Effects of freezing methods on physicochemical properties, protein/fat oxidation and odor characteristics of surimi gels with different cross-linking degrees.

This work investigated the effects of liquid nitrogen immersion freezing (LNF), -35 °C air freezing (AF-35℃) and -18 °C air freezing (AF-18℃) on the physical and chemical characteristics and flavor quality of surimi gels with different cross-linking degrees. Compared to AF-35 °C and AF-18 °C, LNF was shown to considerably delay the texture deterioration and water migration of frozen gels, as well as the accumulation of thiobarbituric acid reactive substance values and carbonyl contents. Additionally, an appropriate increase of cross-linking degree (45.83 to 62.99%) was found able to improve gel properties and inhibit quality deterioration during freezing. Moreover, LNF-treated gels were closer to fresh gels in the amount of volatile compounds, in contrast to most significant negative aroma changes in AF-18℃-treated gels. Furthermore, 29, 29 and 31 key differential volatile compounds were screened for gels with a cross-linking degree of 29.66, 45.83 and 62.99%, respectively, mainly including aldehydes, alcohols and esters.

Physiological and transcriptomic analyses reveal tea plant (Camellia sinensis L.) adapts to extreme freezing stress during winter by regulating cell wall structure.

Tea plants grown in high-latitude areas are often damaged by extreme freezing temperatures in winter, leading to huge economic losses. Here, the physiological and gene expression characteristics of two tea cultivars (Xinyang No. 10 (XY10), a freezing-tolerant cultivar and Fudingdabaicha (FDDB), a freezing-sensitive cultivar) during overwintering in northern China were studied to better understand the regulation mechanisms of tea plants in response to natural freezing stress. Samples were collected at a chill (D1), freezing (D2) and recovery (D3) temperature in winter. TEM analysis of integrated leaf ultrastructure at D2 revealed lower malondialdehyde and relative electrical conductivity in XY10 than in FDDB, with serious cell structure damage in the latter, indicating XY10 was more resistant to freezing stress. Differential gene expression analysis among the different samples over winter time highlighted the following gene functions in cell wall metabolism (CesAs, COBLs, XTHs, PGs, PMEs), transcription factors (ERF1B and MYC2), and signal transduction (CDPKs and CMLs). The expression pattern of cellulose and pectin-related genes suggested higher accumulation of cellulosic and pectic materials in the cell wall of XY10, agreeing with the results of cell wall and its components. These results indicated that under the regulation of cell wall genes, the freezing-resistant tea cultivar can better maintain a well-knit cell wall structure with sufficient substances to survive natural freezing damage. This study demonstrated the crucial role of cell wall in tea plant resistance to natural freezing stress and provided important candidate genes for breeding of freezing-resistant tea cultivars.

High pressure freezing and cryo-sectioning can be used for protein structure determination by electron diffraction.

Electron diffraction of three-dimensional nanometer sized crystals has emerged since 2013 as an efficient technique to solve the structure of both small organic molecules and model proteins. However, the major bottleneck of the technique when applied to protein samples is to produce nano-crystals that do not exceed 200 to 300 nm in at least one dimension and to deposit them on a grid while keeping the minimum amount of solvent around them. Since the presence of amorphous solvent around the crystal, necessary to preserve its integrity, increases the amount of diffuse scattering, thus degrading the signal-to noise ratio of the diffraction signal, other sample preparation strategies have been developed. One of them is the milling of thin crystal lamella using focused ion beam (FIB), which was successfully applied to several protein crystals. Here, we present a new approach that uses cryo-sectioning after high pressure freezing of dextran embedded protein crystals. 150 to 200 nm thick cryo-sections of hen egg white lysozyme tetragonal crystals where used for electron diffraction experiments. Complete diffraction data up to 2.9 Å resolution have been collected and the lysozyme structure has been solved by molecular replacement and refined against these data. Our data demonstrate that cryo-sectioning can preserve protein structure at high resolution and can be used as a new sample preparation technique for 3D electron diffraction experiments of protein crystals. The different orientations found in the crystal chips and their large number resulting from the cryo-sectioning make the latter an attractive approach as it combines advantages from both blotting approaches (number of crystals) and FIB-milling (controlled thickness and absence of solvent around the crystal).

