Freezing is widely used for microbial preservation; however, it promotes ice crystal formation, which may compromise cellular integrity. Given the variability among cryopreservation protocols used in laboratories and reported in the literature, this study evaluated microbial responses to successive freeze-thaw cycles using different glycerol concentrations in Luria-Bertani (LB) broth and two storage temperatures. Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Saccharomyces cerevisiae were selected as representatives of a Gram-negative bacterium, a Gram-positive bacterium, a spore-forming bacterium, and a yeast, respectively. Microorganisms were cryopreserved at -20 °C or -70 °C with 10%, 20%, 35%, or 50% glycerol and evaluated over nine successive freeze-thaw cycles. Viability was assessed by loop inoculation recovery and total viable cell counts determined by surface plating. Repeated freeze-thaw cycles significantly affected microbial viability, with responses varying among the evaluated species. E. coli and S. aureus remained stable throughout storage, whereas B. subtilis showed a progressive decline and S. cerevisiae exhibited variable responses. Storage at -70 °C showed a trend toward greater cell viability, although the overall effect of temperature was not significant. Glycerol concentration did not significantly affect microbial survival. A positive correlation was observed between loop inoculation recovery and microbial load, indicating that higher numbers of viable cells increased the likelihood of successful recovery. These findings demonstrate that microbial survival during cryopreservation depends primarily on species-specific characteristics rather than glycerol concentration, highlighting the importance of tailoring preservation protocols to different microorganisms.

Congenital heart diseases, including semilunar valve (SV) abnormalities, remain a leading cause of mortality in the pediatric population. SV defects present a particular challenge in children because currently available prosthetic valves lack growth potential and often require repeated reinterventions as the patient matures. Partial heart transplantation (PHT) has recently emerged as a novel strategy to overcome this limitation, whereby viable donor SVs, isolated from hearts unsuitable for orthotopic heart transplantation, are implanted with the capacity to grow with the recipient. Given the relative structural simplicity of the SV tissue, multiple preservation strategies may be feasible. Among these, vitrification has demonstrated particular promise, offering the potential for long-term storage while preserving structural integrity, cellular viability, and reducing immunogenicity. This review summarizes the progress to date in applying vitrification to SV preservation and discusses the future applications in PHT.

Silkworm sperm exist as two types: eupyrene and apyrene. Apyrene sperm lack nuclei and are incapable of fertilization. However, they play a crucial role in facilitating fertilization by assisting nucleated sperm in migrating from the bursa copulatrix to the spermatheca. Additionally, apyrene sperm exhibit lower cryotolerance compared with eupyrene sperm, resulting in significantly reduced fertility following cryopreservation. Conventionally, the fertility of cryopreserved semen has been restored by mixing it with semen from sterile triploid males, which contains functional apyrene sperm and nonfunctional eupyrene sperm. However, collecting semen from triploid males is labor-intensive. To reduce this effort, we investigated whether mating with triploid males could improve the fertility of cryopreserved semen without artificial mixing with semen from triploid males. The results of this study demonstrated that females mated with triploid males and artificially inseminated with cryopreserved semen produced more fertilized eggs compared with unmated females. Furthermore, females mated with triploid males after artificial insemination (AI) with cryopreserved semen laid more fertilized eggs and exhibited a significantly higher fertilization rate than those mated before AI with cryopreserved semen. Notably, injecting 5 μL of cryopreserved semen produced more fertilized eggs than 10 μL when females were mated with triploid males after AI. These findings suggest that mating with triploid males eliminates the need for semen collection and permits AI with smaller volumes of cryopreserved semen.

The use of wild rodents as biological resources is hindered by the difficulties of live transport and primary culture contamination caused by skin bacteria. To address this, we developed a "tail-degloving" method to physically remove the contaminated external integument. Using the large Japanese field mouse (Apodemus speciosus), this method suppressed contamination compared to conventional biopsies, yielding cells within 5-7 days that were capable of subculturing and cryopreservation. The protocol withstood 4°C storage for up to 7 days and enabled contamination-free cell establishment from a large rodent. This tail-degloving protocol eliminates live transport risks and effectively establishes primary cells after a multi-day transport delay.

