Cryoprotectant Solution Research Articles

Ice recrystallization inhibitors enable efficient cryopreservation of induced pluripotent stem cells: A functional and transcriptomic analysis

The successful use of human induced pluripotent stem cells (iPSCs) for research or clinical applications requires the development of robust, efficient, and reproducible cryopreservation protocols. After cryopreservation, the survival rate of iPSCs is suboptimal and cell line-dependent. We assessed the use of ice recrystallization inhibitors (IRIs) for cryopreservation of human iPSCs. A toxicity screening study was performed to assess specific small-molecule carbohydrate-based IRIs and concentrations for further evaluation. Then, a cryopreservation study compared the cryoprotective efficiency of 15 mM IRIs in 5 % or 10 % DMSO-containing solutions and with CryoStor® CS10. Three iPSC lines were cryopreserved as single-cell suspensions in the cryopreservation solutions and post-thaw characteristics, including pluripotency and differential gene expression were assessed. We demonstrate the fitness-for-purpose of 15 mM IRI in 5 % DMSO as an efficient cryoprotective solution for iPSCs in terms of post-thaw recovery, viability, pluripotency, and transcriptomic changes. This mRNA sequencing dataset has the potential to be used for molecular mechanism analysis relating to cryopreservation. Use of IRIs can reduce DMSO concentrations and its associated toxicities, thereby improving the utility, effectiveness, and efficiency of cryopreservation.

Assessment of lyophilized microencapsulated multi strain probiotic for improved shelf life and stability

A Multi species Probiotics (MSP) of calf gut origin was encapsulated either with chitosan (ECMSP) or without chitosan (EMSP). Further, the probiotic was encapsulated and lyophilized (ELMSP) using three types of cryoprotectant solutions sucrose (ELMSP(S)), skim milk (ELMSP(SM)) and sucrose + skim milk combination (ELMSP (SSM)). The encapsulation yield was good and ranged from 75 % to 80 % for MSP products. These MSP products were stored at refrigeration temperature (4 °C), deep freeze and room temperature for 12 weeks. The shelf life (mean CFU) of encapsulated microcapsules was significantly better (P < 0.001) at the refrigeration temperature (9.44 Log10 CFU/g) than deep freeze (7.42 Log10 CFU/g) and room temperature (6.67 Log10 CFU/g). At the end of the 12 weeks storage period, EMSP maintained 25.1 %, 49.6 % and 32.6 % viable counts at room temperature, refrigeration and deep freeze condition respectively. For the lyophilized-encapsulated MSP, the mean probiotic counts of ELMSP(S) and ELMSP (SSM) were significantly (P < 0.001) higher (9.62 Log10 CFU/g) than those of ELMSP(SM) ((9.36 Log10 CFU/g) at room temperature. At refrigeration temperature, the mean probiotic counts for ELMSP (SSM. (10.1 Log10 CFU/g)) were significantly higher (P < 0.001) than ELMSP(S, (9.97 Log10 CFU/g) and ELMSP(SM, (9.86 Log10 CFU/g). Further, a 12 weeks storage study for ELMSP (SSM) showed that the microcapsules stored at refrigeration temperature (4°C) showed only 6.4 % drop in viability which was significantly lower (P < 0.001) than that of room temperature showing 15.3 % drop and deep freeze storage with 17.1 % drop in viability. It was appreciable that even at room temperature; there was only one log drop in the viability of MSP showing that it can be comfortably stored at room temperature for longer periods at field level.

Efficacy assessment of different cryoprotectants for preserving the viability of Enterobacterales strains at − 20 °C

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.

Coenzyme Q10 Improves the Post-Thaw Sperm Quality in Dwarf Surfclam Mulinia lateralis.