Identification and functional analysis of differentially expressed proteins in high and low freezing tolerance sheep sperm.

The purpose of this study was to identify proteins associated with differences in the freezing tolerance of sheep sperm and to analyze their functions. Qualified fresh semen from four breeds of rams, the Australian White, white-head Dorper, Black-head Dorper, and Hu sheep breeds, were selected for cryopreservation. The sperm freezing tolerance was investigated by evaluation of the overall vitality, progressive vitality, and rapidly advance vitality of the sperm. A differential model of sperm freezing tolerance was constructed for sheep breeds showing significant differences. Differentially expressed proteins associated with sperm freezing tolerance were identified using iTRAQ and the protein functions were analyzed. It was found that sperm freezing tolerance was best in Hu sheep and worst in white-head Dorper sheep. These two breeds were used for the construction of a model based on differences in freezing tolerance and the identification of sperm proteins expressed differentially before freezing and after thawing. A total of 128 differentially expressed proteins (88 up-regulated and 40 down-regulated) were identified before freezing and after thawing in Hu sheep sperm (fresh/frozen Hu sheep sperm referred to as HL vs. HF), while 219 differentially expressed proteins (106 up-regulated and 113 down-regulated) were identified in white-head Dorper sheep (fresh/frozen white-head Dorper sheep sperm referred to as WL vs. WF). A comparison of these differentially expressed proteins showed that 57 proteins overlapped between the two breeds while 71 were only expressed in Hu sheep and 162 were only expressed in white-head Dorper sheep. Functional annotation and enrichment analyses of differentially expressed proteins down-regulated in Hu sheep involved in phosphorylation of phosphatidylinositol phosphate kinases, regulation of GTPase activity and glycolysis/gluconeogenesis signaling pathway. Up-regulated proteins of Hu sheep participated in oxidoreductase activity and oxidative phosphorylation process of sperm freezing. Furthermore, down-regulated in white-head Dorper sheep involved in the metabolic regulation of carbohydrate and nuclear sugar metabolism. Up-regulated proteins of white-head Dorper sheep involved in the ferroptosis and oxidative phosphorylation pathways. Collectively, These proteins were found to participate mainly in oxidative phosphorylation as well as phosphorylation and metabolic processes in the mitochondria to affect the freezing tolerance of sheep sperm.

Challenges and processing strategies to produce high quality frozen meat.

Freezing is an effective means to extend the shelf-life of meat products. However, freezing and thawing processes lead to physical (e.g., ice crystals formation and freezer burn) and biochemical changes (e.g., protein denaturation and lipid oxidation) in meat resulting in loss of quality. Over the last two decades, several attempts have been made to produce thawed meat with qualities similar to that of fresh meat to no avail. This is due to the fact that no single technique exists to date that can mitigate all the quality challenges caused by freezing and thawing. This is further confounded by the consumer perception of frozen meat as lower quality compared to equivalent fresh-never-frozen meat cuts. Therefore, it remains challenging for the meat industry to produce high quality frozen meat and increase consumer acceptability of frozen products. This review aimed to provide an overview of the applications of novel freezing and thawing technologies that could improve the quality of thawed meat including deep freezing, high pressure, radiofrequency, electro-magnetic resonance, electrostatic field, immersion solution, microwave, ohmic heating, and ultrasound. This review will also discuss the development in processing strategies such as optimising the ageing of meat pre- or post-freezing, and the integration of freezing and thawing in one process/regime to collapse the difference in quality between thawed meat and fresh-never-frozen equivalents.

Comparison of angiogenic potential in vitrified vs. slow frozen human ovarian tissue.

