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.

Antifreeze protein type I in the vitrification solution improves the cryopreservation of immature cat oocytes.

Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 µg/mL (G0, 0 µM); 0.5 µg/mL (G0.5, 0.15 µM), or 1 µg/mL (G1, 0.3 µM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 µg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes.

Cryopreservation of Immune Cells: Recent Progress and Challenges Ahead.

Cryopreservation of immune cells is considered as a key enabling technology for adoptive cellular immunotherapy. However, current immune cell cryopreservation technologies face the challenges with poor biocompatibility of cryoprotection materials, low efficiency, and impaired post-thaw function, limiting their clinical translation. This review briefly introduces the adoptive cellular immunotherapy and the approved immune cell-based products, which involve T cells, natural killer cells and etc. The cryodamage mechanisms to these immune cells during cryopreservation process are described, including ice formation related mechanical and osmotic injuries, cryoprotectant induced toxic injuries, and other biochemical injuries. Meanwhile, the recent advances in the cryopreservation medium and freeze-thaw protocol for several representative immune cell type are summarized. Furthermore, the remaining challenges regarding on the cryoprotection materials, freeze-thaw protocol, and post-thaw functionality evaluation of current cryopreservation technologies are discussed. Finally, the future perspectives are proposed toward advancing highly efficient cryopreservation of immune cells.

Effect of different freezing conditions on ice crystal formation behavior and ice-growth inhibition by cryoprotectants.

BACKGROUND: The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long-term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. RESULTS: The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low-temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze-thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice-growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice-growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. CONCLUSION: The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry.

Development of Macromolecular Cryoprotectants for Cryopreservation of Cells.

Cryopreservation is a common way for long-term storage of therapeutical proteins, erythrocytes, and mammalian cells. For cryoprotection of these biosamples to keep their structural integrity and biological activities, it is essential to incorporate highly efficient cryoprotectants. Currently, permeable small molecular cryoprotectants such as glycerol and dimethyl sulfoxide dominate in cryostorage applications, but they are harmful to cells and human health. As acting in the extracellular space, membrane-impermeable macromolecular cryoprotectants, which exert remarkable membrane stabilization against cryo-injury and are easily removed post-thaw, are promising candidates with biocompatibility and feasibility. Water-soluble hydroxyl-containing polymers such as poly(vinyl alcohol) and polyol-based polymers are potent ice recrystallization inhibitors, while polyampholytes, polyzwitterions, and bio-inspired (glyco)polypeptides can significantly increase post-thaw recovery with reduced membrane damages. In this review, the synthetic macromolecular cryoprotectants are systematically summarized based on their synthesis routes, practical utilities, and cryoprotective mechanisms. It provides a valuable insight in development of highly efficient macromolecular cryoprotectants with valid ice recrystallization inhibition activity for highly efficient and safe cryopreservation of cells.

Design and lyophilization of mRNA-encapsulating lipid nanoparticles.

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.

Exploiting tropical fruit processing coproducts as circular resources to promote the growth and maintain the culturability and functionality of probiotic lactobacilli.

This study evaluated the use of acerola (Malpighia glabra L., CACE), cashew (Anacardium occidentale L., CCAS), and guava (Psidium guayaba L., CGUA) fruit processing coproducts as substrates to promote the growth, metabolite production, and maintenance of the viability/metabolic activity of the probiotics Lactobacillus acidophilus LA-05 and Lacticaseibacillus paracasei L-10 during cultivation, freeze-drying, storage, and exposure to simulated gastrointestinal digestion. Probiotic lactobacilli presented high viable counts (≥8.8 log colony-forming units (CFU)/mL) and a short lag phase during 24 h of cultivation in CACE, CCAS, and CGUA. Cultivation of probiotic lactobacilli in fruit coproducts promoted sugar consumption, medium acidification, and production of organic acids over time, besides increasing the of several phenolic compounds and antioxidant activity. Probiotic lactobacilli cultivated in fruit coproducts had increased survival percentages after freeze-drying and during 120 days of refrigerated storage. Moreover, probiotic lactobacilli cultivated and freeze-dried in fruit coproducts had larger subpopulations of live and metabolically active cells when exposed to simulated gastrointestinal digestion. The results showed that fruit coproducts not only improved the growth and helped to maintain the viability and metabolic activity of probiotic strains but also enriched the final fermented products with bioactive compounds, being an innovative circular strategy for producing high-quality probiotic cultures.

