Cryopreservation In Liquid Nitrogen Research Articles

Semen cryopreservation and storage in liquid nitrogen: Impact on chromatin compaction.

Sperm cryopreservation is a consolidate option for long-term male fertility preservation. The freezing/thawing procedure causes detrimental effects to spermatozoa, including damage to viability, motility, membrane composition, and DNA, whereas the effect on sperm chromatin compaction is less studied. The primary aim of this study was to investigate the impact of cryopreservation on sperm chromatin compaction. Furthermore, the effect of cryopreservation on sperm parameters (motility, viability, chromatin compaction, and DNA fragmentation) was also assessed in relation to the storage time in liquid nitrogen. Semen samples, collected from 126 (92 normozoospermic and 34 oligozoospermic) patients undergoing routine semen analysis in the Andrology Laboratory of Careggi University Hospital of Florence, were frozen by conventional fast vapor freezing method. Sperm motility, viability, kinematic parameters (by computer-aided sperm analysis [CASA]), chromatin compaction (by staining with both aniline blue [AB] and Chromomycin A3 [CMA3]), and sperm DNA fragmentation (sDF, by TUNEL/Propidium Iodide [PI]) were evaluated before freezing and after thawing at different timepoints. After 7 days of storage, a significant decline in sperm motility, viability, and kinematics parameters, as well as a significant increase in the percentage of sperm positivity to CMA3, AB, and sDF, were observed. It is noteworthy that while motility and viability decreased in almost all subjects, the increase in CMA3 and AB positivity was observed in 68.0% and 79.2% of samples, respectively. A progressive deterioration of sperm motility and viability, less evident for chromatin structure, was observed at longer times of storage (28 and 180 days). Our results indicate that freezing/thawing procedures can alter chromatin structure. A reduction in protamine content and/or a modification in chromatin assembly can be hypothesized. Furthermore, the length of storage in liquid nitrogen appears to progressively affect sperm parameters, although it should be confirmed in larger cohort of subjects. Current sperm cryopreservation protocols need to be improved with new strategies and personalized procedures aimed to minimize the damage.

Effects of oxidative stress on the changes of viability of Paeonia lactiflora seeds with different water content before and after cryopreservation

Cryopreservation in liquid nitrogen (LN) is an efficient long - term seed preservation strategy and water content is one of the main factors affecting seed viability after cryopreservation. In this study, Paeonia lactiflora seeds with varying water content were used as materials to determine changes in the antioxidant system before and after cryopreservation. When the water content of P. lactiflora seeds was 15.96 %–10.12 %, the viability of P. lactiflora seeds decreased significantly compared with that of the seeds without cryopreservation, but at the water content of 8.14 %–7.56 % there were no significant differences. However, when the water content of P. lactiflora seeds decreased to 6.01 %–5.19 % the viability was slightly increased compared to the seeds without cryopreservation. The content of superoxide anion (O2−), hydroxyl radical (·OH), hydrogen peroxide (H2O2), malondialdehyde (MDA) and protein carbonyl group (PCO) of seeds with high water content (15.96 %–10.12 %) were significantly increased after cryopreservation. However, superoxide anion (O2−), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) did not change significantly in seeds with low water content (6.01 %–5.19 %) after cryopreservation, while oxidative stress indexes MDA and PCO decreased. These three substances were significantly negatively correlated with seed viability. In terms of antioxidant substances, the contents of catalase (CAT), ascorbic acid (AsA) and glutathione (GSH) decreased and the activities of glutathione reductase (GR) and dehydroascorbate reductase (DHAR) increased during the cryopreserved process of seeds with varying water content. These changes were significantly correlated with ROS content and the changes of MDA and PCO, among which AsA content, GSH content and CAT activity were positively correlated with seed viability. The changes of GR and DHAR activity were negatively correlated with seed viability. In summary, when the water content of the seeds ranged from 8.14 % to 5.19 %, ROS content did not increase significantly after cryopreservation compared with that of before preservation. The changes of MDA and PCO contents before and after cryopreservation, it was inferred that no obvious oxidative damage occurred in seeds, so the viability of seeds did not decrease compared with that of before cryopreservation. Therefore, the optimum water content of P. lactiflora seeds for cryopreservation is 8.14 %–5.19 %.