Vitrification of ovarian tissue is a promising alternative approach to the traditional slow freezing method. Few empirical investigations have been conducted to determine the angiogenic profiles of these two freezing methods. In this study we aimed to answer the question whether one of the cryopreservation methods should be preferred based on the secretion of angiogenic factors. Tissue culture with reduced oxygen (5%) was conducted for 48 h with samples of fresh, slow frozen/thawed and vitrified/rapid warmed ovarian cortex tissue from 20 patients. From each patient, tissue was used in all three treatment groups. Tissue culture supernatants were determined regarding cytokine expression profiles of angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin binding epidermal growth factor, hepatocyte growth factor, Leptin, Platelet-derived growth factor B, placental growth factor and vascular endothelial growth factor A via fluoroimmunoassay. Apoptotic changes were assessed by TUNEL staining of cryosections and supplemented by hematoxylin and eosin and proliferating cell nuclear antigen staining. Comparing the angiogenic expression profiles of vitrified/rapid warmed tissue with slow frozen/thawed tissue samples, no significant differences were observed. Detection of apoptotic DNA fragmentation via TUNEL indicated minor apoptotic profiles that were not significantly different comparing both cryopreservation methods. Vitrification of ovarian cortical tissue does not appear to impact negatively on the expression profile of angiogenic factors and may be regarded as an effective alternative approach to the traditional slow freezing method.

[Effect of pasteurization and freezing on the content of IgA1 and IgA2 subtypes in breast milk]. / Reducción del contenido de IgA1 e IgA2 en la leche materna por efecto de la pasteurización y congelación.

OBJECTIVE: To determine the effect of pasteurization and freezing on the content of IgA1 and IgA2 in breast milk. METHODS: Observational, retrospective study, carried out in women who had been breastfeeding their newborn for more than 30 days, and could donate 50 mL of milk. The concentration of IgA1 and IgA2 was determined by turbidimetry, before and after being subjected to pasteurization and freezing, every 15 days for 2 months. Freezing was at -20°C. A total IgA content of 1598.5 mg/dL was found. RESULTS: 10 breast milk donors were selected. The initial concentration of IgA1 and IA2 was 651 and 945.7 mg/dL, respectively; At the end of the freezing times, the content of both immunoglobulins decreased: IgA1 of 74% and IgA2 of 86%. After the treatments, the immunoglobulin content decreased dramatically, with a significant difference of p < 0.05. CONCLUSIONS: Pasteurization and freezing significantly affect the content of IgA1 and IgA in breast milk; therefore, breast-feeding remains the best way to offer full immunological protection to the infant.

OBJECTIVO: Determinar el efecto de la pasteurización y congelación en el contenido de IgA1 e IgA2 en la leche materna. MÉTODOS: Estudio observacional y retrospectivo, llevado a cabo en mujeres que tuvieran más de 30 días de ofrecer lactancia a su neonato, y pudieran donar 50 mL de leche. Se determinó la concentración de IgA1 e IgA2 mediante turbidimetría, antes y después de someterse a pasteurización y congelación, cada 15 días durante 2 meses. La congelación fue a ­20°C. Se encontró un contenido total de IgA de 1598,5 mg/dL. RESULTADOS: Se seleccionaron 10 donantes de leche materna. La concentración inicial de IgA1 e IA2 fue de 651 y 945.7 mg/dL, respectivamente; al finalizar los tiempos de congelación disminuyó el contenido de ambas inmunoglobulinas: IgA1 del 74% e IgA2 del 86%. Después de los tratamientos, el contenido de inmunoglobulinas disminuyó de forma contundente, con diferencia significativa de p < 0.05. CONCLUSIONES: La pasteurización y la congelación afectan de manera importante el contenido de IgA1 e IgA en la leche materna; por tanto, la alimentación al seno materno sigue siendo la mejor forma de ofrecer toda protección inmunológica al lactante.

Effects of frozen storage conditions and freezing rate on the stability of coagulation proteins in human plasma.