Enhancing cell cryopreservation with acidic polyamino acids integrated liquid marbles.

Cryopreservation is highly desired for long-term maintenance of the viability of living biosamples, while effective cell cryopreservation still relies heavily on the addition of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). However, the intrinsic toxicity of DMSO is still a bottleneck, which could not only cause the clinical side effect but also induce cell genetic variants. In the meantime, the addition of FBS may bring potentially the risk of pathogenic microorganism contamination. The liquid marbles (LMs), a novel biotechnology tool for cell cryopreservation, which not only have a small volume system that facilitated recovery, but the hydrophobic shell also resisted the harm to cells caused by adverse environments. Previous LM-based cell cryopreservation relied heavily on the addition of FBS. In this work, we introduced acidic polyaspartic acid and polyglutamic acid as cryoprotectants to construct LM systems. LMs could burst in an instant to facilitate and achieve ultrarapid recovery process, and the hydrophilic carboxyl groups of the cryoprotectants could form hydrogen bonds with water molecules and further inhibit ice growth/formation to protect cells from cryoinjuries. The L929 cells could be well cryopreserved by acidic polyamino acid-based LMs. This new biotechnology platform is expected to be widely used for cell cryopreservation, which has the potential to propel LMs for the preservation of various functional cells in the future.

Morphological evaluation of adult domestic cat testicular biopsy after vitrification.

Testicular biopsies (9 mm3) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher's post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO's negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.

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

BACKGROUND: The 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. RESULTS: In 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 NOVELTY: By 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.

Formulation optimization of lyophilized aptamer-gold nanoparticles: Maintained colloidal stability and cellular uptake.

Anti-nucleolin (NCL) aptamer AS1411 is the first anticancer aptamer tested in clinical trials. Gold nanoparticles (AuNP) have been widely exploited for various biomedical applications due to their unique functional properties. In this study, we evaluated the colloidal stability and targeting capacity of AS1411-funtionalized AuNP (AuNP/NCL-Apt) against MCF-7 breast cancer cell line before and after lyophilization. Trehalose, mannitol, and sucrose at various concentrations were evaluated to determine their cryoprotection effects. Our results indicate that sucrose at 10 % (w/v) exhibits the best cryoprotection effect and minimal AuNP/NCL-Apt aggregation as confirmed by UV-Vis spectroscopy and dynamic light scattering (DLS) measurements. Moreover, the lyophilized AuNP/NCL-Apt at optimized formulation maintained its targeting and cytotoxic functionality against MCF-7 cells as proven by the cellular uptake assays utilizing flow cytometry and confocal laser scanning microscopy (CLSM). Quantitative PCR (qPCR) analysis of nucleolin-target gene expression also confirmed the effectiveness of AuNP/NCL-Apt. This study highlights the importance of selecting the proper type and concentration of cryoprotectant in the typical nanoparticle lyophilization process and contributes to our understanding of the physical and biological properties of functionalized nanoparticles upon lyophilization.

A brief review of the methodology and cryoprotectants in selected fish and mammalian species.

Cryopreservation is a valuable technique used to assist in the genetic improvement of cultured stocks and provide a continuous supply of good-quality semen for artificial insemination. Conserving semen by cryopreservation serves several purposes (e.g. artificial reproductive technologies and species conservation) and is also used in the clinical treatment of human infertility. However, the lifespan of cryopreserved semen is influenced by a range of factors, including storage temperature, cooling rate, chemical composition of the extender, the concentration of cryoprotectant, reactive oxygen species, seminal plasma composition and hygienic control. The choice of cryoprotectant is a vital factor underlying the success of animal semen cryopreservation. In this regard, extensive research has been carried out on various cryoprotectants, such as egg yolk, dimethyl sulfoxide, methanol, ethylene glycol and dimethylacetamide. Recent studies have also described the use of a range of new cryoprotectants for cryopreservation, including compounds of plant origin (soy), amino acids, antifreeze proteins, carbohydrates and cyclodextrins. Moreover, semen cryopreservation and storage require the use of liquid nitrogen or ultralow refrigeration methods for both long- and short-term storage. This review summarizes the general methods used for freezing semen and discusses the use of traditional and newly emerging cryoprotectants (permeable and non-permeable) for the cryopreservation of semen in selected fish and mammalian species.

