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Vitrification is a promising approach for ice-free cryopreservation of biological material, but progress is hindered by the limited set of experimental tools for studying processes in the interior of the vitrified matter. Isochoric cryopreservation chambers are often metallic, and their opacity prevents direct visual observation. In this study, we introduce photon counting X-ray computed tomography (CT) to compare the effects of rigid isochoric and unconfined isobaric conditions on vitrification and ice formation during cooling of two aqueous solutions: 50 wt% DMSO and a coral vitrification solution, CVS1. Previous studies have only compared vitrification in isochoric systems with isobaric systems that have an exposed air-liquid interface. We use a movable piston to replicate the surface and thermal boundary conditions of the isochoric system yet maintain isobaric conditions. When controlling for the boundary conditions we find that similar ice and vapor volume fractions form during cooling in isochoric and isobaric conditions. Interestingly, we observe distinct ice and vapor cavity morphology in the isochoric systems, possibly due to vapor outgassing or cavitation as rapid cooling causes the pressure to drop in the confined systems. These observations highlight the array of thermal-fluid processes that occur during vitrification in confined aqueous systems and motivate the further application of imaging techniques such as photon counting X-ray CT in fundamental studies of vitrification.

Liver biobanking is a promising approach that saves the lives of patients with end-stage liver disease. Cryopreservation based on vitrification enables semi-permanent organ preservation, contributing to overcome the shortage of donors for liver transplants. A technical challenge in cryopreservation of transplantable organs lies in thawing methodology, and conventional convective warming cannot maintain the glassy state during thawing because of the large temperature gradient between the inner and outer parts of the organs, leading to ice formation and damage of cells in the organ. Nanowarming, in which magnetic nanoparticles are dispersed in a vitrification solution and heated by exposure of alternating magnetic field, can achieve uniform and rapid heating of organs. Herein, we report that amphiphilic phospholipid polymers composed of 2-methacryloyloxyethyl phosphorylcholine and n -butyl methacrylate can function as a cryoprotectant for nanowarming. The amphiphilic phospholipid polymers enhanced the viability of primary rat hepatocytes after vitrification. Moreover, the polymers enhanced the dispersion stability of magnetic nanoparticles in vitrification solution, and the perfusion of the vitrification solution with magnetic nanoparticles into rat livers through portal vein provided uniform distribution of the nanoparticles in the liver. After perfusion, the vitrified liver was successfully thawed rapidly and uniformly by nanowarming, which maintained tissue integrity and cell viability. [ABSTRACT FROM AUTHOR]

Cardiotoxicity associated with hepatic metabolism and drug–drug interactions is a serious concern. Predicting drug toxicity using animals remains challenging due to species and ethical concerns, necessitating the need to develop alternative approaches. Drug cardiotoxicity associated with hepatic metabolism cannot be detected using a cardiomyocyte-only evaluation system. Therefore, we aimed to establish a system for evaluating cardiotoxicity via hepatic metabolism by co-culturing cryopreserved human hepatocytes (cryoheps) and human iPS cell-derived engineered heart tissues (hiPSC-EHTs) using a stirrer-based microphysiological system. We investigated candidate media to identify a medium that can be used commonly for hepatocytes and cardiomyocytes. We found that the contraction length was significantly greater in the HM Dex (-) medium, the medium used for cryohep culture without dexamethasone, than that in the EHT medium used for hiPSC-EHT culture. Additionally, the beating rate, contraction length, contraction speed, and relaxation speed of hiPSC-EHT cultured in the HM Dex (-) medium were stable throughout the culture period. Among the major CYPs, the expression of CYP3A4 alone was low in cryoheps cultured in the HM Dex (-) medium. However, improved oxygenation using the InnoCell plate increased CYP3A4 expression to levels comparable to those found in the human liver. In addition, CYP3A4 activity was also increased by the improved oxygenation. Furthermore, expression levels of hepatic function-related gene and nuclear receptors in cryoheps cultured in HM Dex (-) medium were comparable to those in the human liver. These results suggest that the HM Dex (-) medium can be applied to co-culture and may allow the evaluation of cardiotoxicity via hepatic metabolism. Moreover, CYP induction by typical inducers was confirmed in cryoheps cultured in the HM Dex (-) medium, suggesting that drug–drug interactions could also be evaluated using this medium. Our findings may facilitate the evaluation of cardiotoxicity via hepatic metabolism, potentially reducing animal testing, lowering costs, and expediting drug development. [ABSTRACT FROM AUTHOR]

Simple Summary: The Czech Golden Spotted Hen is the only original Czech hen breed included in the National Genetic Reserve Program. The objective of this program is to preserve the genetic resources of native breeds in the Czech Republic through reproductive biotechnologies, such as artificial insemination or the cryopreservation of sperm. The objective of the present study was to investigate the impact of varying concentrations of the non-penetrating cryoprotectant trehalose on sperm fertilization capacity. This was achieved by assessing sperm functional and kinematic parameters, as well as fertility rates. The established methodology was used to optimize the cryopreservation procedure for the National Program of Genetic Reserves of the Czech Golden Spotted Hen breed. The objective of this study was to evaluate the impact of the supplementation of varying concentrations of the impermeable disaccharide trehalose on the in vitro and in vivo fertilization capacity of cryopreserved rooster spermatozoa in the original Czech Golden Spotted Hen breed. The control trehalose concentration was 0 mM, while TRE50 (50 mM), TRE100 (100 mM), and TRE200 (200 mM) were used as experimental trehalose concentrations. The kinematic and functional parameters of frozen/thawed spermatozoa were evaluated in vitro using mobile computer-assisted sperm analysis and a flow cytometer. The addition of 100 mM trehalose demonstrated the most favorable results for total (34.17%) and progressive (3.57%) motility after thawing. A statistically significant difference was found for these kinetic parameters compared to the other monitored concentrations. This experimental group was also found to have a significantly higher percentage of spermatozoa without plasma membrane or acrosome damage (33.37%) compared to the TRE50 group (30.74%; p < 0.05) and the TRE200 group (29.05%; p < 0.05). In vivo, artificial insemination was performed to verify fertilization ability. Hens (n = 40) were artificially inseminated twice (10 hens/treatment) with a 3-day interval between inseminations. In conclusion, the addition of 100 mM trehalose significantly improved total and progressive motility after thawing and preserved plasma membrane and acrosome integrity (p < 0.05). The fertilization rate of eggs fertilized with semen frozen with the addition of 100 mM trehalose was not significantly different from the other concentrations tested or the control group but was numerically higher (23.21% vs. 15.20% of fertilized eggs in this group). [ABSTRACT FROM AUTHOR]