OBJECTIVES: Degradation of coagulation proteins in frozen plasma may influence assay results. The aims of this study were to explore the changes in coagulation parameters in patient plasma and internal quality control (IQC) after different freezing and storage conditions during the short-term and long-term periods. METHODS: Platelet poor plasma was prepared from citrated peripheral blood collected from a group of healthy donors. The plasma was pooled, frozen and stored in a variety of freezing and storage conditions. The changes were monitored using routine coagulation assays, as well as factor VIII (FVIII) and protein S (PS) assays. RESULTS: Plasma stored in liquid nitrogen (LN 2 ) or in -80°C showed long-term stable values for routine tests for a period of over 12 months, and 6 months for FVIII. Interestingly, the activated partial thromboplastin time (aPTT) showed a temporary significant prolongation over the first two weeks. Plasma frozen and stored in -40°C is not viable for aPTT and FVIII testing, otherwise it can be used for other parameters for up to 4 months. PS showed a significant increase in all frozen samples. Freezing rate has a significant impact on plasma quality and the final storage temperature influences the long-term stability. CONCLUSION: The optimal storage conditions are ultra-low temperatures (LN 2 or -80°C) and the highest freezing rate possible. However, frozen plasma is not viable for IQC of aPTT during a period of two weeks after freezing. This study is unique in its conception as a practical guide for the handling of frozen plasma samples in modern laboratory settings.

Body yield and quality of fresh and post-freezing filet of Nile tilapia (Oreochromis niloticus) genetic groups.

The aim of this study was to evaluate the body yield and quality of fresh and post-freezing filet of male and female fish of inbred and non-inbred AquaAmérica genetic group and the hybrid between the AquaAmérica and Tilamax varieties. Forty fish (20 males and 20 females) of each genetic group were housed in four 48-m3 hapa net cages, getting 120 fish per cage. The fish were housed at 51 days of age and farmed for 269 days. Pre-slaughter weight was higher (P<0.05) in the AquaAmérica × Tilamax males (0.805±0.204 kg) than in the inbred AquaAmérica male (0.643±0.115 kg). Filet yield percentage was higher (P<0.05) in the AquaAmérica × Tilamax males (32.14±4.72%) than in the inbred AquaAmérica (28.15±2.67%) and non-inbred AquaAmérica (29.06±2.80%) males. Head and viscera yield percentages, pH, color values (L*, a* and b*), shear force, drip loss and cooking loss did not differ significantly between the genetic groups and sexes. Alterations in meat quality were observed after freezing. In conclusion, inbreeding in the AquaAmérica variety resulted in reduced slaughter weight for males; AquaAmérica × Tilamax males have a higher filet yield; and filet quality is not influenced by crossing, inbreeding, or sex, but is changed after freezing.

Nonlinear shear characteristics of frozen loess-concrete interface.

Under the different temperature environment, the precast pile-soil interface characteristics has an important impact on the safety and long-term stability for pile foundation. A large precast pile-soil shear experimental device is used to carry out the direct shear test of concrete-loess interface with different moisture contents under different freezing temperatures. The variation laws of shear strength parameters are revealed with influencing factors, and the shear mechanism of interface is discussed. The stress-strain constitutive equation of interface is proposed, and the shear strength criterion is established with considering the effects of temperature and moisture content on cohesion and internal friction angle. The results show the curve of shear stress and shear displacement can be divided into three stages: elastic deformation stage, plastic deformation stage and sliding failure stage, which macroscopically reflects the shear failure mechanism of the frozen soil-concrete interface. The shear strength of the interface is affected by the test temperature, sample moisture content and normal stress. The lower the test temperature, the greater the shear strength of the interface; With the increase of normal stress, the shear strength of interface increases; With the increase of moisture content, the shear strength of the interface increases and then decreases. The relationship of shear stress and shear displacement of frozen soil-concrete interface can be well described by the piecewise combination of hyperbolic function and linear function.

Strong Inhibition of Ice Growth by Biomimetic Crowding Coacervates.