Improvement of bovine oocyte vitrification focuses mainly on the cooling step, while warming steps receive little attention. Recent studies report improved in vitro embryo production (IVEP) efficiency in vitrified rodent embryos warmed rapidly at high temperatures (50-60°C). This effect has not yet been tested on bovine oocytes; therefore, this study aimed to evaluate rapid warming's influence on mitochondrial functionality, nuclear DNA integrity, and IVEP efficiency. Three groups of in vitro matured oocytes were included: 1) control, 2) SW - vitrified oocytes warmed slowly, and 3) RW - vitrified oocytes warmed rapidly. Oocytes were assessed in two experiments for: (1) embryo production and (2) mitochondrial functionality and DNA integrity using MitoTracker® Red CMXRos and Hoechst33342 dyes. All vitrified-warmed oocytes were examined for cytoplasm expansion and rupture after warming. In Experiment 1, cleavage rates differed significantly among all groups, with RW showing higher rates than SW. In Experiment 2, the strongest MitoTracker® Red CMXRos fluorescence was observed in the control group, followed by SW and RW. No significant differences were found between SW and RW in DNA degeneration, cytoplasm expansion, or rupture. Although mitochondrial mass was lowest in RW, higher temperature did not increase DNA damage or affect cytoplasm expansion compared to SW. Moreover, RW achieved the best cleavage and blastocyst rates among vitrified-warmed groups. These findings indicate that rapid warming does not compromise oocyte integrity and may improve IVEP outcomes. The data suggests potential for incorporating rapid warming into vitrification protocols for mature bovine oocytes.

T-Cells are key components of the adaptive immune system and engineered (e.g CAR-T) T-cells are deployed in immunotherapy, particular for haematologic malignancies. Methods to replace DMSO and serum during T-cell cryopreservation are crucial for both therapies and basic research to improve recovery and function. Glycerol and osmolytes are promising alternatives owing to their lower cytotoxicity than DMSO but often fail to give robust post-thaw recovery. Here we explore the combination of glycerol and betaine for the cryopreservation of Jurkat cells and demonstrate a significantly improved cryopreservation process that is suitable for both low-volume multi-well plate and vial-based freezing with no added serum. The optimised formulation of 10% glycerol and 2% betaine gave >90% post-thaw recovery, but crucially this was achieved when linked with a 3 °C/min freezing rate, and it was observed that faster freezing in general outperformed slower (1 °C/min) which is in contrast to standard protocols which rely on passive cooling devices. The increased rate of cooling appeared to mitigate delayed nucleation damage, which is typically associated with multi-well plate freezing, and the solutions were shown to not impact nucleation in droplet freezing assays. These results demonstrate an easily useable formulation for routine banking, particularly in multiwell-plates which is essential for assay-ready/high-throughput screening approaches as well as vials for larger-volume applications. The removal of serum may also facilitate shipping, where country-specific guidelines for animal-based serum prevent import/export as well as reducing costs.

Giant clams (Tridacna spp.) are keystone reef organisms that play important ecological roles in Indo-Pacific coral reef ecosystems. However, their populations have declined substantially because of overexploitation, habitat degradation, and climate change. Cryopreservation offers a potential strategy for preserving genetic resources and supporting future conservation and restoration efforts. In this study, we evaluated the feasibility of cryopreserving D-stage larvae of Tridacna crocea and T. maxima using a two-step freezing protocol. The effects of different cryoprotectants, concentrations, and equilibration times on post-thaw larval integrity and motility were assessed. Larval development was monitored using microscale culture techniques and microscopic observations. The results demonstrated species-specific responses to cryopreservation treatments, with T. maxima generally exhibiting greater cryotolerance than T. crocea. Ethylene glycol produced the highest larval integrity percentage (LIP) in both species. In addition, glycerol and propylene glycol yielded relatively high LIP values in T. crocea, whereas methanol and dimethyl sulfoxide (DMSO) performed more effectively in T. maxima. Post-thaw motility was observed only in a limited number of treatment groups, with the highest motility recorded in larvae treated with 0.75 M glycerol for 30 min and 0.5 M DMSO for 20 min. In contrast, non-cryopreserved control larvae exhibited a progressive decline in motility during culture, decreasing to 12.1% in T. maxima and 4.0% in T. crocea after 24 h. To our knowledge, this study represents the first investigation of cryopreservation in tridacnid D-stage larvae. Although post-thaw motility remained limited, the successful preservation of larval structural integrity demonstrates the potential of cryopreservation as a conservation tool for giant clams. Further optimization of species-specific cryopreservation protocols will be necessary to improve post-thaw recovery and support long-term genetic resource banking.