Fuzi polysaccharide isolated from Aconitum carmichaeli protects against liquid nitrogen cryopreservation-induced damage in rat abdominal aorta by enhancing autophagy

ObjectiveTo investigate the potential protective mechanisms of aconite polysaccharide (FZPS-1) during cryopreservation, with a particular emphasis on morphological changes in autophagy in rat abdominal aorta. MethodsThirty-six male SD rats were divided into the control group, the cryopreserved model group, and the FZPS-1 intervention group treated with different concentrations of FZPS-1. The structural changes of the abdominal aortic wall were assessed via Masson staining, while cytolysosomes were identified using transmission electron microscopy (TEM). The expression of Beclin-1, LC3-II, and P62 was detected by immunohistochemistry (IHC) and western blot separately. Bcl-2 and Bax mRNA expression was measured by RT-qPCR. ResultsCompared with the control group, the abdominal aortic wall in the model group was severely damaged. Contrarily, FZPS-1 10 mg/mL and 20 mg/mL groups had relatively normal structure of the blood vessel wall, higher cytolysosome counts, and increased Beclin-1 and LC3-II expression compared with the model group (all P<0.05); P62 expression also increased in the FZPS-1 20 mg/mL group (P<0.05). Compared with the control group, the mRNA expression of Bcl-2 in the cryopreservation model group was reduced (P<0.05), while Bax was increased (P<0.05). Compared with the cryopreservation model group, the mRNA expression of Bcl-2 was upregulated, while Bax was downregulated in the FPS 10 mg/L group (P<0.05). ConclusionDuring liquid nitrogen cryopreservation, autophagy is inhibited in the rat abdominal aorta, and the blood vessel wall structure is damaged. FZPS-1, as a cryoprotectant, can enhance autophagy and mitigate blood vessel wall damage in the rat abdominal aorta.

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an “off-the-shelf” bioartificial liver system

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

Establishment, characterization and application of Largemouth bronze gudgeon (Coreius guichenoti) fin and skin cell lines in disease resistance

Largemouth bronze gudgeon (Coreius guichenoti) is an economically important fish species in the upstream Yangtze River. However, C. guichenoti has been listed in the National Key Wild Animal List given that its wild population dramatically declined under the influence of hydroelectric projects, intensive fishing, and environmental pollution. To protect this endangered species, C. guichenoti fin (CGF) and skin (CGSK) cell lines were established in present study. Both cell types exhibited epithelial morphology, and the optimal culture conditions for CGF and CGSK were cultured at 28 °C in L-15 medium with 20% serum. Currently, CGF and CGSK have been sub-cultured up to 60 passages and 30 passages, respectively. Gene identification results of nd1, 16 s, 12 s, cytb, and coi indicated that these two cells originated from the C. guichenoti, with a chromosomal number of 2n = 50. After cryopreservation in liquid nitrogen for 6 months, cell viability remained above 70%. Subsequently, predominant expressed genes by transcriptome analysis implied that CGSK cells mainly consisted of dermis fibroblast, while CGF cell types primarily comprised myofibroblasts. The successful transfection of siRNA and pEGFP-N1 into CGF and CGSK cells were observed. Furthermore, both cell lines exhibited significant cytopathic effects (CPE) when stimulated with spring viremia of carp virus (SVCV). Therefore, the establishment of CGF and CGSK cell lines provides a solid foundation for the recovery and conservation of genetic resources, as well as immunological and toxicological studies of C. guichenoti.

Long-Term Cryopreservation May Cause Genomic Instability and the Premature Senescence of Cells.

Cryopreservation is an essential step for utilizing various cell types for biological research and medical purposes. At the same time, there is a lack of data on the effect of cryopreservation, especially when prolonged, on the karyotype of cells. In the present work, we analyzed the genetic stability of cells subjected to a cryopreservation procedure. The objects were immortalized Chinese hamster lung fibroblasts (CHL V-79 RJK line) and human endometrial mesenchymal stem/stromal cells (eMSCs). We showed that short-term cryopreservation in liquid nitrogen for up to 6 months did not affect the karyotype stability of CHL V-79 RJK and eMSCs. On the contrary, karyotyping of G-banded metaphase chromosomes in cells underwent 10-year cryopreservation, which revealed genomic instability in both cell lines associated with the variability of chromosome number in cells, random chromosomal rearrangements, and condensation disorder in homologs. In addition, we found out that long-term cryopreservation of eMSCs does not affect the expression of their typical surface markers and morphology, but results in a significant reduction in proliferative potential and early manifestation of cellular senescence features upon eMSCs culturing. Thus, we concluded that the long-term cryopreservation of cells of different types and biological origin can lead to irreversible changes of their karyotype and acceleration of cellular senescence.