The freezing of biological fluids is intensively studied but remains elusive as it is affected not only by the various components but also by the crowding nature of the biological fluids. Herein, we constructed spherical crowders, fibrous crowders, and coacervates by various components ranging from surfactants to polymers and proteins, to mimic three typical crowders in biological fluids, i.e., globular proteins, fibrous networks, and condensates of biomolecules. It is elucidated that the three crowders exhibit low, moderate, and strong ice growth inhibition activity, respectively, resulting from their different abilities in slowing down water dynamics. Intriguingly, the coacervate consisting of molecules without obvious ice growth inhibition activity strongly inhibits ice growth, which is firstly employed as a highly-potent cryoprotectant. This work provides new insights into the survival of freezing-tolerant organisms and opens an avenue for the design of ice-controlling materials.

Impact of prepubertal bovine ovarian tissue pre-freeze holding duration on follicle quality.

Ovarian tissue cryopreservation prior to gonadotoxic treatment is the only recommended option for fertility preservation in prepubertal girls. Due to the technical complexity of this technique, limited number of centres across the world are equipped to offer the facility. Hence, the retrieved ovarian tissue needs to be maintained at hypothermic temperature (4 °C) for long time during shipment. The time taken between tissue retrieval and cryopreservation could influence the functionality of cells during fertility restoration. This study explored the tissue integrity and follicle quality of ovarian cortical slices subjected to pre-freeze holding for various time durations in vitro. Prepubertal bovine ovarian tissue from < 12 months old animals were handled at hypothermic holding (4 °C) for 0, 24, 48 and 72 h. The tissues were assessed for follicle viability through confocal analysis of live-dead labelled samples, and follicle quality and tissue integrity through histology. Results have shown that follicle viability, and overall follicle quality were not significantly affected at the end of 72 h hypothermic holding. Though, the observation reassures extended hypothermic holding prior to freezing, findings need to be validated in human tissue prior to use in clinical fertility preservation programs.

Detoxification of arsenite by iodide in frozen solution.

The oxidation of arsenite (As(III)) to arsenate (As(V)) has received significant attention because it helps mitigate the hazardous and adverse effects of As(III) and subsequently improves the effectiveness of arsenic removal. This study developed an efficient freezing technology for the oxidative transformation of As(III) based on iodide (I-). For a sample containing a very low concentration of 20 µM As(III) and 200 µM I- frozen at -20 °C, approximately 19 µM As(V) was formed after reaction for 0.5 h at pH 3. This rapid conversion has never been achieved in previous studies. However, As(V) was not generated in water at 25 °C. The acceleration of the oxidation of As(III) by I- in ice may be attributed to the freeze-concentration effect. During freezing, all components (i.e., As(III), I-, and protons) are highly concentrated in the ice grain boundary regions, resulting in thermodynamically and kinetically favorable conditions for the redox reaction between As(III) and I-. The efficiency of the oxidation of As(III) using I- increased at high I- concentrations and low pH values. The low freezing temperature (below -20 °C) hindered the oxidative transformation of As(III) by I-. The efficiency of the oxidation of As(III) significantly increased using a fixed initial concentration of I- by subjecting the system to six freezing-melting cycles. The outcomes of this study suggest the possibility of the self-detoxification of As(III) in the natural environment, indicating the potential for developing an eco-friendly method for the treatment of As(III)-contaminated areas in regions with a cold climate. It also demonstrates radical remediation to almost completely remove a very small amount of As(III) that was input in As(III)-contaminated wastewater detoxification, a benchmark that existing methods have been unable to achieve.

Permafrost thaw causes large carbon loss in boreal peatlands while changes to peat quality are limited.