Previous studies have shown that post-thaw sperm performance is affected by multiple stressors during cryopreservation, such as those induced by physical, chemical, mechanical and physiological changes. One of these is the balance disturbance between the antioxidant defense system and reactive oxygen species (ROS) production. This study investigated whether this disturbance could be alleviated by the addition of different antioxidants to cryoprotective solution [8% dimethyl sulfoxide (DMSO) in 1 µm filtered seawater] optimized for the sperm in dwarf surf clam Mulinia lateralis, the model bivalve species used in many different types of studies. Results showed that the addition of 20 μM coenzyme Q10 (Q10) to 8% DMSO achieved a D-stage larval rate similar to that of the fresh control at a sperm-to-egg ratio at least 50% less than the 8% DMSO treatment alone. The addition of other antioxidants (glycine, melatonin and polyvinylpyrrolidone) did not have any positive effects. The improvement in post-thaw sperm quality by Q10 could be due to its ability to significantly decrease ROS production and lipid peroxidation and significantly increase the motility, plasma membrane integrity, mitochondrial membrane potential, acrosome integrity, DNA integrity and activities of catalase and glutatione. In this study, 37 fatty acids (FAs) were quantified in dwarf surf clam sperm, with 21 FAs being significantly impacted by the cryopreservation with 8% DMSO. Thirteen of these 21 FAs were changed due to the addition of 20 μM Q10 to 8% DMSO, with approximately half of them being improved significantly toward the levels of fresh control, while the remaining half extended further from the trends shown with 8% DMSO treatment. However, no significant difference was found in the percentage of each FA category sum and the ratio of unsaturated/saturated FAs between the two treated groups. In conclusion, the antioxidant Q10 has shown the potential to further improve the sperm cryopreservation technique in bivalves.

Protective Effect of Bio-Scaffold Against Vitrification Damage in Mouse Ovarian Tissue.

Ovarian tissue cryopreservation is regarded as useful method for fertility preservation. This study aimed to preserve most of the follicular reserve from the destructive effects of cryoprotectant solutions and liquid nitrogen. For this purpose, 48 female NMRI mice (8 weeks old) were randomly divided into six groups: Fresh (not vitrified), Vitrification (not encapsulated), Alginate 1 (encapsulated in 1% alginate hydrogel before placing in vitrification solutions), Alginate 2 (encapsulated in 1% alginate hydrogel before placing in liquid nitrogen), Aloe vera 1 (encapsulated in Aloe vera pieces before placing in vitrification solutions), Aloe vera 2 (encapsulated in Aloe vera pieces before placing in liquid nitrogen). After vitrification and warming, the histological evaluation showed that the average number of intact primordial follicles decreased significantly in all groups compared to the Fresh group. (P < 0.05). Results of evaluating the expression of apoptosis-related genes showed that the ratio of Bax/Bcl2 and P53 significantly decreased in the Alginate 2 group compared with the vitrification group. The level of Kit gene (KIT proto-oncogeni receptor tyrosine kinase gene) expression was either the same or lower in the experimental groups than in the vitrification group, but there was no statistically significant difference. Levels of tissue nitric oxide (NO) and malondialdehyde (MDA) in Alginate groups 1 and 2 showed a significant decrease compared with the vitrification group (P < 0.05). To conclude, Encapsulation of ovaries in 1% alginate hydrogel before immersion in liquid nitrogen may reduce the damage caused by cryopreservation.

Quantitative determination of liposomal irinotecan and SN-38 concentrations in plasma samples from children with solid tumors: Use of a cryoprotectant solution to enhance liposome stability

Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT-11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11-d10 and SN-38-d3, were chromatographically separated on a reversed-phase C18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10–5000 ng/mL for T-CPT-11, 2.5–250 ng/mL for NE-CPT-11, and 1–500 ng/mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde®) in pediatric patients with recurrent solid malignancies or Ewing sarcoma.

The characteristics of frozen-thawed rooster sperm using various intracellular cryoprotectants

