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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.

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Frozen saliva samples are often used for later determination of salivary glucocorticoids in research studies on stress and endocrine disorders. We studied the stability of cortisol and cortisone in saliva after six years of storage at -80 °C by repeated analysis of 153 stored aliquots, collected with Salivette®, using liquid chromatography tandem mass spectrometry. We found a very high agreement between the first and the repeated measurement after six years at -80 °C, for both cortisol and cortisone concentrations (rs= 0.96 and rs= 0.98, respectively). Passing-Bablok regression equations were y = 0.02 + 1.00x and y = 0.02 + 1.14x for cortisol and cortisone, respectively. We conclude that salivary cortisol and cortisone concentrations remain essentially unaltered after six years of storage at -80 °C.

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In regenerative medicine, ongoing advancements in cell culture techniques, including isolation, expansion, banking, and transport, are crucial for clinical success. Cryopreservation ensures off-the-freezer availability of living cells, enabling long-term storage and transport. Customizing cryopreservation techniques and cryoprotective agents (CPAs) for specific cell types is crucial for cell source quality, sustainability, safety, and therapeutic intervention efficiency. As regenerative medicine progresses, it becomes imperative that the scientific community and industry provide a comprehensive, cell-specific landscape of available and effective cryopreservation techniques, preventing trial-and-error approaches and unlocking the full potential of cell-based therapies. Open-sharing data could lead to safer, more efficient cell therapies and treatments. Two decades of dermal progenitor cell use for burn wound treatment and Good Manufacturing Practice-compliant technology transfers have highlighted the need for further cryopreservation optimization in manufacturing workflows. In this paper, we present experimental data assessing 5 different cryopreservation formulae for long-term storage of clinical-grade FE002 primary progenitor fibroblasts, emphasizing the crucial difference between DMSO-based and DMSO-free CPAs. Our findings suggest that CryoOx, a DMSO-free CPA, is a promising alternative yielding cell viability similar to that of established commercial CPAs. This research highlights the importance of secure, robust, and efficient cryopreservation techniques in cell banking for maximizing quality, ensuring patient safety, and advancing regenerative medicine.

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Tape is a type of trace evidence commonly seen in forensic science. Little to no research has been published regarding the optimal storage material for pressure-sensitive adhesive (PSA) tape. The purpose of this project was to determine the substrate with the least amount of interference for storing PSA tape. Interferences such as difficulty removing tape from the substrate or substrate components leaching into the adhesive can affect the analysis. This was a long-term study in which samples were stored on various substrates for nearly 10 years. Ten different tape samples were placed on eight different substrates for a total of 80 samples. The substrates included typical materials currently used for tape storage as well as other, less common materials. The tape samples were evaluated for ease of removal at various time intervals. The adhesives from each tape were analyzed at similar time intervals by Fourier transform infrared spectroscopy (FTIR). In addition, the adhesives were analyzed by pyrolysis gas chromatography-mass spectrometry (PyGC-MS), scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS), and x-ray fluorescence (XRF) at the last time interval (112 months) and compared to the adhesive tape samples from the original rolls of tape to determine if there was any adverse effect from the substrates during storage. The FedEx backing was the only substrate that had no adverse effects regarding ease of removal. The substrates that had the least effect on the adhesive for both short-term and long-term storage included FedEx backings, adhesive sheet backings, and polyester transparency sheets.

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Aflatoxins are a group of high toxic mycotoxins in food chain. Recent studies showed that aflatoxins might contaminate post-fermented tea, but the result remains controversial. Here, Aspgergillus flavus growth and aflatoxin production were characterized in Puerh tea, peanut and polished rice at different initial water activity (aw) values for long-term storage. As a result, food initial aw value was the critical factor for A. flavus growth and aflatoxin production, and A. flavus almost not grew on foods at aw value lower than 0.8. A. flavus grew best in peanut, followed by rice, but growth on Puerh tea was limited. A. flavus growth was inhibited significantly by adding tea to Potato Dextrose Agar (PDA). Accordingly, aflatoxins produced dramatically in peanut, followed by rice at the first 90 days storage. However, aflatoxin neither produced in Puerh tea nor on tea modified PDA, indicating tea components inhibited A. flavus growth and aflatoxins synthesis.