This study aimed to examine the effect of adding different concentrations of sorbitol to the Ringer’s lactate-egg yolk (RLEY) diluent on the quality of Gaga roosters’ semen during cold storage. A completely randomized design was used with 10 replicates and 4 levels of diluent treatment, including control, 1%, 2%, and 3% sorbitol. Semen was collected using a massage method, followed by fresh semen evaluation and dilution with a ratio of 1:5. Semen was stored at a cold temperature (5 ºC) and observed after 0, 24, 48, and 72 hours. The variables observed were semen pH, motility, kinematics, viability, plasma membrane integrity, acrosome integrity, malondialdehyde (MDA), and intracellular calcium ion (Ca2+) concentrations of sperm. All data were analyzed using analysis of variance (ANOVA). Subsequently, the evaluation of sperm mitochondrial activity and DNA damage was carried out, and the data were analyzed using the T-test. The results showed that treatment with sorbitol in the diluent had no effect on semen pH, kinematics (except straightness), acrosomal cap integrity, DNA damage, mitochondrial activity, and MDA concentration. However, sorbitol concentrations significantly affect (p<0.05) total motility, progressive motility, static motility, STR, viability, plasma membrane integrity, and intracellular Ca2+. In conclusion, the addition of 1-3% sorbitol preserves the quality of Gaga roosters’ semen during 72 hours of cold storage. [ABSTRACT FROM AUTHOR]

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]

What can't freezing hold still? This article surveys the history of substances used to protect cells and organisms from freezing damage, known as cryoprotectants. Dimethyl sulfoxide (DMSO) has since 1959 been the most widely used of these agents in cryopreservation. Here, its evolution from pulp and paper waste byproduct to wonder drug to all-but-invisible routine element of freezing protocols is used to trace the direct arc from protection to toxicity in theories of how and why cryoprotectants work, from the 1960s to today. The power of these agents to simultaneously protect and degrade is shown to reside in manipulation of chemical time via hydrogen bonding and electron exchange, thereby reframing freezing as a highly active and transformational process. Countering long-held assumptions about cryopreservation as an operation of stasis after which the thawed entity is the same as it was before, this article details recent demonstrations of effects of cryoprotectant exposure that are nonlethal but nonetheless profoundly impactful within scientific and therapeutic practices that depend on freezing infrastructures. Understanding the operationalization of chemical time in the case of cryoprotectants is broadly relevant to other modern technologies dedicated to shifting how material things exist and persist in human historical time. [ABSTRACT FROM AUTHOR]

An essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs. A DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5–10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis. MSCs had an average viability of 94.3% (95% CI: 87.2–100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03–9.0%; P : 0.049) and 11.4% (95% CI: 6.9–15.8%; P < 0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7–100.0%), and it was lower by 5.6% (95% CI: 1.3–9.8%, P < 0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90, and CD105 with no significant difference in global gene expression profiles. MSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution. [ABSTRACT FROM AUTHOR]

Cold brew is a method of coffee extraction that uses low temperature, preserving the volatile compounds of coffee. Freeze-drying allows the preservation of coffee features and nutritional value. The aim of this study was to evaluate the effects of different cryoprotectants in cold brew extracts as a basis for freeze-dried coffee production. Thus, the Coffea arabica extracts and the soluble coffee were characterized concerning caffeine content, antioxidant capacity, total phenolic compounds, and antimicrobial activity to verify the potential of this method. The extracts did not show antimicrobial activity with a high soluble solid content. It was observed that the cold extraction methods were efficient regarding the caffeine content, antioxidant capacity, and total phenolic compounds. Freeze-dried coffees also did not show antimicrobial activity, and they maintained the water and humidity activity standards. In general, cryoprotectants displayed an unfavorable influence on the extract and freeze-dried coffee in the analyses performed. The coffee extract without cryoprotectants had a higher antioxidant capacity (88.12%) and content of phenolic compounds (7.74 mg AG/mL of the coffee extract). Only for the analyses of soluble solids, the cryoprotectants mannitol and fructose showed promising results (14.03 °Brix, 14.40 °Brix, 11.33 °Brix, respectively). Thus, for the analyses conducted, the cryoprotectants did not lead to significant advantages for this process. [ABSTRACT FROM AUTHOR]

During cryopreservation, a substantial portion of spermatozoa undergoes apoptosis due to cryoinjury, resulting in decreased fertility. Boar spermatozoa are highly sensitive to temperature, with low temperature triggering reactive oxygen species (ROS) generation, leading to oxidative stress and apoptosis. Sulforaphane (SFN), a potent natural compound found in cruciferous vegetables, is efficacious in mitigating oxidative stress. We here supplemented different SFN concentrations (0, 1.25, 2.5, 5, 10, and 20 μM) into the freezing extender to explore its effect on boar sperm during cryopreservation and determine the optimal SFN concentration. Supplementation of 5 μM SFN exhibited the highest sperm motility, motion performance, plasma membrane integrity, acrosome integrity, and antioxidant properties (total antioxidant capacity (T-AOC) and antioxidant enzyme activity) after freezing and thawing. Then, RT group, C group and C + SFN group were established to explore the effect of SFN on the cryopreservation-induced sperm apoptosis level and fertilizing capacity of post-thawed sperms. SFN effectively rescued the apoptosis and fertilizing capacity of post-thawed sperms. Mechanistically, SFN activated the redox-sensitive nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) by promoting adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. This activation improved antioxidant defenses, ultimately improving cryoinjury in boar spermatozoa. In summary, SFN suppressed cryopreservation-induced apoptosis of spermatozoa by activating antioxidant defenses and the AMPK/NFE2L2 signaling pathway. These findings suggest a novel approach for augmenting the cryoprotective efficiency and spermatozoa fertility after cryopreservation. • SFN improved the quality of boar spermatozoa after cryopreservation. • SFN increased the antioxidant capacity of boar spermatozoa after cryopreservation. • SFN attenuated boar spermatozoa apoptosis induced by cryopreservation. • SFN exerts the beneficial effects by activating the NFE2L2/AMPK signaling pathway. [ABSTRACT FROM AUTHOR]

In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines. • There are differences in follicular cryotolerance according to the cryopreservation technique used. • Primordial and immature follicles are less prone to degeneration when cryopreserved by slow freezing. • Mature follicles are more sensitive to cryodamage and better preserved by vitrification. [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]

The cryopreservation of gametes and embryos has increased notably over the past 20 years and is now an essential part of assisted reproductive technologies (ARTs). However, because the cryopreservation process is un-physiological for human cells, gametes, and embryos, cryobiologists have suggested diverse methods to successfully cryopreserve human gametes and embryos in order to maintain their viability and assure successful pregnancy. During the first period of early development, major waves of epigenetic reprogramming—crucial for the fate of the embryo—occur. Recently, concerns relating to the increased incidence of epigenetic anomalies and genomic-imprinting disorders have been reported after ARTs and cryopreservation. Epigenetic reprogramming is particularly susceptible to environmental and un-physiological conditions such as ovarian stimulation, embryo culture, and cryopreservation that might collectively affect epigenetics dysregulation. Additionally, recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, osmotic shock, oxidative stress, rapid temperature and pH changes, and cryoprotectants; it is therefore critical to have a more comprehensive understanding of the potential induced perturbations of epigenetic modifications that may be associated with vitrification. The aim of this paper is to present a critical evaluation of the association of gamete and embryo cryopreservation, use of cryoprotectants, and epigenetic dysregulations with potential long-term consequences for offspring health. [ABSTRACT FROM AUTHOR]