Dynamics of Organic Acids during the Droplet-Vitrification Cryopreservation Procedure Can Be a Signature of Oxidative Stress in Pogostemon yatabeanus.

Cryopreservation in liquid nitrogen (LN, -196 °C) is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. In previous studies, a systematic approach was used to develop a droplet-vitrification (DV) cryopreservation protocol for Postemon yatabeanus shoot tips that includes preculture with 10% sucrose, osmoprotection with C4-35%, cryoprotection with A3-80% vitrification solution, and a three-step regrowth starting with the ammonium-free medium. The tricarboxylic acid (TCA) cycle is a crucial component of plant cell metabolism as it is involved in redox state regulation and energy provision. We hypothesized that organic acids (OAs) associated with the TCA and its side reactions indirectly indicate metabolism intensity and oxidative stress development in shoot tips under the cryopreservation procedure. In this study, the contents of 14 OAs were analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS) in P. yatabeanus shoot tips in a series of treatments including individual steps of the DV procedure, additional stress imposed by non-optimum protocol conditions (no preculture, no osmoprotection, various vitrification solution composition, using vials instead of aluminum foils, etc.) and regrowth on different media with or without ammonium or growth regulators. The possible relation of OA content with the total cryoprotectant (CPA) concentration and shoot tips regeneration percentage was also explored. Regeneration of cryopreserved shoot tips reduced in descending order as follows: standard protocol condition (91%) > non-optimum vitrification solution (ca. 68%) > non-optimum preculture (60-62%) > regrowth medium (40-64%) > no osmoprotection, cryopreservation in vials (28-30%). Five OAs (glycolic, malic, citric, malonic, and lactic) were the most abundant in the fresh (control) shoot tips. The dynamic pattern of OAs during the DV procedure highly correlated (r = 0.951) with the total CPA concentration employed: it gradually increased through the preculture, osmoprotection, and cryoprotection, peaked at cooling/rewarming (6.38-fold above control level), and returned to the fresh control level after 5 days of regrowth (0.89-fold). The contents of four OAs (2-hydroxybutyric, 3-hydroxypropionic, lactic, and glycolic) showed the most significant (10-209-fold) increase at the cooling/rewarming step. Lactic and glycolic acids were the major OAs at cooling/rewarming, accounting for 81% of the total OAs content. The OAs were categorized into three groups based on their dynamics during the cryopreservation protocol, and these groups were differently affected by protocol step modifications. However, there was no straightforward relationship between the dynamics of OAs and shoot tip regeneration. The results suggest that active modulation of OAs metabolism may help shoot tips to cope with osmotic stress and the chemical cytotoxicity\ of CPAs. Further intensive studies are needed to investigate the effect of cryopreservation on cell primarily metabolism and identify oxidative stress-related biomarkers in plant materials.

Assessing the Benefits of Antioxidant and Antibiotics Treatments for Cryopreservation of the Model Alga Ectocarpus Siliculosus and the Endemic Alga Acinetospora Asiatica