Freezing-induced ice recrystallization is a major limitation in preserving the quality of high-moisture fruits because of its adverse effects on cellular structure, texture, and nutritional stability. This study investigated vacuum-assisted sucrose impregnation as a cryostabilization approach for improving the quality of frozen pineapple stored at -18 °C for 21 days. Pineapple cubes were subjected to untreated control, atmospheric sucrose impregnation (AI; immersion in 60% (w/w) sucrose solution for 30 min at atmospheric pressure), and vacuum-assisted impregnation (VI; 31.8 kPa for 20 min followed by 10 min relaxation). Mass transfer, thermal characteristics, physicochemical properties, color, texture, and microstructural changes were evaluated. Vacuum treatment significantly enhanced solute incorporation, increasing cryoprotectant uptake to 11.2%. Differential scanning calorimetry revealed that VI reduced the onset freezing temperature from -1.8 to -3.6 °C and decreased melting enthalpy by approximately 52% compared with untreated samples. Freezable water content decreased from 72.4% to 38.6%, while bound water increased from 27.6% to 61.4%, indicating enhanced water immobilization within the fruit matrix. During frozen storage, VI-treated samples exhibited lower ion leakage (9.1%), greater retention of total phenolic content (67.5 mg GAE/100 g), higher antioxidant activity determined by DPPH scavenging (73.9%) and FRAP (581.3 μmol Fe[2+] equivalents/100 g fresh weight), and improved textural stability, maintaining hardness at 21.4 N compared with 10.2 N in control samples after 21 days. SEM observations further confirmed reduced tissue disruption and preservation of cellular architecture. These findings demonstrate that vacuum-assisted sucrose impregnation effectively improves cryostability by regulating water distribution and preserving structural integrity in frozen pineapple.

Oocyte vitrification is widely used in fertility preservation and assisted reproductive technologies (ART), but current protocols rely on sequential cryoprotectant exposure steps that are labor-intensive and operator-dependent. This study evaluated the feasibility of a simplified one-step vitrification and warming protocol using mouse metaphase II oocytes. An initial optimization phase using 716 oocytes identified the optimal exposure time to the newly developed vitrification and warming solutions. Subsequently, 876 oocytes were allocated to fresh controls, standard multi-step vitrification, or the optimized one-step protocol. Outcomes included post-warming survival, meiotic spindle integrity, chromosome alignment, reactive oxygen species (ROS) levels, mitochondrial distribution, intracytoplasmic sperm injection (ICSI) outcomes, blastocyst development, total blastocyst cell counts, and full-term development after embryo transfer. An one-step exposure time of 2 min achieved the highest post-warming survival (100%), comparable to the standard protocol (98.9%; p = 0.080). Immunofluorescence analysis demonstrated normal spindle morphology, chromosome alignment, oxidative stress levels, and mitochondrial distribution across groups. Survival after warming and ICSI, as well as blastocyst formation rates, did not differ significantly between vitrification methods and fresh controls (p > 0.05). Although blastocyst total cell numbers were slightly reduced in vitrified groups, full-term development rates were comparable among all groups, and offspring developed normally to adulthood. These findings demonstrate that one-step vitrification and warming can preserve oocyte developmental competence while substantially simplifying cryopreservation procedures, supporting its potential as a practical alternative to conventional vitrification protocols in ART.