The biological products used in orthopedics include musculoskeletal allografts—such as bones, tendons, ligaments, and cartilage—as well as biological therapies. Musculoskeletal allografts support the body's healing process by utilizing preserved and sterilized donor tissue. These allografts are becoming increasingly common in surgical practice, allowing patients to avoid more invasive procedures and the risks associated with donor site morbidity. Bone grafting is one of the most frequently used procedures in orthopedics and traumatology. Biologic approaches aim to improve clinical outcomes by enhancing the body's natural healing capacity and reducing inflammation. They serve as an alternative to surgical interventions. While preliminary results from animal studies and small-scale clinical trials have been promising, the field of biologics still lacks robust clinical evidence supporting their efficacy. Biological therapies include PRP (platelet-rich plasma), mesenchymal stem cells (MSCs)/stromal cells/progenitor cells, bone marrow stem/stromal cells (BMSCs), adipose stem/stromal cells/progenitor cells (ASCs), cord blood (CB), and extracellular vesicles (EVs), including exosomes. The proper preservation and storage of these cellular therapies are essential for future use. Preservation techniques include cryopreservation, vitrification, lyophilization, and the use of cryoprotective agents (CPAs). The most commonly used CPA is DMSO (dimethyl sulfoxide). The highest success rates and post-thaw viability have been achieved by preserving PRP with a rate-controlled freezer using 6% DMSO and storing other cellular treatments using a rate-controlled freezer with 5% or 10% DMSO as the CPA. Extracellular vesicles (EVs) have shown the best results when lyophilized with 50 mM or 4% trehalose to prevent aggregation and stored at room temperature. [ABSTRACT FROM AUTHOR]

The permeation of cryoprotectants into insect embryos is critical for successful cryopreservation. However, the permeability of silkworm embryos to cryoprotectants and the effects of cryopreservation remain poorly studied. In this study, we evaluated the permeability and toxicity of four cryoprotective agents (CPAs) as well as the vitrification effect on the viability of silkworm embryos. Among the four CPAs, propylene glycol (PG) showed the best permeability. Ethylene glycol (EG) and PG were the least toxic CPAs, but glycerol (GLY) and dimethyl sulfoxide (DMSO) were more toxic. Moreover, we examined several factors including the kind and the concentration of CPAs, exposure time, embryonic stage, and silkworm strains. Embryos at the earlier phases of stage 25 were more tolerant to vitrification using EG. We found that over 21% of embryos treated with EG at the early 2 phase of stage 25: 163 h after egg laying (AEL) developed and progressed to serosa ingestion after vitrification and rewarming. The result was the same in other strains as well. Our results are valuable for the development of new cryopreservation protocols of silkworm embryos. [ABSTRACT FROM AUTHOR]

Melatonin was discovered in plants in 1995, and so-called phytomelatonin. Its characteristic qualities as an antioxidant molecule and with amphiphilic nature make it an excellent candidate to use in plant biotechnological techniques. In plant physiology, melatonin has demonstrated with multiple studies great possibilities of application in crop improvement in aspects such as germination, rooting, plant growth, leaf-senescence, floration, fruit-set, parthenocarpy, fruit ripening and cut flower conservation, among others. In this work, we reviewed many studies on the possible role of melatonin to improve the different plant tissue culture goals, among them: the role of melatonin in callus culture induction and its effects in plant biomass production; the possible action of melatonin as an inductor or elicitor of different secondary metabolite generation in in vitro cultures; the interesting possibilities of melatonin as a protective agent (safener) against abiotic stressors present in in vitro cultures; the potential of melatonin in regulating plant organogenesis, modulating shooting/rooting responses; at last, the studies on the relevant role of melatonin in plant material cryopreservation and the improvement of cell transformation using A. tumefaciens have been discussed. [ABSTRACT FROM AUTHOR]

Cryopreservation is a reliable technique for the long-term storage and preservation of embryogenic cells, maintaining their viability without loss of their embryogenic capacity. However, the large-scale conservation of grapevine embryogenic lines in cryobanks remains limited. A significant challenge is understanding somatic cell rejuvenation. Here, we investigate the encapsulation/dehydration and encapsulation/vitrification for cryopreserving embryogenic material. Cell rejuvenation and enhanced embryogenic competence were observed after cryopreservation, as evidenced through structural cellular changes observed by histology and electron scanning microscopy. Results showed that cryopreserved samples of 110-Richter, Riesling, and Tempranillo using encapsulation/dehydration had better survival rates, averaging 81%, 62%, and 48%, respectively, while encapsulation/vitrification yielded lower survival rates, averaging 58%, 42%, and 32%, respectively. Cryopreservation also improved post-thaw recovery and regeneration efficiency assessed through regrowth of proembryogenic masses and somatic embryo conversion reaching 54–72% against 11–17% in control samples. Cryopreservation triggered changes in gene expression patterns and exhibited considerable increase at genotype-specific basis of 1.5- to 4.5-fold in SERK1, BBM, and WOX associated to embryogenic competence as well as in ChitIV and LEA involved in stress response. Membrane stability index, hydrogen peroxide, and proline contents were used as indicators of oxidative stress uncovering a key role of an osmotic trans-priming effect leading to cryotolerance. Our finding highlighted that cryopreservation enhances embryogenic capacity in senescent callus and probably acts as a screening process allowing safe maintenance of proembryogenic cells and promoting their recovery. This study provides a high throughput innovation to set up cryolines for cell rejuvenation of grapevine and other important plant species. [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 spermatogonia could be a useful tool to preserve the genetic resources of fish, which could be further restored via germ cell transplantation. In this study, the protocol for the cryopreservation of the spermatogonia of Asian sea bass (Lates calcarifer), an economically important fishery resource in the Indo-West Pacific, was optimised. The impact of the cryopreservation technique on cell viability and apoptosis, expression of several genes related to immature germ cell markers, transplantability in allogeneic recipients, and global DNA methylation was evaluated. The slow-freezing method was performed for the cryopreservation of immature testis tissue, which contains a high proportion of spermatogonia. The optimal condition that yielded the highest recovery rate of post-thawed spermatogonia included a cryomedium containing Leibovitz's (L-15) medium and 10 % dimethyl sulfoxide, ice equilibration for 60 min before freezing, and subsequent thawing at 4 °C for 8 min. Moreover, a higher number of early and late apoptotic cells was detected in the cryopreserved than in the fresh testes, suggesting that apoptosis could result in reduced viability. The expression levels of dazl decreased in the cryopreserved testes; however, there were no significant differences in the expression levels of nanos2 or nanos3 between the fresh and cryopreserved testes. Although qRT-PCR showed lower vasa expression in cryopreserved testicular cells, in situ hybridisation showed expressed vasa in the cryopreserved testicular cells. Post-thawed spermatogonia could be incorporated into the genital ridge of allogeneic recipients, suggesting that cryopreserved spermatogonia exhibit transplantability characteristics. Compared with fresh testes, significant changes in the proportion of DNA methylation (decreased 5-mC and 5-caC) were observed in cryomedium-free testicular cells, whereas those of the cryopreserved cells were not significantly different. Therefore, the method we developed for the cryopreservation of the spermatogonia of Asian sea bass enabled post-thaw cells to retain several stemness characteristics and maintain their epigenetic stability. • Cryopreservation of the spermatogonia of Asian sea bass was optimised. • Cryopreserved spermatogonia exhibit transplantability characteristics. • Our method enabled post-thaw cells to retain several stemness characteristics. • Post-thaw cells maintained their epigenetic stability through our method. [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]