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Bacteriophages (phages) have gained considerable attention as effective antimicrobial agents that infect and kill pathogenic bacteria. Based on this feature, phages have been increasingly used to achieve food safety. They are stored in a medium or buffer to ensure stability; however, they cannot be directly applied to food under these conditions due to reasons such as regulatory considerations and concerns about marketability. This study developed a stabilizing solution that allowed the maintenance of phage activity for extended periods at room temperature while being directly applicable to food. The stability of phages stored in distilled water was relatively low. However, adding a stabilizer composed of sugars and salts improved the survival rates of phages significantly, resulting in stability for up to 48 weeks at room temperature. When Escherichia coli O157:H7-contaminated vegetables were washed with tap water containing phages, the phages reduced the pathogenic E. coli count by over 90% compared with washing with tap water alone. Additionally, when pathogenic E. coli-contaminated vegetables were placed in a phage-coated container and exposed to water, the coating of the container dissolved, releasing phages and lysing the pathogenic E. coli. This led to a significant 90% reduction in pathogenic E. coli contamination compared to that after water rinsing. These results suggest an effective and economical method for maintaining phage activity and establishing the potential for commercialization through application in the food industry.

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Flow cytometry of blood samples is a very valuable clinical and research tool to monitor the immune response in human patients. Furthermore, it has been successfully applied in cats, such as for infections with feline immune deficiency virus (FIV). However, if cells are not isolated and frozen, analysis of anticoagulated blood samples requires mostly prompt processing following blood collection, making later analysis of stored full blood samples obtained in clinical studies often impossible. The SMART Tube system (SMART TUBE Inc., California, United States; SMT) allows fixation and long-term preservation of whole blood samples at -80°C. However, this system has so far only been applied to human biological samples. In the present study, a new flow cytometry SMART Tube protocol adapted for feline whole blood samples was successfully established allowing quantification of T-helper cells, cytotoxic T-cells, B-cells, monocytes, and neutrophils up to 2 years post sampling. Results obtained from frozen stabilized and fresh blood samples were compared for validation purposes and correlated to differential blood counts from a conventional hematology analyzer. Clinical applicability of the new technique was verified by using samples from a treatment study for feline infectious peritonitis (FIP). Using the new SMT protocol on retained samples, it could be demonstrated that long-term storage of these SMT tubes is also possible. In summary, the newly adapted SMT protocol proved suitable for performing flow cytometry analysis on stored feline whole blood samples, thus opening up new avenues for veterinary research on a variety of aspects of clinical interest.

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The remarkable success of two FDA-approved mRNA-encapsulating vaccines (Comirnaty® and Spikevax®) indicated the importance of lipid nanoparticles (LNPs) delivery systems in clinical use. Currently, mRNA-encapsulating LNPs (mRNA-LNPs) vaccines are stored as frozen liquid at low or ultralow temperatures. We designed lyophilized LNPs utilizing FDA-approved lipids to expedite the clinical application of our developed lyophilized mRNA-LNPs in the future. The key parameters of sucrose concentration and the selection and molar ratio of the four lipids in these vaccines were optimized for long-term stability with high transfection efficiency after lyophilization. We demonstrated that 8.7% sucrose is the optimal cryoprotectant concentration to maintain the transfection efficiency of lyophilized mRNA-LNPs. Optimal lipid formulations with high transfection efficiency both before and after lyophilization were screened using an orthogonal experimental design. The ratios of distearoylphosphatidylcholine (DSPC)/cholesterol and the selection of the ionizable and PEGylated lipids are the main factors influencing the long-term stability of mRNA-LNPs. Comparative mouse transfection experiments showed that the optimal lyophilized mRNA-LNPs maintained high mRNA expression after lyophilization, predominantly in the spleen or liver, with no expression in the kidneys or eyes. Our studies demonstrated the importance of the sucrose concentration and of the selection and molar ratio of the four lipids composing LNPs for maintaining mRNA-LNP stability under lyophilization and for long-term storage under mild conditions.

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This study aimed to develop an appropriate modified atmosphere packaging (MAP) system for displayed beef steaks following long-term superchilled (-1 °C) storage. After superchilled storage for 0, 2, 8, or 16 weeks, beef loins were fabricated into steaks and displayed with 20%, 50%, or 80% O2-MAP under chilled conditions. At each storage point, after display for 0, 3, 7, or 10 days, instrumental color, myoglobin redox forms percentage, lipid oxidation, total viable count (TVC), and total volatile basic nitrogen (TVB-N) were evaluated. Meat color stability decreased, with prolonged storage period and display time. When the storage period was within 8 weeks, under all the above MAP conditions, the display time for the beef steaks was up to 10 days. Considering 80% O2-MAP promoted lipid oxidation, 50% and 80% O2-MAP were not recommended for displaying steaks for more than 10 and 7 days respectively after 16 weeks of storage. However, 20%, 50%, or 80% O2-MAP could maintain 3 days of microbial shelf-life according to TVC and TVB-N results. Additionally, after long-term superchilled storage for 16 weeks, the various O2 concentrations had minimal impact on microbiota succession during the MAP display period. Furthermore, beef steaks packaged under various MAP systems exhibited similar microbial compositions, with the dominant bacteria alternating between Lactobacillus and Carnobacterium. This study provided practical guidance for improving beef color stability after long-term superchilled storage.