This retrospective single-center study compared recorded postprocedural visual analog scale (VAS) pain scores after microwave ablation (MWA) and cryoablation for pulmonary tumors under routine postoperative care. Patients who underwent CT-guided pulmonary tumor ablation between January 2019 and October 2025 were included. Pain scores were recorded by thoracic surgery ward nurses using a VAS at 8, 16, 24, and 48 h after the procedure, and generalized estimating equations were used to account for repeated observations. A total of 68 ablation sessions, including 41 MWA and 27 cryoablation sessions, were included for pain analysis. Mean VAS scores were lower after cryoablation than after MWA at 8 h (0.5 ± 1.3 vs. 1.5 ± 1.6), 16 h (0.9 ± 1.8 vs. 2.0 ± 1.8), 24 h (0.3 ± 0.6 vs. 1.7 ± 1.8), and 48 h (0.3 ± 0.6 vs. 1.1 ± 1.3). Cryoablation was associated with lower recorded VAS scores than MWA from 8 to 48 h after pulmonary tumor ablation under routine postoperative care. These findings suggest that cryoablation may be associated with lower recorded postoperative pain scores, although MWA remains a feasible treatment option for pulmonary tumors.

Cryogenic liquid nitrogen (LN2) storage containers are widely used for the long-term preservation of cells and tissues, yet they may represent an overlooked source of microbial contamination. Effective contamination control within cryogenic systems and the surrounding cryobank environment is essential, particularly when supporting clinical cell or tissue transplantation. Contamination risk increases in the absence of adequate air filtration and is further amplified by high humidity condensation leading to ice accumulation, frequent container opening, insufficient packaging of stored materials, and improper handling during loading and unloading procedures. This study evaluated microbial contamination in LN2 vapor-phase containers (T ≤ -150 °C) using standard cultivation methods and MALDI-TOF mass spectrometry, with emphasis on the potential spread of microorganisms via LN2 vapor. Contamination levels in LN2 containers were compared with those in mechanical low-temperature freezers (T ≤ -80 °C), alongside microbial profiles detected on protective gloves used during sample manipulation. Microbial growth (2-10[2] CFU/mL), based on analysis of the melting ice, was detected in 5 of 8 LN2 containers. Detected organisms included potential pathogens such as Delftia tsuruhatensis, Acinetobacter spp., Pseudomonas spp., and Staphylococcus aureus, as well as non-pathogenic environmental species. Wall swabs revealed additional bacteria, while all lid swabs remained culture-negative. Mechanical freezers exhibited low-level contamination (1-14 CFU/mL), with microbial profiles overlapping those detected in LN2 systems. Matching organisms found on gloves indicate operator handling as a major contamination route. Microbial presence in frost and meltwater further suggests possible transmission via LN2 vapor. These findings underscore the need for stricter hygiene measures, improved decontamination procedures, and routine microbiological monitoring in cryogenic storage environments.

We evaluated the use of alpaca oocytes vitrified using the Microdrop or Cryotech technique for in vitro embryo production. Grade I-II COCs (n = 1094) were recovered from slaughterhouse ovaries, and in vitro matured for 36 h. Then, MII oocytes were randomly distributed in groups: a) vitrified using two techniques [Microdrop (VM group; n = 250) or Cryotech (VC group; n = 244)], b) just exposed to the corresponding vitrification/thawing solutions (EM group; n = 200 or EC group; n = 200) or c) non-exposed nor vitrified (i.e. fresh oocytes; control group; n = 200). After treatment administration oocytes from all groups were IVF using epididymal sperm and in vitro cultured for up to 8 days. The vitrification technique and the exposure to vitrification solutions of MII oocytes negatively affected (p < 0.001) the cleavage rate compared to control group. On the other hand, exposure to Microdrop vitrification solution (EM group) and vitrification using the Microdrop technique significantly (p < 0.001) reduced the embryo developmental rate at all stages evaluated. Early blastocysts rate was affected (p < 0.001) by treatment, and it was greatest (p < 0.001) for the EC and VC, intermediate (p < 0.05) for the control, and lowest (p < 0.001) for VM and EM groups. Expanded and hatched blastocyst were not observed in the groups where oocytes were subjected to either Cryotech or Microdrop vitrification technique. We conclude that Cryotech is a more suitable vitrification technique for MII alpaca oocytes than Microdrop, under the conditions used in this study (timing and cryoprotectants concentration), allowing the development of early blastocyst at a rate comparable to control group.