Coral reefs are being degraded at alarming rates and decisive intervention actions are urgently needed. One such intervention is coral cryopreservation. Although the cryopreservation of coral sperm and larvae has been achieved, preservation of coral fragments including both its tissue and skeleton, has not. The overarching aim of this study was to understand and assess the physiological stressors that might underlie coral fragment cryopreservation, understand the long-term consequences of these exposures to continued growth, and develop a health metrics scale for future research. Therefore, we assessed small fragments (~1 cm2) from the Hawaiian coral, Porites compressa, examining: (1) chill sensitivity; (2) chemical sensitivity to complex cryoprotectants; (3) methods to safely remove algal symbionts of coral for cryopreservation; (4) continued growth over time of coral fragments exposed to chilling and cryoprotectants; and (5) assessment of health and viability of coral fragments post the applied treatments. Corals were able to withstand chilling to 0 °C for 1 min and after 2 weeks were not significantly different from the live controls, whereas, corals exposed to complex cryoprotectants needed 3 weeks of recovery. Most importantly, it appears that once the coral fragments had surpassed this initial recovery, there was no difference in subsequent growth. Technological advances in cryo-technology promise to support successful coral fragment cryopreservation soon, and its success could help secure much of the genetic and biodiversity of reefs in the next decade. [ABSTRACT FROM AUTHOR]

Simple Summary: There is a growing demand for the development of optimal protocols for ovarian tissue cryopreservation in different animal models to enable the maintenance of cell morphology. Therefore, it is crucial to understand the effects of conservation on ovarian cell proliferation and apoptosis to improve follicular development after in vitro culture and/or ovarian tissue transplantation. This study compared the effects of three concentrations (1, 1.5, and 3 M) of dimethyl sulfoxide (DMSO) during vitrification to identify optimal conditions for preserving bovine preantral follicles and to contribute to the improvement of the technique and its future application in assisted reproduction procedures. Following vitrification, cryopreserved ovarian fragments exhibited similar percentages of intact follicles, with only the 3 M DMSO concentration differing from the control. Upon analyzing the free radical production, the 3 M concentration exhibited higher oxidative stress levels than lower DMSO concentrations. Exposure to 3 M DMSO for 30 min led to a higher rate of follicular degeneration. After in vitro cultivation of the vitrified fragments, the 1 M DMSO concentration showed higher percentages of morphologically intact follicles than the other concentrations. Accordingly, bovine preantral follicles could be cryopreserved in situ with greater efficiency in 1 M DMSO. Ovarian tissue cryopreservation has been widely investigated for preserving female fertility. In the present study, we aimed to compare the effects of three concentrations (1, 1.5, and 3 M) of dimethylsulfoxide (DMSO) on the vitrification of ovarian tissue. The ovarian cortex was divided into control and vitrified groups: (i) 1 M-DMSO, (ii) 1.5 M-DMSO, and (iii) 3 M-DMSO. Follicles from all fragments were analyzed for DMSO-induced deleterious effects, morphological and morphometric aspects, and concentration of reactive oxygen species. Additionally, the fragments were cultured to assess the integrity and return of follicular development post-vitrification. All DMSO concentrations resulted in a higher percentage of degenerated preantral follicles than before the cryopreservation process. After vitrification, the cryopreserved ovarian fragments showed similar percentages of intact follicles; however, the 3 M DMSO concentration differed from the control. Analyzing free radical production, we found that the 3 M DMSO concentration had higher levels of oxidative stress than the lower DMSO. After in vitro cultivation of the vitrified/warmed fragments, the 1 M DMSO concentration exhibited higher percentages of morphologically intact follicles than the other concentrations. Therefore, we suggest that bovine preantral follicles can be cryopreserved in situ with greater efficiency in 1 M DMSO. [ABSTRACT FROM AUTHOR]

Abstract Mesenchymal stem cells (MSCs) are a type of cell capable of regulating the immune system, as well as exhibiting self-renewal and multi-lineage differentiation potential. Mesenchymal stem cells have emerged as an essential source of seed cells for therapeutic cell therapy. It is crucial to cryopreserve MSCs in liquid nitrogen prior to clinical application while preserving their functionality. Furthermore, efficient cryopreservation greatly enhances MSCs’ potential in a range of biological domains. Nevertheless, there are several limits on the MSC cryopreservation methods now in use, necessitating thorough biosafety assessments before utilizing cryopreserved MSCs. Therefore, in order to improve the effectiveness of cryopreserved MSCs in clinical stem cell treatment procedures, new technological techniques must be developed immediately. The study offers an exhaustive analysis of the state-of-the-art MSC cryopreservation techniques, their effects on MSCs, and the difficulties encountered when using cryopreserved MSCs in clinical applications.

This research examines the analytical investigations for radiating Casson nanofluid models with unsteady convecting flow, which are valued by the order of Caputo fractional derivative (CFD). Ethylene glycol (EG) is used as a base fluid, and graphene oxide (GO) and carbon nanotubes (CNTs) are used as nanoparticles. The problem's leading PDEs are nondimensionalized by applying the proper nondimensional variables. The solutions to the dimensionless governing equations are found by using the Fourier sine and Laplace transformation techniques together. For an enormous study of the problem, graphical illustrations and tables are developed by using MATLAB software programming with the help of Euler inversion. We examine the impact on the fractional heat and momentum equation of the α , Gr, Pr, ϕ , R, β , oscillations. Using the properties of the fluid, important discoveries were made that indicated a number of elements for a number of flow parameters as well as fractional parameters. The thermal profiles are increased for R , ϕ decreased for α at τ = 0. 4 and τ = 1. 4. The velocity profiles are increased for R and Gr decreased for β and ϕ at τ = 0. 4 and 1.4. Different shapes of nanoparticles are performed for ordinary fractional parameters, which are increased for temperature as well as velocity. [ABSTRACT FROM AUTHOR]