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

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BACKGROUND QUERCUS: seeds that are recalcitrant to desiccation and freezing temperatures cannot be stored in gene banks under conventional conditions. However, the germplasm of some recalcitrant seeded species can be stored in liquid nitrogen (-196 °C). Unfortunately, for many species, among them for almost the whole genus Quercus, an effective cryostorage method is still unknown. In this study, we propose a successful cryostorage protocol for Quercus petraea (Matt.) Liebl. germplasm using plumules (a shoot apical meristem of an embryo) frozen on aluminium cryo-plates. RESULTS: The plumules isolated from the acorns of ten provenances were prestored in 0.5 M sucrose solution (for 18 h). To form alginate beads (one plumule per bead), the plumules were placed in the wells of a cryo-plate and embedded in calcium alginate gel. For cryoprotection, the encapsulated plumules were immersed in cryoprotectant solution containing 2.0 M glycerol and different concentrations of sucrose (0.8-1.2 M) for 40 min at 25 °C and desiccated under a laminar flow cabinet for 1.0-4.0 h. Cryo-plates with plumules were directly immersed in liquid nitrogen and then cryostored for 30 min. For rewarming, cryo-plates with plumules were immersed in 1.0 M sucrose solution and rehydrated for 15 min at 25 °C. Survival rates varied from 25.8 to 83.4 were achieved after cryoprotection in 1.0 M sucrose solution and the drying of plumules for 2 h. The in vitro regrowth rate of cryopreserved plumules varied among provenances and was 26-77%. CONCLUSIONS: This study presents, for the first time, a successful, simple and effective protocol for the cryopreservation of Q. petraea germplasm that could be used in gene banks. The experiment was successfully repeated on seeds from various provenances, each yielding similar, good results. However, seed quality and storage time after harvesting are important factors in plumule regrowth after cryopreservation.

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The conservation of genetic resources in pig breeds, notably the Iberian pig, is crucial for genetic improvement and sustainable production. Prolonged storage in liquid nitrogen (LN2) is recognized for preserving genetic diversity, but potential adverse effects on seminal quality remain debated. This study aims to assess the impact of ten years of storage at different LN2 levels and to optimize thawing protocols for Iberian pig sperm. Sperm samples from 53 boars were cryopreserved and stored at varying LN2 levels and, a decade later, the samples were thawed at 37 °C for 20 s or at 70 °C for 8 s. Sperm motility, membrane integrity, acrosome status, and DNA fragmentation were evaluated in year 0 and year 10. Overall, no significant differences were observed in post-thaw sperm quality between storage levels in year 0 or year 10. But thawing at 70 °C 8 s showed significant improvements, particularly in samples that were always stored in LN2, in all analyzed parameters except fragmentation, which was not affected by cryostorage. This study suggests that the long-term preservation of Iberian pig sperm does not affect quality over time, regardless of whether the samples were fully submerged in LN2. Furthermore, it is determined that thawing at 70 °C for 8 s maximizes post-thaw sperm quality, especially in those samples stored constantly submerged in LN2.

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Fibre typing by immunohistochemistry on cryosections from human skeletal muscle biopsies is an essential tool in the diagnosis and research of muscular diseases, ageing, and responses to exercise training and disuse. Preserving a good quality in these frozen specimens can be challenging especially if they are stored for longer periods before histological processing, which is often the case in studies with a large number of test subjects and/or repeated sampling separated by multiple years. We demonstrate in this article that both, the morphology and reactivity of epitopes to myosin heavy chain isoforms and dystrophin are well preserved in up to 18-year-stored unfixed and unstained cryosections of human m. vastus lateralis (n = 241). Any variation in staining intensity between samples was unrelated to the age of the biopsy donor or the storage period of the unstained cryosections, and in all cases, the obtained images were appropriate for image analysis, such as the determination of the fibre type composition and the fibre cross-sectional area, and quantitative analysis of muscle capillarisation.