Cryopreservation of rooster semen is essential for conserving genetic resources, genetic improvement, and increasing productivity. However, the nature of avian sperm presents a global issue in ensuring superior frozen semen for artificial insemination. Thus, the present study aimed to evaluate the impact of using dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and ethylene glycol (EG) as cryoprotectants on post-thawed sperm motility, quality, antioxidant indicators, and fertilizing capacity. Twice a week, fresh semen ejaculates were collected from 15 adult roosters and immediately evaluated to constitute a pool from clean and qualified samples. The pooled semen was further diluted at a ratio of 1:2 (v/v) with an extender and then subjected to a freezing protocol in a liquid nitrogen vapor after adding a cryoprotectant solution containing 6% of either DMA, DMSO, or EG, respectively. After thawing, characteristics of sperm motion, quality, antioxidants, and fertilizing ability were evaluated and compared to fresh and cooled semen as controls. The results demonstrated that semen cooling negatively affected some parameters of sperm motility, quality, antioxidant biomarkers, and fertility. In comparison to the DMSO and EG groups, employing DMA considerably (P < 0.05) raised the percentages of sperm progressive motility, viability, plasma membrane intactness, and DNA integrity. The DMA group showed a significant increase in the catalase and glutathione reduced antioxidant enzyme activity and a reduction in nitric oxide and lipid peroxidation. After artificial insemination, the DMA and DMSO groups exhibited considerably (P < 0.05) better rates of hatchability and fertility than the EG group. It is concluded that freezing extenders containing 6% DMA is better than DMSO or EG to improve the post thaw semen quality and fertility in chickens.

L-Proline as a Cryoprotective Agent for the Preservation of Galea Spixii Skin Fibroblasts.

Somatic cell biobanking is a promising strategy for developing reproductive techniques. Although cryopreservation, a technique used for creating biobanks, has been performed on Galea spixii, structural and physiological damage to its cells highlight the need to optimize the cryoprotective solution being used. Therefore, the osmoprotective activity of 5 mM L-proline was evaluated as an alternative cryoprotectant for G. spixii fibroblast conservation. The concentration was defined based on previous studies conducted on mammalian cells. Cells derived from the skin of six individuals were cultured until the fifth passage were cryopreserved under the following treatments: (i) control (non-cryopreserved); (ii) a solution with 10% dimethyl sulfoxide (Me2SO), 10% fetal bovine serum (FBS), and 0.2 M sucrose; (iii) a solution with 10% Me2SO, 10% FBS, and 5 mM L-proline; and (iv) a solution with 10% Me2SO, 10% FBS, 0.2 M sucrose, and 5 mM L-proline. Tests were conducted to analyze cell morphology, viability, metabolism, proliferation, and apoptosis; reactive oxygen species (ROS) levels; and mitochondrial membrane activity (ΔΨm). A reduction in the number of viable cells (72.3% ± 1.2%) was observed in the sucrose-containing group compared to the control (86.7% ± 2.0%) and L-proline (88.4% ± 1.8% and 87.8% ± 2.1%) groups. After apoptotic analysis, a reduction in the number of viable cells was observed in the group with sucrose alone (74.6% ± 4.1%) compared to the control group (88.2% ± 1.1%). The ROS levels (1.03 ± 0.5 and 1.07 ± 0.5, respectively) and ΔΨm values (0.99 ± 0.42 and 1.22 ± 0.73, respectively) observed in the groups with L-proline were similar to that observed in the control group (1.00 ± 0.5 and 1.00 ± 0.4, respectively). Moreover, no difference was observed between groups for cell morphology, metabolism, or proliferation. Thus, L-proline is a cryoprotectant agent that can be used during G. spixii fibroblast cryopreservation, alone or with sucrose. In addition, we developed an adequate biobank for G. spixii, whereby stored cells could be used for reproductive techniques.

Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study

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

Influence of hydrogen bonds on state diagrams of cryoprotectant solutions

BACKGROUND: Transformation of state diagrams of cryoprotectant solutions under the influence of weak intramolecular interactions was considered. MATERIALS AND METHODS: Phase states of aqueous glycerol and DMSO solutions within temperature range +25???−150 °?? were studied using method of volumetric scanning tensodilatometry. Temperatures below which hydrogen bonds significantly affect crystallization-melting kinetics of such solutions were determined. RESULTS: Principles for plotting of state diagram for binary solutions with weak intermolecular interaction of the components were set up. The study demonstrates that in such solutions formation of clusters based on ice microcrystals and cryoprotectant occurs. Based on the obtained results, state diagrams for glycerol and DMSO aqueous solutions were plotted. These diagrams include area of cluster phase existence and differ fundamentally from those describing eutectic crystallization. CONCLUSION: Nanostructures occurring in cryoprotectant solutions during their cooling were analyzed. Difference between these structures and classical solid phase eutectics were demonstrated.