The cryopreservation of cell lines such as primordial germ cells and germ cells is a promising strategy to conserve and reconstitute endangered or commercially important species in aquaculture. In Mexico, the northwest region is the center of the country's most significant fishing and aquaculture production. However, most of the species used in capture fishing are overexploited. Despite this, protocols for the cryopreservation of germ cells are non-existent. Therefore, this work aimed to establish a protocol of isolation, identification, and cryopreservation of germ cells in two species, totoaba (Totoaba macdonaldi) and yellowtail amberjack (Seriola lalandi). Three concentrations of trypsin (0.25%, 0.3%, and 0.5%) were tested for gonadal dissociation. The 0.3% trypsin concentration was the best because it presented the most significant number of viable cells, with 14.35 x 105 for totoaba and 2.96 x 105 for yellowtail amberjack. The immunohistochemistry identification of germ cells in both species was positive for vasa, with 33.30% for totoaba and 34.20% for yellowtail amberjack. The cryoprotectant used was ethylene glycol (1.5 M or 2 M). The ideal temperature for the cryopreservation of gonadal tissue was different for each species, -1°C/min for totoaba and -5°C/min for yellowtail amberjack with 58.42% and 63.48% viable cells after thawing, respectively, with ethylene glycol 1.5 M being the best for both species. The non-controlled rate was the most effective technique to freeze the cell suspension, with 4.20 ± 1.09 x 105/mL viable cells for totoaba and 7.31 ± 2.25 x 105/mL for yellowtail amberjack. In conclusion, the results of the isolation, identification, and cryopreservation protocols for germ cells in totoaba and yellowtail amberjack obtained in this work are the first report for fish species from northwest Mexico, opening the door for the generation of cryobanking of germ cells. Finally, this work would help conserve endangered species and be an alternative to conserving species of commercial importance in aquaculture. [ABSTRACT FROM AUTHOR]

In this review, the physicochemical and conformational changes of myofibrillar proteins (MPs) of freeze-induced mince-based aquatic foods were comprehensively summarized in depth. Studies have demonstrated that temperature fluctuation and long-time freezing negatively affect food quality, resulting in texture alteration, drip fluid, flavor degradation, and nutrition loss due to MPs denaturation, aggregation, and oxidation. Attempts have been made in ice-recrystallization inhibition, freezing point depression, and ice shape and growth control for better cryopreservation. Moreover, to further minimize the quality deterioration, cryoprotectants were acknowledged to reduce the denaturation and aggregation of the MPs effectively. Recently, interest in novel functional ingredients, including oligosaccharides, protein hydrolysates, and natural polyphenols demonstrated excellent cryoprotective effects while avoiding health concerns and undesirable flavor caused by traditional sugar-based or phosphates-based cryoprotectants. Therefore, the present review provides a systematic overview of these low molecular weight multifunctional substances with a particular sequence and highlights their underlying mechanism in the inhibition of ice recrystallization the stabilization of MPs. [ABSTRACT FROM AUTHOR]

Larval cryopreservation techniques have been established in Pacific oysters and Mediterranean mussels. Although initial cryopreservation protocols for both species differed slightly in Ficoll PM 70 (FIC) concentration, better post-thaw larval survivability was produced in mussels than oysters. Furthermore, the post-thaw survivability in the latter could be improved significantly by the addition of lipids and antioxidants to the cryoprotectant agent. These findings have generated a unique opportunity to investigate the cryo-functions of both endogenous and exogenous fatty acids (FAs) in bivalves as mammalian studies indicated that lipid compositions could contribute the interspecific difference in gamete and embryo cryopreservation. Our study showed that the higher survivability of post-thaw larvae in mussels (65% vs. 34% in oysters) could be attributed to their higher proportion of total polyunsaturated fatty acids (PUFAs), especially C22:6, and higher resistance of FA profiles to cryopreservation. In oysters, on the other hand, their FA profiles were very sensitive to cryopreservation, with saturated FAs and PUFAs being significantly increased (from 37 to 41%) and decreased (from 35% to ~ 32%), respectively. Although exogenous lipids could significantly improve the survivability of post-thaw oyster larvae from 34 to 51%, their supplementation did not alter the response pattern of endogenous FA profiles to cryopreservation. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are a type of cell capable of regulating the immune system, as well as exhibiting self-renewal and multi-lineage differentiation potential. Mesenchymal stem cells have emerged as an essential source of seed cells for therapeutic cell therapy. It is crucial to cryopreserve MSCs in liquid nitrogen prior to clinical application while preserving their functionality. Furthermore, efficient cryopreservation greatly enhances MSCs' potential in a range of biological domains. Nevertheless, there are several limits on the MSC cryopreservation methods now in use, necessitating thorough biosafety assessments before utilizing cryopreserved MSCs. Therefore, in order to improve the effectiveness of cryopreserved MSCs in clinical stem cell treatment procedures, new technological techniques must be developed immediately. The study offers an exhaustive analysis of the state-of-the-art MSC cryopreservation techniques, their effects on MSCs, and the difficulties encountered when using cryopreserved MSCs in clinical applications. [ABSTRACT FROM AUTHOR]

Recent years have witnessed significant advancements in the cryopreservation of various tissues and cells, yet several challenges persist. This review evaluates the current state of cryopreservation, focusing on contemporary methods, notable achievements, and ongoing difficulties. Techniques such as slow freezing and vitrification have enabled the successful preservation of diverse biological materials, including embryos and ovarian tissue, marking substantial progress in reproductive medicine and regenerative therapies. These achievements highlight improved post-thaw survival and functionality of cryopreserved samples. However, there are remaining challenges such as ice crystal formation, which can lead to cell damage, and the cryopreservation of larger, more complex tissues and organs. This review also explores the role of cryoprotectants and the importance of optimizing both cooling and warming rates to enhance preservation outcomes. Future research priorities include developing new cryoprotective agents, elucidating the mechanisms of cryoinjury, and refining protocols for preserving complex tissues and organs. This comprehensive overview underscores the transformative potential of cryopreservation in biomedicine, while emphasizing the necessity for ongoing innovation to address existing challenges. [ABSTRACT FROM AUTHOR]

