not significantly affect the NPC viability, whereas exposure to 5 and 10% DMSO (most commonly used concentration) caused cell viability loss (loss of 57% and 68% respectively after 24 h) and cell death in a dose‐ and time‐dependent manner. DMSO increased intracellular and mitochondrial ROS (1.9‐fold and 3.6‐fold respectively after 12 h exposure to 10% DMSO) and downregulated gene expression levels of antioxidant enzymes in a dose‐dependent manner. Tempering ROS through NAC treatment significantly attenuated DMSO‐induced oxidative stress and supported maintenance of cell viability. Conclusions: This study demonstrated dose‐ and time‐dependent cytotoxic effects of DMSO on human NPC. The addition of NAC to the cryopreservation medium ameliorated cell viability loss by reducing DMSO‐induced oxidative stress in the freeze–thawing cycle. These findings may be useful for future clinical applications of whole cells and cellular products. [ABSTRACT FROM AUTHOR]

Gymnocypris przewalskii (Naked carp), a native teleost, plays an important role in maintaining the ecological balance of Lake Qinghai (altitude, 3.2 km), the largest saline lake in China. In this study, a new gill cell line from G. przewalskii was developed using the explant technique and named as GPG. This cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM) (high glucose), supplemented with 15% fetal bovine serum (FBS), and was successfully subcultured up to 32 passages. Meanwhile, this cell line was also authenticated by sequencing the mitochondrial cytochrome C oxidase subunit I (COI) and 16S rRNA genes and by chromosome analysis. With the Cytomegalovirus (CMV) promoter, the GPG cell line could express green fluorescent protein (GFP) at about 5% transfection efficiency. MTT test showed that Clostridium botulinum toxin (BTX) was toxic to the cell line. After cryopreservation with 10% dimethyl sulfoxide (DMSO), this cell line could be successfully revived at an efficiency over 70%. This study revealed that the GPG cell line could be used as materials for physio‐chemical investigation of G. przewalskii and also provided a tool for gene function study and toxicological reaction in vitro. [ABSTRACT FROM AUTHOR]

In this study, the influence of myoinositol (MYO) as an antioxidant on the inhibition of the negative impacts of cryopreservation on sperm quality in men with Asthenospermia was investigated. In this prospective study, each semen sample from 25 cases was separated into three groups: Fresh, Control (with freezing medium), Myoinositol (2 mg/ml). According to the World Health Organization criteria (WHO) (2010), total motility, progressive sperm motility, viability, normal morphology, and DNA integrity were assessed. In addition, the hypo‐osmotic swelling (HOS) test and mitochondrial membrane potential (MMP) were used. Total antioxidant capacity (TAC), malondialdehyde (MDA), and antioxidant enzyme activity were determined by the ELISA method. In contrast to the fresh samples, lipid peroxidation, DNA integrity damage, DNA fragmentation, HOST, and MMP had significant enhancement in the control samples. Sperm quality was significantly decreased (p < 0.05). Mean percentage viability, normal morphology, total motility, progressive motility, and DNA integrity were significantly enhanced in the MYO group in comparison to the control group (p < 0.05). The MDA and TAC levels and DNA damage in the MYO group were significantly lower compared to the control group (p < 0.05). The findings confirm that sperm quality in patients with Asthenospermia is improved by the administration of 2 mg/ml of myoinositol together with the freezing medium after sperm cryopreservation. [ABSTRACT FROM AUTHOR]

Enhancing the cooling rate is often an alternative to increasing concentrations of cryoprotectants to inhibit harmful ice growth during freezing. Processing cell suspensions as dispersed droplets can improve the cooling rate but carries the risk of difficult collection and contamination. As the most readily available cells, human red blood cells (RBCs) are used to test the feasibility of advanced cryopreservation techniques. Herein, a feasible and straightforward method for scalable cryopreservation of RBCs is presented. This method can be easily realized by selecting 5 × 6 mm polytetrafluoroethylene (PTFE) tubes with better cooling capacity and applying the trehalose dehydration technique. Finally, the highest freeze‐thaw recovery rate in the available literature with trehalose alone (93.87% with 0.4 m Tre) and freezing the volume of RBCs from 2 to 20 mL with a higher freeze‐thaw recovery of 95–96% after adding 5% glycerol (0.4 m Tre + 5% Gly) are achieved. A two‐step washing method is also evaluated to remove the cryoprotectants, allowing the process could be completed within 20 min after thawing. Furthermore, the functional expression of postwash RBCs after cryopreservation is examined and all results show that this scalable volume cryopreservation method can be extended to current clinical RBC cryopreservation. [ABSTRACT FROM AUTHOR]

The cold‐adapted bacterium Pseudomonas sp. ID1 produces the extracellular exopolysaccharide ID1 (EPS ID1) with cryoprotective activity. This study was designed to optimize the vitrification/in‐straw warming protocol of in vitro‐produced (IVP) blastocysts by adding EPS ID1 to the vitrification media. Day 7‐expanded blastocysts were vitrified/warmed using the VitTrans device after the addition of 0 or 100 μg/mL EPS ID1 to the vitrification media. Blastocysts vitrified by the Cryotop method and fresh non‐vitrified blastocysts served as controls. Outcomes were assessed in the warmed embryos in terms of survival rates and mRNA relative abundances of BAX, BCL2, GPX1, and CDX2 genes. No differences in survival rates were observed at 3 h post‐warming between vitrification treatments. At 24 h post‐warming, the addition of EPS prior to vitrification with the VitTrans device produced similar survival rates to Cryotop‐vitrified embryos and similar hatching rates to fresh non‐vitrified or Cryotop‐vitrified embryos. No differences emerged in BCL2 gene expression. Lower BAX (p <.05) and higher GPX1 (p <.05) and CDX2 (p <.1) gene expression were observed in expanded and/or hatched blastocysts derived from VitTrans‐EPS‐vitrified embryos when compared to those from the non‐supplemented group. In conclusion, addition of EPS not only promoted blastocyst survival and hatching after VitTrans vitrification/warming but also modified the expression of genes associated with better embryo quality. [ABSTRACT FROM AUTHOR]

The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified‐warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10−9 M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10−9 M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60‐min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2′,7′‐dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p <.01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p <.05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified‐warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10−9 M melatonin during in vitro maturation had no effect on these parameters. [ABSTRACT FROM AUTHOR]