BACKGROUND: Cryopreservation in liquid nitrogen is a suitable technique for preserving seaweeds, a group of photosynthetic organisms with many applications. Although there are some standard protocols for seaweed cryopreservation, most rely on expensive controlled-rate coolers. Moreover, several factors, such as the use of antioxidants or antibiotics, remain unexplored. OBJECTIVE: To test the effect of 2-mercapthoethanol (antioxidant) and antibiotic mixtures on the cryopreservation of the model alga Ectocarpus siliculosus and the endemic brown seaweed Acinetospora asiatica using a low-tech passive rate cooler. MATERIALS AND METHODS: 2mercaptoethanol was added to the cryoprotectant (CPA) solution, while antibiotic mixtures were included in the culture medium during the recovery process. In addition, two CPA solutions were tested on E. siliculosus. RESULTS: After two weeks of recovery, the treatment comprising PSC antibiotic mixture (Penicillin G, streptomycin, and chloramphenicol) showed a significant increase in post-thaw viability. Antioxidant treatment did not improve viability. The highest viabilities for E. siliculosus and A. asiatica were 64-83%, and 83-87%, respectively, using 10% glycerol + 10% proline as CPA solution. CONCLUSION: E. siliculosus and A. asiatica were successfully cryopreserved using a low-tech passive rate cooler, 10% glycerol + 10% proline solution, and antibiotic treatment. The highest post-thaw viabilities (64-87%) reported for PSC antibiotic mixture suggest the potential benefits of using antibiotics during post-thaw recovery of marine macroalgae. This study is the first report on the cryopreservation of A. asiatica.

Effects of Stool Sample Preservation Methods on Gut Microbiota Biodiversity: New Original Data and Systematic Review with Meta-Analysis.

Here, we aimed to compare the effects of different preservation methods on outcomes of fecal microbiota. We evaluated the effects of different preservation methods using stool sample preservation experiments for up to 1 year. The stool samples from feces of healthy volunteers were grouped based on whether absolute ethanol was added and whether they were hypothermically preserved. Besides, we performed a systematic review to combine current fecal microbiota preservation evidence. We found that Proteobacteria changed significantly and Veillonellaceae decreased significantly in the 12th month in the room temperature + absolute ethanol group. The four cryopreservation groups have more similarities with fresh sample in the 12 months; however, different cryopreservation methods have different effects on several phyla, families, and genera. A systematic review showed that the Shannon diversity and Simpson index of samples stored in RNAlater for 1 month were not statistically significant compared with those stored immediately at -80°C (P = 0.220 and P = 0.123, respectively). The -80°C refrigerator and liquid nitrogen cryopreservation with 10% glycerine can both maintain stable microbiota of stool samples for long-term preservation. The addition of absolute ethanol to cryopreserved samples had no significant difference in the effect of preserving fecal microbial characteristics. Our study provides empirical insights into preservation details for future studies of the long-term preservation of fecal microbiota. Systematic review and meta-analysis found that the gut microbiota structure, composition, and diversity of samples preserved by storage methods, such as preservation solution, are relatively stable, which were suitable for short-term storage at room temperature. IMPORTANCE The study of gut bacteria has become increasingly popular, and fecal sample preservation methods and times need to be standardized. Here, we detail a 12-month study of fecal sample preservation, and our study provides an empirical reference about experimental details for long-term high-quality storage of fecal samples in the field of gut microbiology research. The results showed that the combination of -80°C/liquid nitrogen deep cryopreservation and 10% glycerol was the most effective method for the preservation of stool samples, which is suitable for long-term storage for at least 12 months. The addition of anhydrous ethanol to the deep cryopreserved samples did not make a significant difference in the preservation of fecal microbiological characteristics. Combined with the results of systematic reviews and meta-analyses, we believe that, when researchers preserve fecal specimens, it is essential to select the proper preservation method and time period in accordance with the goal of the study.

The effect of cryopreservation in liquid nitrogen on the viability of flax seeds

Background. A promising technique for preserving plant genetic resources is cryopreservation of seeds in liquid nitrogen at –196°C. The lack of information on cryopreservation of flax seeds and on the effect of liquid nitrogen on their viability makes this work relevant for the conservation of valuable flax germplasm.Materials and methods. Seeds of the fiber flax cultivar ‘Orshansky 2’ were used in the experiment. The experiment was carried out in 2 replications; each of them included 6 options. In options 1 and 2, the seeds were stored in paper bags at room temperature (1 – control, seeds without treatment; 2 – treated with a biodegradable polymer of 3-hydroxybutyric acid [P(3GB)]). In options 3, 4, 5 and 6, the seeds were stored in liquid nitrogen without treatment and with P(3GB) treatment (in 3 and 4 in gauze packaging, and in 5 and 6 in laminated foil bags).Results. Laboratory-based germination energy and viability of control seeds were the same in all options of the experiment (99%), and field viability averaged 83%. The lowest germination energy, laboratory-based and field germination capacity were observed after storage in laminated bags, without P(3HB) processing: option 5. stage II (79%); option 5, stage III (74%), and with P(3HB): processing: option 6, stage II (78%); option 6, stage III (82%). A two-factor analysis of variance showed that liquid nitrogen had significant effect on laboratory germination energy, field germination capacity, laboratory germination energy and capacity after 6 months of storage at room temperature: the effect size was 42, 11, 31, and 24%, respectively, while treatment with P(3HB) had no significant effect on any of the options of the experiment with its application.Conclusion. Flax seeds tolerated direct immersion in liquid nitrogen (t = –196°C) best of all in gauze packaging. The biodegradable polymer P(3HB) did not produce a significant effect on seed viability in any option.