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure

BackgroundThe Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process.ResultsExpression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were “carbohydrate metabolic process”, “integral component of membrane” and “chitin binding” for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the “neuroactive ligand receptor interaction”, “endocytosis” and “spliceosome” as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage.ConclusionsThe current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

Comprehensive characterisation and cryopreservation optimisation of buffalo (Bubalus bubalis) adipose tissue-derived mesenchymal stem cells

Over half of the world's buffalo (Bubalus bubalis) inhabit India, and buffaloes frequently encounter health challenges that resist conventional treatments, prompting the exploration of alternative therapeutic strategies. One promising approach is stem cell therapy, particularly multipotent mesenchymal/stromal stem cells (MSCs). These cells have shown significant efficacy in addressing various diseases in livestock that exhibit resistance to conventional therapies. Adipose tissue-derived MSCs (ADSCs) have garnered attention due to their accessibility and robust expansion potential. The current study comprehensively characterises buffalo ADSCs (bADSCs), confirming their identity as MSCs capable of differentiating into diverse cell lineages—the identified characteristics position bADSCs as promising candidates for applications in regenerative medicine, applicable in veterinary contexts. Notably, the study established that a cryoprotective solution comprising 10 % dimethyl sulfoxide and 90 % fetal bovine serum is optimal for preserving bADSCs. This cryoprotective solution maintains vital parameters, including viability, apoptosis, senescence, cell adherence, adherent cell viability, metabolic and clonogenic efficiency, and the activity of reactive oxygen species and trilineage differentiation potential following thawing. These findings lay the foundation for developing a cryo-banking system for bADSCs. Subsequent research efforts are focused on exploring the therapeutic potential of bADSCs in specific disease models and clinical settings. The outcomes of such investigations may pave the way for innovative and effective treatments, further enhancing our understanding of the regenerative capabilities of bADSCs.

Effect of Elamipretide on the Vitrification of Mouse Ovarian Tissue by Freezing.

The importance of ovarian cortical cryopreservation in fertility preservation is receiving increasing attention from reproductive specialists, and mitochondrial dysfunction is an important cause of reduced ovarian tissue cryopreservation. Elamipretide (SS-31) is a novel mitochondria-targeted antioxidant. However, whether it has a protective effect on mouse ovarian tissue cryopreservation remains to be studied. In this study, we examined follicular morphology and viability, mitochondrial function and oxidative stress levels, apoptosis, and culture in vitro after vitrification cryoresuscitation operation by treating ovarian tissues with SS-31 in cryoprotectant resuscitation solution. At the end of the experiment, the addition of 100 μmol/L SS-31 significantly improved follicle quality and oocyte maturation rate in vitro (p < 0.05) and significantly reduced apoptosis (p < 0.05) and oxidative stress levels (superoxide dismutase, catalase, malondialdehyde, p < 0.05). Meanwhile, mitochondrial respiratory chain complex enzyme activity, mtDNA copy number (p < 0.05), and adenosine triphosphate (p < 0.05) content were significantly increased in the 100 μmol/L SS-31-treated group. In addition, the mRNA expression levels of mitochondrial energy metabolism- and biosynthesis-related genes (STRT1, PGC-1a, PPAR-a, TFAM, p < 0.05) were markedly upregulated (p < 0.05) in the 100 μmol/L SS-31 group. In conclusion, SS-31 improved the cryopreservation of ovarian tissues, and 100 μmol/L SS-31 was found to be the most effective.

In situ monitoring of loading and elution processes of cryoprotectants at the single-cell level with Raman spectroscopy