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BACKGROUND: Anisopteromalus calandrae (Howard) is a solitary ectoparasitoid with wide-ranging potential applications as a natural biological control agent against various coleopterous pests in food warehouses. Implementing an effective cold storage program is crucial for extending the shelf life of biological control agents and ensuring their stable and abundant supply. Herein, we attempted to determine the optimal cold storage conditions for Anisopteromalus calandrae by investigating the effect of cold storage at three different temperatures (7, 13, and 19 °C) for 7, 21, and 35 days on four developmental stages (late-instar larvae, early-stage pupae, mid-stage pupae, and 2-day-old adults). Additionally, we explored the maximum cold storage potential by observing early-stage pupae stored at 13 °C for various durations (30, 60, 90, 120, and 150 days). RESULTS: The most suitable cold storage temperature for the early-stage pupae of Anisopteromalus calandrae was 13 °C, and the highest adult emergence rate (98.3%) was after 90 days of storage at 13 °C. Furthermore, we did not find any significant effect on longevity (female: 44.3 days; male: 38.1 days) or fecundity (121.7 wasps). The female ratio ranged from 43.5% to 50.8%. More importantly, cold storage did not adversely affect the developmental duration or fecundity of the offspring. CONCLUSION: This study offers crucial insights for managing Anisopteromalus calandrae populations under laboratory conditions and lays the foundation for potential industrial production and development. © 2023 Society of Chemical Industry.

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Olive leaves as a main byproduct of olive oil and fruit industry are a valuable source of phytochemicals such as polyphenols, with multiple biomedical effects. Apart from leaves, olive branches and stems make up a significant amount of olive waste. It is well known that the drying process and long-term storage affect the stability and concentration of polyphenols present in raw materials. For that matter, two different means of storing olive waste, at room temperature and +4 °C, were compared by determining the content of the polyphenol oleuropein (OLE) in olive leaf, branch, and stem extracts (LE, BE, and SE) by HPLC-DAD method. Total phenols (TPC), o-diphenols (o-DPC), and total flavonoids (TFC) content in extracts were assessed by UV-Vis measurements. LE prepared from leaves stored at +4 °C had the highest OLE content, 30.7 mg g-1 of dry extract (DE). SE from stems stored at +4 °C was the richest in TPC and TFC (193 mg GAE/g DE and 82.9 mg CE/g DE, respectively), due to the higher purity of the extract. The biological activity of extracts was determined on cervical cancer (HeLa), melanoma (A375), metastatic melanoma (A375M) tumor cell lines, and on spontaneously immortalized cell line of keratinocytes (HaCaT), using the MTT assay. The data show that all extracts had a similar dose-dependent effect on cell viability in HeLa cells, while the effect of LE on melanoma A375 and A375M, and HaCaT cells was cell-line dependent.

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This study aimed to develop a long-term pollen storage protocol for Luffa species (L. acutangula, L. cylindrica, L. echinata, and L. graveolens) and assess its potential for crop improvement. The optimal medium for in vitro pollen germination varied by species, with Brewbaker and Kwack (BK) medium with 10% sucrose suitable for L. acutangula, L. cylindrica, and L. echinata, and BK medium with 3% sucrose ideal for L. graveolens. Overestimation in staining tests compared to in vitro pollen germination was observed. The best results for cryopreservation were achieved with desiccation periods of 20, 30, and 40 min, maintaining moisture content between 14.04% and 18.55%. Pollen viability was negatively correlated with storage temperature (25, 4, and -20°C) and duration. Cryopreserved pollen at -196°C exhibited the highest viability over a prolonged period (2 months) and was comparable to fresh pollen in terms of germination, ovule fertilization, and fruit and seed set. This study presents a simple and reproducible pollen cryopreservation protocol applicable across Luffa species, facilitating long-term storage and its use in crop improvement efforts.