Survival and State of Garlic Explants of Two Lithuanian Cultivars after Cryopreservation

Cryopreservation features of Lithuanian garlic (Allium sativum L.) cultivars ‘Žiemiai’ and ‘Dangiai’ were investigated. The survival rate and state of explants, depending on the type of explant, and dehydration conditions were evaluated in the experiment. Explants isolated from cloves and bulbils were dehydrated with a plant vitrification solution 3 (PVS 3) containing 50% w/v glycerol and 50% w/v sucrose in liquid MS medium. Three different exposure durations of 1.5, 2, and 3 h in PVS3 solution were applied at 0 °C. Unloaded stem domes were planted in Petri dishes with MS medium supplemented with 1 mg L−1 naphthaleneacetic acid (NAA) and 6–dimethylallylamino purine (2-iP) on 0.8% plant agar. The obtained results showed that the cryopreservation in liquid nitrogen reduced the survival rate of explants by 20–40%. The average number of surviving explants after freezing reached 64.3% for both cultivars. Cultivar ‘Žiemiai’ showed a 24% higher capability for survival than ‘Dangiai’. The total number of surviving explants of ‘Žiemiai’ reached 76.1%. Explants from bulbils were 23% more effective for cryopreservation compared to cloves. Evaluation of the effect of dehydration duration showed that the survival rate of the explants from bulbils of both cultivars was highest after treatment with PVS3 for 3 h and reached 91%. Treatment in PVS3 solution for 2 h was sufficient for survival of explants from cloves. Obtained results confirm that the efficiency of garlic cryopreservation depends on complex factors.

Criopreservação de sementes de espécies brasileiras do gênero Dyckia (Bromeliaceae)

Cryopreservation of seeds of Brazilian species of the genus Dyckia (Bromeliaceae). The objective of this study was to evaluate different cryoprotectants in cryopreservation in liquid nitrogen (NL) of seeds of Dyckia dusenii, Dyckia kranziana and Dyckia walteriana and to quantify the lipid content of the seeds. The treatments were: control, immersion in NL, glycerol 1 mol.L-1, sucrose 0.4 mol.L-1, PVS1, PVS2, PVS2 + 1% floroglucinol and PVS3. From the seeds, the total lipid content was evaluated. The percentage of germination and abnormal seedlings, germination speed index, length and dry mass of the seedlings were evaluated. After 150 days, the length and dry mass of the aerial and root parts. The cryoprotectants were not effective in the seeds of D. dusenii and D. kranziana, however, for those of D. walteriana it is recommended to use the cryoprotectants glycerol 1 mol.L-1, sucrose 0.4 mol.L-1, PVS1, PVS2 , PVS2 + 1 % floroglucinol or PVS3. Studies of the composition of saturated and unsaturated fatty acids are necessary to establish a relationship with cryopreservation.

Evaluation of the genetic stability of Leptospira reference strains maintained under two conservation methods

Objective. The genetic stability of Strains of Leptospira spp., maintained under two conservation systems, was evaluated. Methodology. The degree of conservation of the 16S rRNA and ompL1 genes of 10 reference serovars from the Leptospira spp. collection, belonging to the Sistema de Bancos de Germoplasma de la Nación para la Alimentación y la Agricultura (SBGNAA), was determined. Results. It was corroborated that the genes evaluated these have not undergone considerable changes, since similarities greater than 99.69 % were evidenced for 16S rRNA and 99.02% for ompL1, in the paired alignments. Conclusion. The genetic stability and purity of the reference strains of Leptospira spp. were verified. spp., kept in cryopreservation in liquid nitrogen at -196 °C and at room temperature for approximately eight years.