Background: Ovarian cryopreservation is a promising technique despite being hindered by damage from freezing and thawing. Melatonin can mitigate this outcome. Objective: This study aimed to evaluate the effect of melatonin on the follicular dynamics of ovarian tissue in a cryopreserved cell culture. Methods: Three-month-old adult female Wistar rats (n = 24) weighing approximately 250 g were oophorectomized and divided into two groups (n = 12): the control group (CG) and the melatonin group (MG). In the CG, slow cryopreservation was performed according to the standard protocol with Medium M2 and dimethyl sulfoxide (DMSO). In MG, melatonin diluted in ethyl alcohol vehicle at a concentration of 0.1 μm was added to the culture medium. In both groups, the ovaries were cryopreserved by slow freezing and kept in liquid nitrogen for 24 h. Subsequently, after thawing, the ovaries were reimplanted in the retroperitoneum, one on each side of the great vessels (inferior vena cava and aorta). After 30 days, the animals were euthanized during the diestrus phase; then, the grafts were removed and processed for histomorphometric and immunohistochemical analyses, whereas the blood was subjected to biochemical analysis. Student's t test was used to assess the difference between the groups. Results: The FSH levels in MG (83.79 ± 32.37) were lower than those in CG (120.52 ± 36.59), p = 0.03. The FSH/AMH ratios were also lower in MG (3.53 ± 1.13) than in CG (6.52 ± 2.85), p = 0.001. The SOD2 immunoexpression was higher in MG than in CG regarding all parameters except for the degenerated follicles (follicular cells and internal thecal cells): CG (16.80 ± 4.80 [13.36–20.24]) and MG (14.91 ± 4.04 [12.01–17.79]) p = 0.351. Statistically, the difference in intact follicles (theca + interstitium) between CG (6.60 ± 2.59 [4.75–8.45]) and MG (9.31 ± 3.09 [7.09–11.51]) was significant (p = 0.049), with a small difference in the expression of regular antral follicles. Conclusions: Melatonin can improve the quality of cryopreserved tissues, as evidenced in this study, and the evaluation of cryopreserved ovarian grafts, as shown in the melatonin group with better hormonal parameters and greater immunohistochemical expression of the SOD2 antioxidant. Thus, damage is reduced during cryopreservation and transplantation is improved. [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: Cryopreservation is a crucial procedure for safeguarding cells or other biological constructs, showcasing considerable potential for applications in tissue engineering and regenerative medicine. Aims: This study aimed to evaluate the effectiveness of different cryopreservation conditions on human cells viability. Methods: A set of cryopreserved data from Department of Tissue Engineering and Regenerative Medicine (DTERM) cell bank were analyse for cells attachment after 24 h being revived. The revived cells were analysed based on different cryopreservation conditions which includes cell types (skin keratinocytes and fibroblasts, respiratory epithelial, bone marrow mesenchymal stem cell (MSC); cryo mediums (FBS + 10% DMSO; commercial medium); storage durations (0 to > 24 months) and locations (tank 1–2; box 1–5), and revival methods (direct; indirect methods). Human dermal fibroblasts (HDF) were then cultured, cryopreserved in different cryo mediums (HPL + 10% DMSO; FBS + 10% DMSO; Cryostor) and stored for 1 and 3 months. The HDFs were revived using either direct or indirect method and cell number, viability and protein expression analysis were compared. Results: In the analysis cell cryopreserved data; fibroblast cells; FBS + 10% DMSO cryo medium; storage duration of 0–6 months; direct cell revival; storage in vapor phase of cryo tank; had the highest number of vials with optimal cell attachment after 24 h revived. HDFs cryopreserved in FBS + 10% DMSO for 1 and 3 months with both revival methods, showed optimal live cell numbers and viability above 80%, higher than other cryo medium groups. Morphologically, the fibroblasts were able to retain their phenotype with positive expression of Ki67 and Col-1. HDFs cryopreserved in FBS + 10% DMSO at 3 months showed significantly higher expression of Ki67 (97.3% ± 4.62) with the indirect revival method, while Col-1 expression (100%) was significantly higher at both 1 and 3 months compared to other groups. Conclusion: In conclusion, fibroblasts were able to retain their characteristics after various cryopreservation conditions with a slight decrease in viability that may be due to the thermal-cycling effect. However, further investigation on the longer cryopreservation periods should be conducted for other types of cells and cryo mediums to achieve optimal cryopreservation outcomes. Highlights: Cryopreservation optimizes cell recovery, viability for tissue engineering. Fibroblasts maintain phenotype in FBS + 10% DMSO, but longer periods need scrutiny. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigated the use of hormones and temperature control to improve breeding success. Injecting Ovaprim significantly increased egg and sperm production. However, temperature played a critical role. A medium temperature (around 22 °C) produced the best results, with more eggs, faster egg release, and healthier sperm. Both low and high temperatures negatively impacted sperm quality and larval fish survival. Using extender E4 (15% DMSO) for cryopreservation improved fertilization rates. Overall, the study highlights the importance of precise hormone control and temperature management for successful goldfish reproduction, benefiting for fish farming. This study investigates the efficacy of hormone-induced artificial reproduction in goldfish (Carassius auratus) under controlled temperatures. Ovaprim injections significantly enhanced ovulation and sperm production compared to controls. Medium temperature (22 °C) produced the highest ovulation rates, fastest ovulation timing, and optimal sperm quality (motility and morphology) compared to high (28 °C) and low (16 °C) temperature groups. The low-temperature group exhibited reduced sperm motility duration and higher rates of sperm and larvae damage. The sperm volume of the high-temperature group was higher, but their post-injection survival rates were lower. Furthermore, the lowest spawning rate and low egg quality were noted in the high temperature. Cryopreservation using extender E4 (15% DMSO) exhibited superior post-thaw sperm motility and achieved higher fertilization rates. Fertilization rates, embryo development, and larval survival were all highest at the medium temperature. Larvae hatched from fresh sperm at medium temperature exhibited faster growth and fewer deformities. These findings suggest that hormone stimulation coupled with a medium temperature regimen is critical for successful artificial reproduction in goldfish. Cryopreservation with extender E4 holds promise for sperm banking; however, further optimization is necessary to improve fertilization success with thawed sperm. Future research could explore the influence of temperature on sperm physiology and refine cryopreservation protocols to enhance fertilization rates. [ABSTRACT FROM AUTHOR]