Rapid, ongoing permafrost thaw of peatlands in the discontinuous permafrost zone is exposing a globally significant store of soil carbon (C) to microbial processes. Mineralization and release of this peat C to the atmosphere as greenhouse gases is a potentially important feedback to climate change. Here we investigated the effects of permafrost thaw on peat C at a peatland complex in western Canada. We collected 15 complete peat cores (between 2.7 and 4.5 m deep) along four chronosequences, from elevated permafrost peat plateaus to saturated thermokarst bogs that thawed up to 600 years ago. The peat cores were analysed for peat C storage and peat quality, as indicated by decomposition proxies (FTIR and C/N ratios) and potential decomposability using a 200-day aerobic laboratory incubation. Our results suggest net C loss following thaw, with average total peat C stocks decreasing by ~19.3 ± 7.2 kg C m-2 over <600 years (~13% loss). Average post-thaw accumulation of new peat at the surface over the same period was ~13.1 ± 2.5 kg C m-2 . We estimate ~19% (±5.8%) of deep peat (>40 cm below surface) C is lost following thaw (average 26 ± 7.9 kg C m-2 over <600 years). Our FTIR analysis shows peat below the thaw transition in thermokarst bogs is slightly more decomposed than peat of a similar type and age in permafrost plateaus, but we found no significant changes to the quality or lability of deeper peat across the chronosequences. Our incubation results also showed no increase in C mineralization of deep peat across the chronosequences. While these limited changes in peat quality in deeper peat following permafrost thaw highlight uncertainty in the exact mechanisms and processes for C loss, our analysis of peat C stocks shows large C losses following permafrost thaw in peatlands in western Canada.

Integrated metabolomics and transcriptomics analysis reveals that the change of apoplast metabolites contributes to adaptation to winter freezing stress in Euonymus japonicus.

Euonymus japonicus, a common urban street tree, can withstand winter freezing stress in temperate regions. The apoplast is the space outside the plasma membrane, and the changes of metabolites in apoplast may be involved in plant adaptation to adverse environments. To reveal the molecular mechanism underlying the winter freezing stress tolerance in E. japonicus, the changes in physiological and biochemical indexes, apoplast metabolites, and gene expression in the leaves of E. japonicus in early autumn and winter were analyzed. A total of 300 differentially accumulated metabolites were identified in apoplast fluids in E. japonicus, which were mainly related to flavone and flavonol biosynthesis, and galactose metabolism, amino acid synthesis, and unsaturated fatty acid synthesis. Integrated metabolomics and transcriptomics analysis revealed that E. japonicus adjust apoplast metabolites including flavonoids such as quercetin and kaempferol, and oligosaccharides such as raffinose and stachyose, to adapt to winter freezing stress through gene expression regulation. In addition, the regulation of ABA and SA biosynthesis and signal transduction pathways, as well as the activation of the antioxidant enzymes, also played important roles in the adaptation to winter freezing stress in E. japonicus. The present study provided essential data for understanding the molecular mechanism underlying the adaptation to winter freezing stress in E. japonicus.

Role of Poloxamer 188 in Preventing Ice-Surface-Induced Protein Destabilization during Freeze-Thawing.

The phase behavior of poloxamer 188 (P188) in aqueous solutions, characterized by differential scanning calorimetry (DSC) and synchrotron X-ray diffractometry, revealed solute crystallization during both freezing and thawing. Sucrose and trehalose inhibited P188 crystallization during freeze-thawing (FT). While trehalose inhibited P188 crystallization only during cooling, sucrose completely suppressed P188 crystallization during both cooling and heating. Lactate dehydrogenase (LDH) served as a model protein to evaluate the stabilizing effect of P188. The ability of P188, over a concentration range of 0.003-0.800% w/v, to prevent LDH (10 µg/mL) destabilization was evaluated. After five FT cycles, the aggregation behavior (by dynamic light scattering) and activity recovery were evaluated. While LDH alone was sensitive to interfacial stress, P188 at concentrations of ≥0.100% w/v stabilized the protein. However, as the surfactant concentration decreased, protein aggregation after FT increased. The addition of sugar (1.0% w/v; sucrose or trehalose) improved the stabilizing function of P188 at lower concentrations (≤0.010% w/v), possibly due to the inhibition of surfactant crystallization. Based on a comparison with the stabilization effect of polysorbate (both 20 and 80), it was evident that P188 could be a promising alternative surfactant in frozen protein formulations. However, when the surfactant concentration is low, the potential for P188 crystallization and the consequent compromise in its functionality warrant careful consideration.