No time to die: Comparative study on preservation protocols for anaerobic fungi.

Anaerobic fungi (AF, phylum Neocallimastigomycota) are best known for their ability to anaerobically degrade recalcitrant lignocellulosic biomass through mechanic and enzymatic means. While their biotechnological potential is well-recognized, applied research on AF is still hampered by the time-consuming and cost-intensive laboratory routines required to isolate, maintain, and preserve AF cultures. Reliable long-term preservation of specific AF strains would aid basic as well as applied research, but commonly used laboratory protocols for AF preservation can show erratic survival rates and usually exhibit only moderate resuscitation success for up to one or two years after preservation. To address both, the variability, and the preservation issues, we have set up a cross-laboratory, year-long study. We tested five different protocols for the preservation of AF. The experiments were performed at three different laboratories (Austria, Germany, Switzerland) with the same three morphologically distinct AF isolates (Anaeromyces mucronatus, Caeocmyces sp., and Neocallimastix cameroonii) living in stable co-culture with their naturally occurring, syntrophic methanogens. We could show that handling greatly contributes to the variability of results, especially in Anaeromyces mucronatus. Cryopreservation of (mature) biomass in liquid nitrogen had the highest overall survival rates (85–100%, depending on the strain and laboratory). Additionally, preservation on agar at 39°C had surprisingly high survival rates for up to 9 months, if pieces of agar containing mature AF thalli were resuscitated. This low-cost, low-effort method could replace consecutive batch cultivation for periods of up to 6 months, while long-term preservation is best done by cryopreservation in liquid nitrogen. Regardless of the method, however, preserving several replicates (>three) of the same strain is highly advisable.

Cryopreservation of Pig Semen Using a Quercetin-Supplemented Freezing Extender.

Reactive oxygen species (ROS) produced during freeze–thaw procedures cause oxidative damage to the sperm, reducing fertility. We aimed to improve the post-thaw quality of pig sperm by quercetin (QRN) supplementation to reduce the cryodamage associated with the freeze–thaw procedure. Four equal aliquots of pooled boar semen were diluted with a freezing extender supplemented with different concentrations of QRN (0, 25, 50, and 100 µM) and then were subjected to cryopreservation in liquid nitrogen. Semen analysis was performed following 7 days of cryopreservation. Results demonstrated that the semen samples supplemented with 50 µM QRN significantly improved the post-thaw sperm quality than those subjected to other supplementations (p < 0.05). Semen samples supplemented with 50 µM QRN showed significantly improved plasma membrane functional integrity (47.5 ± 1.4 vs. 43.1 ± 4.1, 45.3 ± 1.7, and 44.1 ± 1.4) and acrosome integrity (73.6 ± 3.4 vs. 66.3 ± 2.4, 66.7 ± 3.6, and 68.3 ± 32.9) as compared to the control, 25 µM, and 100 µM QRN groups, respectively. The mitochondrial activity of the 50 µM QRN group was greater than control and 25 µM QRN groups (43.0 ± 1.0 vs. 39.1 ± 0.9 and 41.9 ± 1.0) but showed no difference with the 100 µM QRN group. Moreover, the 50 µM QRN group showed a higher sperm number displaced to 1 cm and 3 cm points in the artificial mucus than other groups. Therefore, supplementing the freezing extender with QRN can serve as an effective tool to reduce the magnitude of oxidative damage associated with sperm freezing.