This study aimed to evaluate daily variations in sperm quality, testosterone levels, cryoresistance, and the relocation of AQP3 in plasma membrane domains in wild and domestic goats. Samples were collected from five Murciano-Granadina bucks and seven Iberian ibex at 12:00 h (noon) and 00:00 h (midnight), during the rutting season. Sperm motility, viability, acrosome integrity, DNA fragmentation, and immunolocalization of AQP3 were assessed in fresh and frozen-thawed semen samples. Serum testosterone concentrations were also determined at both times. In domestic goats, kinematic parameters were greater (p < 0.05) in semen collected at midnight, whereas the cryoresistance ratio was greater (p < 0.05) for these variables in semen recovered at noon. In ibexes, fresh semen collected at midnight was greater (p = 0.05) for VCL, VSL, VAP, and ALH values. After freezing-thawing, a significant increase in AQP3 labelling at the acrosomal region was observed in samples collected at midnight for both species, as well as in samples collected at noon for bucks (p < 0.05). An increase in the proportion of spermatozoa with AQP3 labelling in the mid-piece was detected in bucks in frozen-thawed samples collected at noon (p < 0.05). Testosterone concentrations were higher (p < 0.05) at midnight in bucks but no differences were found in ibexes. The findings reveal daily variations in sperm quality in both domestic and wild goats, which do not exactly coincide with the daily changes in cryoresistance, thus calling for attention when processing semen.

Extending the storage of donated livers beyond the current 4-6 h via freezing could significantly improve transplant availability. However, mitigating freezing injury in complex organs remains challenging because conventional cryoprotective agents (CPAs) are difficult to perfuse uniformly and several biologically important injury mechanisms are not well controlled. Ice recrystallization, where larger ice crystals grow at the expense of smaller ones, is one such mechanism. To address this, we evaluated two ice recrystallization inhibitors (IRIs), N-(2-fluorophenyl)-C6-azido-D-gluconamide (2FA) and N-(2-chlorophenyl)-D-gluconamide (4CLA), for hepatocompatibility and tissue permeation during a 4 h portal vein perfusion of rat livers. Hepatic function assessments showed no general toxicity for either IRI. However, 4CLA-perfused livers exhibited biliary stasis and lacked bile production. Conversely, 2FA maintained bile production and, in post-perfusion biopsies subjected to freezing, successfully reduced ice crystal growth within 200 μm of portal venules. These findings demonstrate that 2FA is a highly promising candidate for supporting long-term liver preservation in the frozen state.

Vitrification is a fast, simple and cost-effective cryopreservation technique which has emerged as a promising approach for sperm cryopreservation in a variety of animals, including aquaculture species. However, sperm vitrification in bivalves remains undeveloped, although molluscan sperm cryopreservation has been studied for more than 50 years. This investigation aimed to develop a sperm vitrification technique in dwarf surfclam Mulinia lateralia (a model species in bivalve research) and to assess the cryodamage at different levels. The results showed that the synergistic cryoprotective effect of coenzyme Q10 and ficoll was beneficial for the sperm vitrification with the dimethyl sulfoxide (DMSO) as the base cryoprotectant. The optimal cryoprotective solution for sperm vitrification in dwarf surfclam was 8% DMSO + 20 μM coenzyme Q10 + 5% ficoll. Although the D-stage larval rate achieved in vitrified sperm did not significantly differ from the fresh sperm, a sperm to egg ratio of 200 times higher was required in vitrified sperm than the fresh control at fertilization. This increased sperm requirement was associated with the vitrification-induced cryodamage, including reduced sperm motility, plasma membrane integrity, mitochondrial membrane potential, acrosome integrity, enzyme activity and DNA integrity, together with increased lipid peroxidation. In conclusion, the sperm vitrification technique presents a promising and technically achievable alternative for sperm cryopreservation in marine bivalve species.