Anti-Hebbian plasticity in the motor cortex promotes defensive freezing.

Regional brain activity often decreases from baseline levels in response to external events, but how neurons develop such negative responses is unclear. To study this, we leveraged the negative response that develops in the primary motor cortex (M1) after classical fear learning. We trained mice with a fear conditioning paradigm while imaging their brains with standard two-photon microscopy. This enabled monitoring changes in neuronal responses to the tone with synaptic resolution through learning. We found that M1 layer 5 pyramidal neurons (L5 PNs) developed negative tone responses within an hour after conditioning, which depended on the weakening of their dendritic spines that were active during training. Blocking this form of anti-Hebbian plasticity using an optogenetic manipulation of CaMKII activity disrupted negative tone responses and freezing. Therefore, reducing the strength of spines active at the time of memory encoding leads to negative responses of L5 PNs. In turn, these negative responses curb M1's capacity for promoting movement, thereby aiding freezing. Collectively, this work provides a mechanistic understanding of how area-specific negative responses to behaviorally relevant cues can be achieved.

Cell Therapy Drug Product Development: Technical Considerations and Challenges.

Cell therapy uses living cells as a drug to treat diseases. To develop a cell therapy drug product (DP), cryopreservation plays a central role in extending the shelf life of these living medicines by pausing their biological activities, especially preventing degradation, at a temperature as low as liquid nitrogen. This helps overcome the temporal and geographical gaps between centralized manufacturing and clinical administration, as well as allowing sufficient time for full release testing and flexibility in scheduling patients for administration. Cryopreservation determines or influences several key manufacturing, logistical, or clinical in-use processes, including formulation, filling, controlled rate freezing, cryogenic storage and transportation, thawing, and dose preparation. This article overviews the key technical aspects of cell therapy DP development and elucidates fundamental principles of cryobiology that should be considered when we design and optimize the relevant processes. This article also discusses the challenges that motivate continued innovation for cell therapy drug product development.

Proteomic analysis of high and low-motility frozen-thawed spermatozoa in yak provides important insights into the molecular mechanisms underlying sperm cryodamage.

Sperm cryodamage caused by cryopreservation limits the use of frozen yak spermatozoa in artificial insemination (AI). However, the proteomic changes involved in the cryodamage of yak spermatozoa have not been investigated to date. Therefore, this study aimed to identify proteins related to freezing tolerance. Tandem mass tag (TMT) were used in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for identifying differentially expressed proteins (DEPs) between high-motility (HM) and low-motility (LM) frozen-thawed yak spermatozoa. A total of 116 DEPs were identified (>1.5-fold, P < 0.05); of which, 104 proteins were upregulated in HM spermatozoa and 12 proteins were upregulated in LM spermatozoa. The results of functional annotation analysis revealed that the DEPs were mainly involved in metabolic processes. A total of 20 DEPs that were abundantly expressed in HM spermatozoa were strongly associated with carbohydrate metabolism. The results of KEGG analysis revealed that the DEPs were enriched in glycolysis/gluconeogenesis, PPAR signaling pathway, and Ras signaling pathway. In addition, many antioxidant enzymes such as superoxide dismutase (SOD1), peroxiredoxin-6 (PRDX6), and Parkinson disease protein 7 (PARK7) were upregulated in HM spermatozoa, suggesting that these enzymes affect the motility of spermatozoa by regulating the levels of reactive oxygen species (ROS) in frozen-thawed spermatozoa. Altogether, the findings of this study elucidate the mechanisms through which cryopreservation affects the movement of yak spermatozoa and offer a novel basis for refining freezing techniques and modifying cryopreservation extender components.

Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation.

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.