Comprehensive evaluation of the effects of long-term cryopreservation on peripheral blood mononuclear cells using flow cytometry

Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

Malt fly antifreeze cocktail more than the sum of its parts

Even though water is a prerequisite for life, ice is a killer. Forming crystals in frozen tissue, ice ruptures cells and causes irreversible damage. But a select band of creatures have evolved strategies for avoiding the perils of freezing, and the larvae of the malt fly, Chymomyza costata, are possibly some of the most resilient creatures on the planet. ‘Overwintering cold-acclimated larvae in diapause can survive freezing to –75°C and even survive long-term (18 months) cryopreservation in liquid nitrogen’, says Vladimír Koštál from the Czech Academy of Sciences, Czech Republic. Knowing that the robust insects flood their bodies with an amino acid antifreeze, proline, for protection as temperatures plummet, Koštál and colleagues from several Czech institutions – including the Czech Academy of Sciences, the University of South Bohemia and the Crop Research Institute in Praha – set out to identify additional compounds which could offer the hardy larvae protection from a bitter winter.Simulating winter conditions in the lab by cutting the larvae's day length from 16 h at 18°C to 12 h, the team then dropped the temperature to 11°C, while plunging the insects into complete darkness for a week before chilling the larvae further to 4°C to trigger an extreme form of suspended animation, known as diapause. Then the team collected haemolymph – insect blood – from the dormant insects, as well as portions of various tissues, including muscle, fat and the midgut, to find out which protective components accumulated in the insects’ bodies, and where, as they prepared for winter.In addition to a colossal (∼10-fold) increase in the amount of proline circulating in the insect's haemolymph (from 35 mmol l−1 to 313.2 mmol l−1), the larvae also accumulated two other amino acids (asparagine more than doubling to 25.8 mmol l−1 and glutamine rising from 33.6 to 55.2 mmol l−1). The antifreeze sugar trehalose also increased more than twice, up to 107.5 mmol l−1, while several other trace compounds that are known to protect cells from freezing and dehydration increased by more than 50%. But how well does this antifreeze cocktail protect the dormant insects from frost damage?The team investigated the impact of the five most abundant components (proline, trehalose, glutamine, asparagine and betaine) on the insects’ haemolymph as the temperature dropped and realised that instead of forming ice crystals, the fluid became like a solid glass. And when the team tracked how the different components of the cocktail protected the insects as the temperature fell, it was clear that the sugar trehalose was a key player, driving the transformation of body fluids to form glass. Meanwhile, the amino acid proline allowed the haemolymph to remain fluid at temperatures down to –30°C, accumulating between delicate tissues and areas packed with ice crystals forming a protective bumper.But could the antifreeze cocktail protect unprepared malt fly larvae from the damaging effects of ice formation at low temperatures? Koštál and colleagues fed 17-day-old larvae a diet supplemented with the antifreeze cocktail's main components (proline, trehalose, glutamine, asparagine and betaine, mixed in the correct proportions) for 3 days and were amazed that 58% of the larvae survived freezing in liquid nitrogen for an hour, with almost 20% going on to develop into adults. However, when the team tested the survival of larvae fed individual components alone, only proline seemed to provide a little protection from freezing. ‘Simple addition of the individual effects of the components was not sufficient to explain the effect of the complete cryoprotective cocktail, suggesting a synergy and division of labour between the components’, says Koštál.

A mixture of innate cryoprotectants is key for freeze tolerance and cryopreservation of a drosophilid fly larva.

Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on the mechanisms of individual CP interactions, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here, we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata, which survives long-term cryopreservation in liquid nitrogen. The CPs proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine and sarcosine accumulate in hemolymph in a ratio of 313:108:55:26:6:4:2.9:0.5 mmol l-1. Using calorimetry, we show that artificial mixtures, mimicking the concentrations of major CPs in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it probably forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.

Cryopreservation of human amniotic membrane for ocular surface reconstruction: a comparison between protocols.

To compare the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -180°C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80°C. Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -180°C, dimethyl sulfoxide at -80°C, dextran-based medium at -180°C, dextran-based medium at -80°C and dry freezing at -80°C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. Switching from liquid nitrogen cryopreservation to -80°C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.

The review of fungi and omycota long-term storage methods

Fungi are extremly diverse and beneficial organisms both for environment and industry; thus we are obliged to secure it’s biodiversity. There are several laboratory methods for fungi longterm preservation. The main goal is to secure their vitality and biological properties. Some of them are simple but the same time laborious, other demand specific equipment. Among the most popular methods we find: freez-drying, cryopreservation at –80°C and in liquide nitrogen. The most reliable is cryopreservation in liquid nitrogen. However even this method is not universal and siutable for all cultures.