BackgroundThe analysis of cell membrane permeability plays a crucial role in improving the procedures of cell cryopreservation, which will affect the specific parameter settings in loading, removal and cooling processes. However, existing studies have mostly focused on deriving permeability parameters through osmotic theoretical models and cell volume response analysis, and there is still a lack of the direct experimental evidence and analysis at the single-cell level regarding the migration of cryoprotectants. ResultsIn this work, a side perfusion microfluidics chips combined with Raman spectroscopy system was built to monitor in situ the Raman spectroscopy of extracellular and intracellular solution during loading and elution process with different cryoprotectant solution systems (single and dual component). And it was found that loading a high concentration cryoprotectant solution system through a single elution cycle may result in significant residual protective agent, which can be mitigated by employing a multi-component formula but multiple elution operations are still necessary. Furthermore, the collected spectral signals were marked and analyzed to was perform preliminary relative quantitative analysis. The results showed that the intracellular concentration changes can be accurately quantified by the Raman spectrum and are closely related to the extracellular solution concentration changes. Significance and noveltyBy using the method of small flow perfusion (≤20 μL/min) in the side microfluidic chip after the gravity sedimentation of cells, the continuous loading and elution process of different cryoprotectants on chip and the spectral acquisition can be realized. The intracellular and extracellular concentrations can be quantified in situ based on the ratio of spectral peak intensities. These results indicate that spectroscopic analysis can be used to effectively monitor intracellular cryoprotectant residues.

Cryoprotectant-Mediated Cold Stress Mitigation in Litchi Flower Development: Transcriptomic and Metabolomic Perspectives.

Temperature is vital in plant growth and agricultural fruit production. Litchi chinensis Sonn, commonly known as litchi, is appreciated for its delicious fruit and fragrant blossoms and is susceptible to stress when exposed to low temperatures. This study investigates the effect of two cryoprotectants that counteract cold stress during litchi flowering, identifies the genes that generate the cold resistance induced by the treatments, and hypothesizes the roles of these genes in cold resistance. Whole plants were treated with Bihu and Liangli cryoprotectant solutions to protect inflorescences below 10 °C. The soluble protein, sugar, fructose, sucrose, glucose, and proline contents were measured during inflorescence. Sucrose synthetase, sucrose phosphate synthetase, antioxidant enzymes (SOD, POD, CAT), and MDA were also monitored throughout the flowering stage. Differentially expressed genes (DEGs), gene ontology, and associated KEGG pathways in the transcriptomics study were investigated. There were 1243 DEGs expressed after Bihu treatment and 1340 in the control samples. Signal transduction pathways were associated with 39 genes in the control group and 43 genes in the Bihu treatment group. The discovery of these genes may contribute to further research on cold resistance mechanisms in litchi. The Bihu treatment was related to 422 low-temperature-sensitive differentially accumulated metabolites (DAMs), as opposed to 408 DAMs in the control, mostly associated with lipid metabolism, organic oxidants, and alcohols. Among them, the most significant differentially accumulated metabolites were involved in pathways such as β-alanine metabolism, polycyclic aromatic hydrocarbon biosynthesis, linoleic acid metabolism, and histidine metabolism. These results showed that Bihu treatment could potentially promote these favorable traits and increase fruit productivity compared to the Liangli and control treatments. More genomic research into cold stress is needed to support the findings of this study.

Cryopreservation of sessile oak (Quercus petraea (Matt.) Liebl.) plumules using aluminium cryo-plates: influence of cryoprotection and drying

Background Quercusseeds 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.ResultsThe 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%.ConclusionsThis 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.

Engineering Magnetic Nanoclusters for Highly Efficient Heating in Radio-Frequency Nanowarming.

Effective thawing of cryopreserved samples requires rapid and uniform heating. This is achievable through nanowarming, an approach that heats magnetic nanoparticles by using alternating magnetic fields. Here we demonstrate the synthesis and surface modification of magnetic nanoclusters for efficient nanowarming. Magnetite (Fe3O4) nanoclusters with an optimal diameter of 58 nm exhibit a high specific absorption rate of 1499 W/g Fe under an alternating magnetic field at 43 kA/m and 413 kHz, more than twice that of commercial iron oxide cores used in prior nanowarming studies. Surface modification with a permeable resorcinol-formaldehyde resin (RFR) polymer layer significantly enhances their colloidal stability in complex cryoprotective solutions, while maintaining their excellent heating capacity. The Fe3O4@RFR nanoparticles achieved a high average heating rate of 175 °C/min in cryopreserved samples at a concentration of 10 mg Fe/mL and were successfully applied in nanowarming porcine iliac arteries, highlighting their potential for enhancing the efficacy of cryopreservation.