Evaluation of Kappa-carrageenan supplementation in extender for post-thaw Kajli ram sperm quality.

Cryopreservation is one of the reliable techniques for long-term storage of sperm. The success of this technique depends on the choice of cryoprotectant; therefore, a plethora of literature has reported the effects of different cryoprotective agents so far. Kappa-carrageenan (κ-carrageenan) is a hydrocolloid polysaccharide extracted from red marine seaweed. Its unique property makes it a promising option as a non-colligative cryoprotectant. The current study aims to evaluate the cryoprotective effect of k-carrageenan along with glycerol on ram sperm quality both after equilibration and freezing. Nine Kajli rams were utilized in this experiment for semen collection through an artificial vagina maintained at 42°C. Qualified samples were diluted in tris egg yolk glycerol (TEYG) extender containing different concentrations of k-carrageenan as 0 mg/mL (control), 0.2, 0.5, 0.8 and 1 mg/mL. Post-thaw assessment was done at 37°C after 24 h of storage, which showed a significant improvement (p < .05) in sperm viability, motility, membrane and acrosome integrity in an extender containing k-carrageenan at a concentration of 0.5 mg/mL compared to control. It is concluded from the current study that the combination of glycerol and 0.5 mg/mL concentration of k-carrageenan improved the sperm post-thaw quality.

Technology for Biobanking of Epididymal Spermatozoa from Patient with Obstructive Azoospermia: Case Report about Baby Born after Conventional Freezing Only with a Nonpermeable Cryoprotectant 360 kDa Polyvinylpyrrolidone.

This publication reports, for the first time, the birth of a healthy child after intracytoplasmic sperm injection (ICSI) of motile spermatozoa after conventional ("slow") freezing of epididymal spermatozoa using 5% polyvinylpyrrolidone (PVP) of high molecular weight (360 kDa). Cryopreservation solution with 10% PVP was added to 30 µL of spermatozoa suspension in a 1:1 ratio, with a final PVP concentration of 5%. Then, polycarbonate capillaries for oocyte denudation with a diameter of 170 µm were filled with 60 µL of the resulting sperm suspension. After that, the capillaries were placed for 10 minutes at a height of 15 cm above liquid nitrogen and immersed into liquid nitrogen. To warm the spermatozoa, the capillaries were immersed in a water bath at a temperature of 40°C for 30 seconds. Oocyte fertilization was performed by ICSI. Zygotes were cultured in vitro for 5 days to the blastocyst stage. More than 100 spermatozoa were obtained after percutaneous epidydimal sperm aspiration, of which 80% were motile. After cryopreservation, storage for 3 months in liquid nitrogen, and thawing, 72% of the total sperm cells remained motile. Ten oocyte-cumulus complexes were found after follicle puncture, and eight metaphase II stage oocytes were fertilized using ICSI. After 18 hours, two pronuclei were found in seven (88%) of the oocytes. An analysis of the morphological characteristics of 5-day-old embryos showed that four (57%) of them reached the blastocyst stage. One embryo was transferred, and the remaining embryos were cryopreserved (vitrified). The onset of pregnancy was detected on the 14th day after embryo transfer, and one healthy girl (3300 g) was born at term.

Reexamining the diverse functions of arginine in biochemistry.

Arginine in a free-state and as part of peptides and proteins shows distinct tendency to form clusters. In free-form, it has been found useful in cryoprotection, as a drug excipient for both solid and liquid formulations, as an aggregation suppressor, and an eluent in protein chromatography. In many cases, the mechanisms by which arginine acts in all these applications is either debatable or at least continues to attract interest. It is quite possible that arginine clusters may be involved in many such applications. Furthermore, it is possible that such clusters are likely to behave as intrinsically disordered polypeptides. These considerations may help in understanding the roles of arginine in diverse applications and may even lead to better strategies for using arginine in different situations.

The association of resveratrol and AFPI did not enhance the cryoresistance of ram sperm.