Simple Summary: Spermatozoa cryopreservation technology has been widely used to preserve high-quality male spermatozoa and has to some extent solved the problem of spermatozoa supply in yellow catfish (Pelteobagrus fulvidraco). However, cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, it is necessary to study cryo-injury, which is beneficial for further optimizing the spermatozoa cryopreservation protocol. In this experiment, we investigated the effects of adding or not adding 10% DMSO on spermatozoa motility, ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish. Using 10% DMSO as a cryoprotectant can improve spermatozoa motility (75.64 ± 1.12), significantly alleviate injuries to organelles and membrane structures, improve SOD (7.99 × 104 ± 5.24 nmol/mgprot) and T-AOC (3.7 × 105 ± 0.06 nmol/mgprot) enzyme activity, reduce MDA (1.09 ± 0.45 nmol/mgprot) enzyme activity, and mitigate the degree of oxidative damage. It also greatly reduces the DNA fragmentation rate (59 ± 1.56%). The research results provide data support for the optimization of the cryopreservation protocol for the spermatozoa of yellow catfish, which is beneficial for the preservation of the germplasm resources of yellow catfish and the effective utilization of resources during large-scale production. Spermatozoa cryopreservation protocols have been established for yellow catfish (Pelteobagrus fulvidraco), but cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, the aim of this paper was to evaluate the effects of adding or not adding cryoprotectants during low-temperature storage on the ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish spermatozoa. The mixed semen of three male yellow catfish was divided into a fresh spermatozoa group, a frozen spermatozoa group (DMSO+) with a cryoprotectant (10% DMSO), and a frozen spermatozoa group without a cryoprotectant (DMSO−). Ultrastructural of the spermatozoa after thawing were observed under an electron microscope and the spermatozoa were assayed for SOD, MDA, and T-AOC enzyme activities, as well as for DNA integrity. In terms of movement parameters, compared with DMSO−, the addition of DMSO has significantly improved sperm motility, curve line velocity (VCL), and straight line velocity (VSL). The ultrastructural results showed that although thawed spermatozoa exhibited increased damage than fresh spermatozoa, 10% DMSO effectively reduced the damage to the plasma membrane, mitochondria, and flagellum of spermatozoa by cryopreservation, and most of the spermatozoa were preserved with intact structure. The results of oxidative damage showed that compared with frozen spermatozoa, 10% DMSO significantly increased the activities of SOD and T-AOC enzymes and clearly reduced the activity of the MDA enzyme. The antioxidant capacity of spermatozoa was improved, lipid peroxidation was reduced, and the oxidative damage caused by cryopreservation was mitigated. The DNA integrity of spermatozoa showed that 10% DMSO clearly reduced the DNA fragmentation rate. In conclusion, 10% DMSO can effectively reduce the ultrastructural damage, oxidative damage, and DNA damage of yellow catfish spermatozoa during cryopreservation; it can also further optimize the cryopreservation protocol for yellow catfish spermatozoa. Meanwhile, it also provides a theoretical basis for the future optimization of the cryopreservation protocols. [ABSTRACT FROM AUTHOR]

Cryopreservation is essential for the broad clinical application of mesenchymal stem cells (MSCs), yet its impact on their cellular characteristics and cardiomyogenic differentiation potential remains a critical concern in translational medicine. This study aimed to evaluate the effects of cryopreservation on the biological properties and cardiomyogenic capacity of rat adipose-derived MSCs (AD-MSCs). We examined their cellular morphology, surface marker expression (CD29, CD90, CD45), trilineage differentiation potential (adipogenic, osteogenic, chondrogenic), and gene expression profiles for the pluripotency marker REX1 and immunomodulatory markers TGFβ1 and IL-6. After inducing cardiomyocyte differentiation, we assessed cardiac-specific gene expressions (Troponin I, MEF2c, GSK-3β) using quantitative RT-qPCR, along with live/dead cell staining and immunofluorescence for cardiac-specific proteins (Troponin T, α-actinin, Myosin Heavy Chain). Cryopreserved AD-MSCs preserved their morphology, surface markers, and differentiation potential, but exhibited a reduced expression of REX1, TGFβ1, and IL-6. Additionally, cryopreservation diminished cardiomyogenic differentiation, as indicated by the lower levels of Troponin I, MEF2c, and GSK-3β seen compared to non-cryopreserved cells. Despite this, high cell viability (>90%) and maintained cardiac protein expression were observed post-cryopreservation. These findings highlight the necessity of optimizing cryopreservation protocols to ensure the full therapeutic potential of AD-MSCs, particularly in applications related to cardiac regenerative medicine. [ABSTRACT FROM AUTHOR]

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis. [Display omitted] • Increases in inner cell mass cell numbers are achieved by supplementing recombinant human interleukin-6. • Positive effects of human interleukin-6 supplementation on both the inner cell mass and trophectoderm were detected after embryo cryopreservation. • Human interleukin-6 may is effective in bovine embryos. [ABSTRACT FROM AUTHOR]

Controlling the formation and growth of ice is essential to successfully cryopreserve cells, tissues and biologics. Current efforts to identify materials capable of modulating ice growth are guided by iterative changes and human intuition, with a major focus on proteins and polymers. With limited data, the discovery pipeline is constrained by a poor understanding of the mechanisms and the underlying structure-activity relationships. In this work, this barrier is overcome by constructing machine learning models capable of predicting the ice recrystallisation inhibition activity of small molecules. We generate a new dataset via experimental measurements of ice growth, then harness predictive models combining state-of-the-art descriptors with domain-specific features derived from molecular simulations. The models accurately identify potent small molecule ice recrystallisation inhibitors within a commercial compound library. Identified hits can also mitigate cellular damage during transient warming events in cryopreserved red blood cells, demonstrating how data-driven approaches can be used to discover innovative cryoprotectants and enable next-generation cryopreservation solutions for the cold chain. Controlling ice formation is crucial for cryopreservation. Here, authors developed machine learning models to predict ice recrystallisation inhibition by small molecules, showcasing a data-driven approach for cryoprotectant discovery. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma. [ABSTRACT FROM AUTHOR]

Background: Hematopoietic stem cell transplantation (HSCT) is an approach that has significantly improved the prognosis and survival of hematological patients. However, ovarian dysfunction and infertility following HSCT have gained increasing attention. Live births have been reported following ovarian tissue cryopreservation prior to HSCT and subsequent retransplantation of these tissues. Still, the feasibility of ovarian tissue cryopreservation (OTC) following graft failure (GF) of HSCT remains unknown. In this study, we report the first case of OTC following a GF of allogenic HSCT (allo-HSCT), as well as the cryopreservation of four MII oocytes via in vitro maturation with informed consent. Despite the lack of clinical outcomes after cryopreserved ovarian tissue retransplantation, we documented an interesting case in a woman after GF of allo-HSCT exhibiting functional ovaries and emphasized a clinical dilemma: whether OTC should be offered to women suffering from GF of HSCT. Case presentation: A 22-year-old woman with severe aplastic anemia who had suffered GF of allo-HSCT from her sibling brother [HLA allele match (7/10)] with a reduced dose conditioning regimen including fludarabine, cyclophosphamide, and antithymocyte globulin came to our reproductive center for fertility preservation, as she was about to receive the second allo-HSCT. We evaluated the ovarian reserve of this patient. Hormone assessments showed an antiMüllerian hormone level of 3.921 ng/mL, a follicle-stimulating hormone level of 5.88 IU/L, a luteinizing hormone level of 10.79 IU/L, and an estradiol level of 33.34 pg/mL. Antral follicle counts accessed transvaginally showed 12–15 follicles. All assessments indicated a well-protected ovarian reserve. Due to the urgency of the second allo-HSCT, the patient decided to undergo ovarian cryopreservation. Laparoscopic surgery proceeded. Ovarian tissues were successfully cryopreserved using vitrification technology, and histologic evaluation demonstrated a follicle density of 20 per 2 × 2 mm² biopsy with good viability. Four MII oocytes were obtained via in vitro maturation technology and cryopreserved. After the second HSCT, the patient relieved from aplastic anemia but suffered iatrogenic premature ovarian failure as predicted. Conclusion: OTC is applicable to fertility preservation in those undergoing GF of HSCT with benign hematological disorders and especially those who are about to receive the second HSCT. [ABSTRACT FROM AUTHOR]