Investigation on recrystallization of cryoprotectant solutions during warming under alternating electric fields

In the field of cryopreservation, recrystallization during rewarming is a significant factor leading to cryo-injury, causing ice crystal-induced mechanical injury to biological samples. Currently, methods, such as adding ice recrystallization inhibiting agents and increasing warming rates, have been adopted to reduce recrystallization injury. These methods also greatly increase the potential toxicity risk and engineering difficulty of cryopreservation. Accumulating research has progressively demonstrated that application of an electromagnetic field during cryopreservation can influence the preservation efficacy. However, the mechanism of action of the electric field has remained a subject of ongoing debate. We observed the recrystallization phenomenon of cryoprotectant solutions under a polarized light microscope and discovered promising experimental results. The electric field strength did not exhibit a discernible impact on the ice crystal size, while electric field frequency exerted a more significant impact. We found that at specific frequencies, the ice crystal size and growing rate were significantly affected during annealing. Compared to no electric field, an alternating electric field with a voltage of 10 V and a frequency of 5 × 104 Hz increased the recrystallization areas of a 15% (w/v) propylene glycol solution by 57%. And an alternating electric field with a voltage of 3 V and a frequency of 5 × 108 Hz decreased the recrystallization area of a 0.9% (w/v) NaCl aqueous solution by 9.9%. Different types of cryoprotectants exhibit varying responses to alternating electric fields frequency. These findings prompt us to reevaluate the role and mechanism of alternating electric fields in the cryopreservation of biological materials.

Analysis of the Nonequilibrium Phase Change Behaviors of the Cryoprotectant Solutions for Cryopreservation of Human Red Blood Cells with Low-Concentration Glycerol.

Recently, we proposed a low-glycerol cryoprotectant formulation (consisting of 0.4 M trehalose and 5% glycerol) for cryopreservation of human red blood cells (RBCs), which greatly reduced the concentration of glycerol, minimized intracellular ice damage, and achieved high recovery. Although this study was successful in cellular experiments, the nonequilibrium phase transition behaviors of the cryoprotective agent solution have not been systematically analyzed. Therefore, it is essential to provide reliable thermodynamic data to substantiate the viability of this cryopreservation technique. In this study, the phase change behaviors and thermal properties of typical trehalose and/or glycerol solutions quenched in liquid nitrogen were investigated using differential scanning calorimetry and cryomicroscopy. It was found that the glass transition temperatures of both the trehalose aqueous solution (<1.0 M) and glycerol aqueous solution (<40% w/v) did not vary apparently with the concentration at low concentrations, while they increased significantly with increasing concentration at high concentrations. Moreover, it was revealed that the inhibitory effect of trehalose on ice growth was affected by glycerol. We further found that the addition of low concentrations of glycerol facilitates the partial glass transition of trehalose solutions at low concentrations. The results of this work provide reliable thermodynamic data to support the cryopreservation of human RBCs with unusually low concentrations of glycerol.

Cryoprotective Effect of Pectin Tanacetan from Tanacetum vulgare L.

We researched the ability of tanacetan pectin from inflorescences of common tansy Tanacetum vulgare L. to change the osmolarity and freezing point of water in solutions of cryoprotectants: glycerol-3.5%, dimethyl sulfoxide (DMSO)-10%, dimethylacetamide-10% (DMAC), and 1.2-propanediol (1.2-PD)-10%, as well as the effect of solutions of tanacetan (0.2%, 0.4%) on the kinetics of crystallization processes and the nature of crystal formation. We used a combination of protector and pectin that we tested earlier, which provided effective protection for human leukocytes and platelets, as well as bovine spermatozoa, at temperatures below freezing (-20°C and -80°C). It has been established that tanacetan slows down the process of water freezing in glycerol, but not in DMSO, DMAC, and 1.2-PD, promotes deeper supercooling of the medium, and affects the morphological structure of ice. The addition of pectin to the cryosolution increases the activity of the main cryoprotectant glycerol even at its low concentrations. The combination of glycerol and tanacetan can be effective in freezing biological materials, which is confirmed by the preservation of leukocytes at -20°C and -80°C for 7 days, platelets at -80°C for 30 days, and spermatozoa at -80°C within 1 day. A comprehensive analysis of the chemical, physicochemical, and cryoprotective properties of tanacetan indicates the prospect of using pectin in the cryopreservation of biological objects at temperatures of electric freezers.