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Hydrogen is recognized as a significant potential energy source and energy carrier for the future. On the one hand, storing hydrogen is a challenging task due to its low volumetric density, on the other hand, a particular type of hydrogen in the form of a liquid can be used to store large quantities of hydrogen at ambient conditions in thermoplastic tanks. But storing hydrogen in this form for a long time in polymer tanks affects the physical and chemical properties of the liner. In the current automotive industry, high-density polyethylene (HDPE) has already been used in existing fuel tank applications. However long-term exposure to fuels leads to the permeation of hydrocarbons into the polymers, resulting in a loss of mechanical properties and reducing the efficiency of fuel cells (FC) in automotive applications. Additionally, facing material shortages and a limited supply of resin leads to an increase in the cost of the material. Therefore, an alternative material is being searched for, especially for hydrogen fuel tank applications. In this study, two semi-crystalline thermoplastics, HDPE and polyketone (POK), were compared, which were exposed to a selected liquid organic hydrogen carrier (LOHC) at 25 °C and 60 °C for up to 500 h in an enclosed chamber, to measure their fuel up-take. A short analysis was carried out using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and mechanical testing to understand the influence of the LOHC on the polymer over time. Fuel sorption and tensile properties showed a plasticizing effect on HDPE. The material degradation was more pronounced for the aged samples of HDPE in comparison to POK. As expected, thermal aging was increased at 60 °C. The fuel absorption of POK was lower compared to HDPE. A slight increase in crystallinity was observed in POK due to the aging process that led to changes in mechanical properties. Both HDPE and POK samples did not show any chemical changes during the aging process in the oven at 25 °C and 60 °C.

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With the widespread application of embryo cryopreservation in assisted reproductive techniques, it is necessary to assess the safety of long-term cryopreservation of human embryos and it is unclear whether storage time has an impact on the DNA methylation profiles of human embryos. Nine women who received IVF treatment were recruited for this study. The retrieved eight-cell human embryos were classified into three groups including fresh embryos, cryopreserved embryos stored for 3 years, and cryopreserved embryos stored for 8 years. Single-cell whole-genome bisulfite sequencing (scWGBS) was conducted. The genome-wide methylation pattern of the fresh and two cryopreserved groups were similar. In addition, the methylation level in different genomic regions showed comparable patterns and no significant differences were observed in the methylation level of imprinted genes among the three groups. A total of 587 differentially methylated regions (DMRs) in the 3-year group and 540 DMRs in the 8-year group were identified comparing to fresh group. However, they were not enriched in promoters and had a similar genome-wide distributions, suggesting that these DMRs may not contribute to the changes in corresponding gene expressions. Our study illustrated that long-term cryopreservation will not affect the DNA methylation profiles of human eight-cell embryos at single-cell level.

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Studies of structural changes in mAbs under forced stress and storage conditions are essential for the recognition of degradation hotspots, which can be further remodeled to improve the stability of the respective protein. Herein, we used diethyl pyrocarbonate (DEPC)-based covalent labeling mass spectrometry (CL-MS) to assess structural changes in a model mAb (SILuMAb). Structural changes in the heat-stressed mAb samples were confirmed at specific amino acid positions from the DEPC label mass seen in the fragment ion mass spectrum. The degree of structural change was also quantified by increased or decreased DEPC labeling at specific sites; an increase or decrease indicated an unfolded or aggregated state of the mAb, respectively. Strikingly, for heat-stressed SILuMAb samples, an aggregation-prone area was identified in the CDR region. In the case of longterm stress, the structural consequences for SILuMAb samples stored for up to two years at 2-8 °C were studied with SEC-UV and DEPC-based CL-MS. While SEC-UV analysis only indicated fragmentation of SILuMAb, DEPC-based CL-MS analysis further pinpointed the finding to structural disturbances of disulfide bonds at specific cysteines. This emphasized the utility of DEPC CL-MS for studying disulfide rearrangement. Taken together, our data suggests that DEPC CL-MS can complement more technically challenging methods in the evaluation of the structural stability of mAbs.

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Caenorhabditis elegans is an exceptional model organism. More than twenty thousand different strains have been developed, increasing knowledge on countless topics. However, the traditional method to cryopreserve this nematode, based on slow freezing, usually reaches recovery rates of around 35% for the L1 and L2 larval stages. Here, we propose two alternative methods to cryopreserve this nematode based on vitrification that are applicable in common laboratories and allow the selective individual cryopreservation of this organism. These new methods require ultra-high warming rates, which are achieved by employing very thin capillaries as the nematode container, and a very low final concentration of cryoprotectants, which, as compared to slow freezing, reduce toxicity damage. The recovery rate was 98.5% for larvae (L1 - L4) and 84.3% for adults. Given these results, our procedures offer an alternative to cryopreserve this nematode (larvae and adults) with higher recovery rates, avoiding expensive requirements. Indeed, it only needed a container with liquid nitrogen and a warming bath for water at 37 °C. The high performance of this approach has been revealed by preserving the long-term memory and, probably, the connectome of this nematode.

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