Cryoprotectants are required to reduce damage caused to the cells due to low temperatures during the cryopreservation. Antifreeze proteins (AFP) have a well-known role in cell membrane protection, while resveratrol is a potent antioxidant. This study assessed the effect of the association of resveratrol concentrations and AFP I in a ram semen extender. Pooled semen of four rams was allocated into six treatments in a factorial arrangement: (CONT, only the semen extender); only AFP I (ANT: 0.1 µg/mL of AFP I), only resveratrol, one treatment with two levels (10 µM/mL or 50 µM/mL of resveratrol); and two treatments with the interactions, with one AFP I and one of the two levels of resveratrol (0.1 µg/mL of AFP I with 10 µM/mL resveratrol; 0.1 µg/mL of AFP I with 50 µM/mL resveratrol). No interaction between factors was observed on sperm kinetics, plasma membrane integrity, hypo-osmotic test, and mitochondrial activity parameters. There was a high probability (P = 0.06) of reducing sperm cells with functional membrane percentage in the hypo-osmotic test and increasing the percentage of sperm with high mitochondrial activity (P = 0.07) was observed in AFP presence. An interaction of AFP and resveratrol was observed in non-capacitated sperm (P = 0.009), acrosomal reaction (P = 0.034), and sperm binding (P = 0.04). In conclusion, the association of resveratrol and AFP did not improve the quality of frozen-thawed semen and even promoted deleterious effects compared to their single addition in the semen extender. The supplementation of 50 µM/mL of resveratrol improved the outcomes of frozen-thawed ram sperm, being a potential cryoprotectant.

Morphological and biochemical responses of a neotropical pest insect to low temperatures.

As traditionally cold areas become warmer due to climate change, temperature could no longer be a barrier to the establishment of non-native insects. This is particularly relevant for pest insects from warm and tropical areas, mainly those with some tolerance to moderately low temperatures, which could expand their range into these new locations. From this perspective, in this work we studied the morphological and biochemical responses of the Neotropical pest Paysandisia archon to low temperatures, as part of a possible strategy to colonize new areas. To that end, wild larvae were exposed for 7 days to either low (1 and 5 °C) or ambient (23 °C) temperatures. We then quantified the inner and outer morphological changes, by X-Ray Computer Tomography and Digital Holographic Microscopy, as well as the accumulation of metabolites acting as potential endogenous cryoprotectants, by Spectrophotometry. We found that Paysandisia archon developed a cold-induced response based on different aspects. On the one hand, morphological changes occurred with a significant reduction both in fluids susceptible to freezing and fat body, together with the thickening, hardening and increased roughness of the integument. On the other hand, we found an increase in the hemolymph concentration of cryoprotective substances such as glucose (6-fold) and glycerol (2-fold), while trehalose remained unchanged. Surprisingly, this species did not show any evidence of cold-induced response unless the environmental temperature was remarkably low (1 °C). These results could be useful to improve models predicting the possible spread of such a pest, which should incorporate parameters related to its resistance to low temperatures.

Clinical and Preclinical Methods of Heat-Stabilization of Human Vaccines.

Vaccines have historically faced challenges regarding stability, especially in regions lacking a robust cold chain infrastructure. This review delves into established and emergent techniques to improve the thermostability of vaccines. We discuss the widely practiced lyophilization method, effectively transforming liquid vaccine formulations into a solid powdered state, enhancing storage and transportation ability. However, potential protein denaturation during lyophilization necessitates alternative stabilization methods. Cryoprotectants, namely, starch and sugar molecules, have shown promise in protecting vaccine antigens and adjuvants from denaturation and augmenting the stability of biologics during freeze-drying. Biomineralization, a less studied yet innovative approach, utilizes inorganic or organic-inorganic hybrids to encapsulate biological components of vaccines with a particular emphasis on metal-organic coordination polymers. Encapsulation in organic matrices to form particles or microneedles have also been studied in the context of vaccine thermostability, showing some ability to store outside the cold-chain. Unfortunately, few of these techniques have advanced to clinical trials that evaluate differences in storage conditions. Nonetheless, early trials suggest that alternative storage techniques are viable and emphasize the need for more comprehensive studies. This review underscores the pressing need for heat-stable vaccines, especially in light of the increasing global distribution challenges. Combining traditional methods with novel approaches holds promise for the future adaptability of vaccine distribution and use.

Is post-hypertonic lysis of human red blood cells caused by excessive cell volume regulation?