The preservation of microorganisms is pivotal in microbiological practice. Currently, cryopreservation is assumed to be an effective and inexpensive approach for the storage of microorganisms, including bacteria. The key point of cryopreservation is optimal cryoprotectant selection. In the present study, different cryoprotectant compositions were tested for long-term storage of 15 Enterobacterales bacterial strains at − 20 °C. The survival rates of the bacterial strains were evaluated in four different cryoprotectant solutions containing 70% glycerin only (cryoprotectants 1 and 4), 10% dimethyl sulfoxide (DMSO) with 70% glycerin (cryoprotectant 2), and 10% DMSO (cryoprotectant 3). In addition, cryoprotectants 1 and 2 contained peptone and yeast extract as nutritional supplements. The general survival rates of the bacterial strains were evaluated after 12 months of storage. After 12 months, the survival rates of the different cryoprotectants were as follows: cryoprotectant 1—88.87%; cryoprotectant 2—84.85%; cryoprotectant 3—83.50%; and cryoprotectant 4—44.81%. Thus, the composition of cryoprotectant 1 (70% glycerin with nutrient supplements) was optimal for preserving 15 tested strains of the order Enterobacterales. Despite these findings, the biochemical properties of the tested strains changed after cryopreservation for 12 months in the presence of 1 or 3 cryoprotectants. Alterations in the biochemical profile could be related to changes in environmental conditions and cold adaptation. We assume that the composition of cryoprotectant 1 can be optimal for storing the order Enterobacterales at − 20 °C. However, further investigations are needed to elucidate the problem of cryopreservation and to support our assumption. [ABSTRACT FROM AUTHOR]

Cryopreservation is pivotal for conserving genetic diversity. This study aimed to optimize cryopreservation techniques for Dandarawi rooster sperm through two experiments. Experiment I explored the optimal levels of two cryoprotectants, with or without sucrose, in two trials. Semen from 20 Dandarawi males was pooled, diluted, and divided into five aliquots. In trial 1, one aliquot remained fresh (control), while others were frozen in straws using glycerol (GLY) at concentrations of 8% and 11%, with or without 5% sucrose. In trial 2, one aliquot served as a control, while the others were frozen in pellets using a cryo-diluent containing 5% or 7% dimethylacetamide (DMA), with or without sucrose. Danadarawi hens (n=10 per group) were inseminated with fresh (control) or frozen-thawed sperm, with six repetitions of semen collection and insemination. In Experiment II, pooled semen was similarly divided. Aliquot 1 served as a control, while aliquots 2 and 3 were frozen in pellets with 5% DMA cryo-diluent, with and without 5% sucrose, respectively. Aliquots 4 and 5 were frozen in straws with 8% GLY, with and without 5% sucrose. Transmission electron microscopy (TEM) evaluated spermatozoa for cryodamage. GLY level did not significantly affect motility, fertility, or hatchability, but sucrose notably improved hatchability with 8% and 11% GLY. DMA did not significantly impact motility, although 7% DMA demonstrated the highest fertility. Sucrose enhanced hatchability with 5% DMA but not with 7% DMA. TEM analysis revealed higher levels of acrosomal damage in spermatozoa frozen with GLY compared to DMA, with sucrose consistently mitigating acrosomal injuries. Moderate damage was observed in the tail, sparing the nucleus and midpiece. In conclusion, the choice of freezing method and the inclusion of sucrose significantly influenced sperm freezing outcomes, demonstrating their critical roles in preserving sperm quality. Sucrose was particularly effective across all treatments, notably in protecting the acrosome region. [ABSTRACT FROM AUTHOR]

The effect of immersion freezing on water distribution and calcium ion migration in pears was studied. The samples were frozen at -20 °C and -40 °C. Results showed that the -40 °C combined immersion freezing (-40IF-T) was better in inhibiting sample browning and sensory quality, compared with other groups. Water activity and freezable water content were closer to that of the unfrozen sample. Compared with -20IF, the freezing time of -40IF-T was reduced by 74.90 %. Both the drip loss and calcium ion content showed that -40IF-T was better than other groups. Low-field nuclear magnetic resonance results showed that -40IF-T reduced water migration during freezing, indicating a more uniform distribution of water in -40IF-T tissues. The results of relative conductivity showed that -40IF-T could effectively reduce the damage of cell membrane caused by freezing. In conclusion, low temperature and cryoprotective fluid can be used as effective methods to maintain sample quality. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl 2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl 2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. A buffer consisting of 10% trehalose-20 mM MgCl 2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing. [ABSTRACT FROM AUTHOR]

Objective: This research was conducted to analyze the effect of cryopreservation of sperm using modified cryoprotectants on embryo development through in vitro fertilization (IVF). In this research, three types of cryoprotectant combinations were compared, including Nakagata, modified cryoprotectant, and commercial (Kitazato). Materials and Methods: Several parameters, namely sperm concentration, motility, morphology, plasma membrane integrity, cryo-survival rate, and viability rate, were measured and compared before vitrification and warming. Embryo development was also observed on the first and third days of development based on the cell number, cell size, and fragmentation rate. Results: Sperm cryopreservation exhibited a negative influence on embryo quality. Both Nakagata cryoprotectants and modified cryoprotectants attained good-quality embryos. In terms of embryonic development, this research revealed a modified cryoprotectant superior to Nakagata's cryoprotectant, although Kitazato was superior to the Nakagata cryoprotectant and modified cryoprotectant. Significant differences were found in the three cryoprotectants on observations on day 1 and day 3, all of them with p-value < 0.05. Conclusion: Modified cryoprotectant was found to be better than Nakagata but less significant than Kitazato in terms of embryonic development quality. Therefore, modified cryoprotectants could be a better alternative compared to commercial (Kitazato) cryoprotectants for improving embryo quality. [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]

Simple Summary: In the last years, many domesticated and wild animal species, as well as local and transboundary breeds, have been threatened with genetic depletion and extinction. The application of conservation strategies to sheep breeds is necessary to safeguard the productive characteristics but also the historical and cultural value that they exert on their territory and inhabitants. Despite many advances that have been made in gamete cryopreservation, in vitro embryo production (IVEP) and juvenile in vitro production (JIVET), the efficiency of these technologies is still low, and the application is restricted in the ovine species. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating cryoprotectants (CPAs), were applied to pre-pubertal lamb immature cumulus–oocyte complexes (COCs) and evaluated on nuclear and cytoplasmic parameters after in vitro maturation (IVM). Slightly more encouraging results were observed with the LC-SE vitrification protocol, leading to the hypothesis that low CPA concentrations in association with prolonged exposure times could be more promising to pursue in order to improve pre-pubertal lamb immature COC vitrification. Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further. [ABSTRACT FROM AUTHOR]