Human red blood cells (RBC) exposed to hypertonic media are subject to post-hypertonic lysis - an injury that only develops during resuspension to an isotonic medium. The nature of post-hypertonic lysis was previously hypothesized to be osmotic when cation leaks were observed, and salt loading was suggested as a cause of the cell swelling upon resuspension in an isotonic medium. However, it was problematic to account for the salt loading since the plasma membrane of human RBCs was considered impermeable to cations. In this study, the hypertonicity-related behavior of human RBCs is revisited within the framework of modern cell physiology, considering current knowledge on membrane ion transport mechanisms - an account still missing. It is recognized here that the hypertonic behavior of human RBCs is consistent with the acute regulatory volume increase (RVI) response - a healthy physiological reaction initiated by cells to regulate their volume by salt accumulation. It is shown by reviewing the published studies that human RBCs can increase cation conductance considerably by activating cell volume-regulated ion transport pathways inactive under normal isotonic conditions and thus facilitate salt loading. A simplified physiological model accounting for transmembrane ion fluxes and membrane voltage predicts the isotonic cell swelling associated with increased cation conductance, eventually reaching hemolytic volume. The proposed involvement of cell volume regulation mechanisms shows the potential to explain the complex nature of the osmotic response of human RBCs and other cells. Cryobiological implications, including mechanisms of cryoprotection, are discussed.

Vitrification Preservation of Oocyte Hydrogel Microspheres Prepared by Microfluidics / 生物化学与生物物理进展

ObjectiveThis study aimed to develop a novel method for encapsulating oocytes in sodium alginate hydrogel using microfluidics, then to vitrify these encapsulated oocytes in a single-step process with low concentrations of cryoprotectants. MethodsWe utilized a flow-focusing microfluidic chip to generate sodium alginate hydrogel microspheres. The influence of various parameters, including throat structure, cross-linking method, sodium alginate concentrations, and flow rate ratios on the stability diameter, and coefficient of variation of microspheres were examined. To further investigate the cold-resistance of these microspheres, we used cryomicroscopy to observe changes in volume and morphology of microspheres during cooling and warming processes. We used microfluidic chip to encapsulate oocytes in sodium alginate hydrogel microspheres, the empty rate of microspheres and loss rate of oocytes were determined. After releasing from microspheres and parthenogenetic activation with cytochalasin B and strontium chloride, the survival, cleavage and blastocyst rates were evaluated during in vitro maturation. Finally, oocytes encapsulated in sodium alginate microspheres were vitrified with low concentrations of cryoprotectants. We compared the survival and development capability of the oocytes with the Cryotop method. ResultsWhen the throat of the microfluidic chip measures 300 μm in length and 120 μm in width, microspheres can be uniformly formed at the throat of the chip. Sodium alginate generates microspheres with a wide size distribution when cross-linking outside the chip, while internal cross-linking within the chip results in more uniform microspheres. The stability of microsphere formation is significantly improved with the use of a three-channel internal cross-linking chip. At a flow rate of 2 μl/min and with 1% sodium alginate, the microfluidic chip can consistently and uniformly produce microspheres. Under flow rate ratios of 10, 15, and 20, the average microsphere diameters are 262.71 μm, 193.63 μm, and 156.63 μm, respectively. The sodium alginate hydrogel microspheres maintained their volume and structural integrity during the cooling and warming processes. Using a three-channel internal cross-linking microfluidic chip to encapsulate oocytes, at a flow rate ratio of 10, the empty rate is 32.28%, and the cell loss rate is 11.09%. After encapsulation and subsequent release, the oocyte survival rate (96.99%), cleavage rate (88.71%), and blastocyst formation rate (26.29%) showed no significant differences compared to the fresh group. After the microspheres were vitrified using a low concentration of cryoprotectant (10% DMSO+10% ehylene glycol (EG)+0.5 mol/L trehalose), the survival rate, cleavage rate, and blastocyst rate were 92.48%, 70.80%, and 20.42%, respectively. No significant difference was observed when compared to the Cryotop method using a higher concentration of cryoprotectant solution (15% DMSO+15% EG+0.5 mol/L trehalose). ConclusionWe designed and fabricated a microfluidic system with three-channel internal cross-linking chips used for oocyte vitrification preservation. The microfluidic system can generate oocytes-loaded sodium alginate hydrogel microspheres with uniform size, low empty rate, and good cold-resistance. The method successfully reduced the concentration of cryoprotectants in a single-step vitrification process, the developmental capability of oocytes during in vitro maturation were comparable with Cryotop method. Unlike the Cryotop method, the oocytes encapsulated in hydrogel does not come into contact with liquid nitrogen, eliminating the risk of cross-contamination. This study provides a novel approach to oocyte vitrification.