Relevance of controlled cooling and freezing phases in T-cell cryopreservation.

When cells are cryopreserved, they go through a freezing process with several distinct phases (i.e., cooling until nucleation, ice nucleation, ice crystal growth and cooling to a final temperature). Conventional cell freezing approaches often employ a single cooling rate to describe and optimize the entire freezing process, which neglects its complexity and does not provide insight into the effects of the different freezing phases. The aim of this work was to elucidate the impact of each freezing phase by varying different process parameters per phase. Hereto, spin freezing was used to freeze Jurkat T cells in either a Me2SO-based or Me2SO-free formulation. The cooling rates before ice nucleation and after total ice crystallization impacted cell viability, resulting in viability ranging from 26.7% to 52.8% for the Me2SO-free formulation, and 22.5%-42.6% for the Me2SO-based formulation. Interestingly, the degree of supercooling upon nucleation did not exhibit a significant effect on cell viability in this work. However, the rate of ice crystal formation emerged as a crucial factor, with viability ranging from 2.4% to 53.2% for the Me2SO-free formulation, and 0.3%-53.2% for the Me2SO-based formulation, depending on the freezing rate. A morphological study of the cells post-cryopreservation was performed using confocal microscopy, and it was found that cytoskeleton integrity and cell volume were impacted, depending on the formulation-process parameter combination. These findings underscore the importance of scrutinizing all cooling and freezing phases, as each phase impacted post-thaw viability in a distinct way, depending of the specific formulation used.

Beyond Excision: Cryotherapy as a Non-surgical Treatment for Palatal Solitary Neurofibroma.

Palatal solitary neurofibromas (SNFs), not linked to neurofibromatosis type 1, are uncommon. A 45-year-old female with a palatal SNF underwent non-surgical treatment using liquid nitrogen cryotherapy (LNC). The lesion, initially 9 x 8 x 3 mm, was treated with two 1-2 minute freeze-thaw cycles, progressively extended to two 2-2 minute freeze-thaw cycles to address the refractoriness. After four LNC sessions, the lesion resolved without recurrence at five months. This case demonstrates LNC's efficacy as a surgical alternative for palatal SNF, offering a non-invasive option for patients declining surgery. The positive outcome warrants further research into LNC's role in managing similar benign lesions.

Fluctuation of flavor quality in roasted duck: The consequences of raw duck preform's repetitive freeze-thawing.

This study aimed to investigate the changes in flavor quality of roasted duck during repetitive freeze-thawing (FT, -20 ℃ for 24 h, then at 4 ℃ for 24 h for five cycles) of raw duck preforms. HS-SPME/GC-MS analysis showed that more than thirty volatile flavor compounds identified in roasted ducks fluctuated with freeze-thawing of raw duck preforms, while hexanal, nonanal, 1-octen-3-ol, and acetone could as potential flavor markers. Compared with the unfrozen raw duck preforms (FT-0), repetitive freeze-thawing increased the protein/lipid oxidation and cross-linking of raw duck preforms by maintaining the higher carbonyl contents (1.40 âˆ¼ 3.30 nmol/mg), 2-thiobarbituric acid reactive substances (0.25 âˆ¼ 0.51 mg/kg), schiff bases and disulfide bond (19.65 âˆ¼ 30.65 µmol/g), but lower total sulfhydryl (73.37 âˆ¼ 88.94 µmol/g) and tryptophan fluorescence intensity. Moreover, A lower protein band intensity and a transformation from α-helixes to ß-sheets and random coils were observed in FT-3 âˆ¼ FT-5. The obtained results indicated that multiple freeze-thawing (more than two cycles) of raw duck preforms could be detrimental to the flavor quality of the roasted duck due to excessive oxidation and degradation.

Effect of freezing rates on α-amylase enzymatic susceptibility, in vitro digestibility, and technological properties of starch microparticles.

The aim of this study was to evaluate the effect of freezing rates using direct (LF: Liquid nitrogen) and indirect (RF: Cryogenic refrigerator and UF: ultra-freezer) methods at temperatures of (-20, -80, and - 196 °C) on the enzymatic susceptibility with α-amylase for microparticles. In vitro digestibility parameters and technological properties were also analyzed. Lower rates resulted in larger ice crystals, damaging the starch structure. Hydrolysis was more pronounced at slower rates RF: 0.07 °C/min and UF: 0.14 °C/min, yielding maximum values of RDS: 37.63% and SDS: 59.32% for RF. Type A crystallinity remained unchanged, with only a noted increase in crystallinity of up to 6.50% for FR. Starch pastes were classified as pseudoplastic, with RF exhibiting superior textural parameters and apparent viscosity. (RF: 7.18 J g-1 and UF: 7.34 J g-1) also showed lower values of gelatinization enthalpy. Freezing techniques were viable in facilitating the diffusion of α-amylase and reducing RS by up to 81%.

Lipid mixtures (from a liposome kit) and melatonin improve post-thawed Angora goat sperm parameters.

Semen freezing and storing has been widely used in reproductive biotechnology, being applied to certain males of livestock breeds or animal species with economic value such as the Angora goat. The development of a semen extender with the cryoprotective agents can prevent the deterioration of sperm parameters after thawing. This study aimed to investigate lipid mixtures (from a liposome kit, Lps) and melatonin (Mel) at different doses to prevent the deterioration of sperm parameters and to provide the cryoprotective effects on sperm DNA. The Angora goat ejaculates were collected and pooled. They were divided into seven equal volumes, and each of them was diluted with the extenders of the experimental groups with additives (Lps 321.99 µg/mL, Lps 841.33 µg/mL, Mel 0.25 mM, Mel 1 mM, Lps 321.99 µg/mL + Mel 1 mM, Lps 841.33 µg/mL + Mel 0.25 mM) and no additives (control group). After the freeze-thawing process, motility, viability, acrosome integrity, DNA double-strand breaks, and abnormal DNA integrity were assessed for different extender groups. It was determined that the use of Lps alone at low dose or the combination of Lps and Mel had significant cryoprotective effects on motility, viability, acrosome integrity, and DNA damage in Angora goat sperm. This study will help us to understand the effects of Lps and Mel used alone or in combination at different doses and which doses give the optimum spermatological parameter rates following the freeze-thawing process, and hence it will shed light on further studies.

Effects of ultrasonication and freeze-thaw pretreatments on the vacuum freeze-drying process and quality characteristics of apricot (Prunus armeniaca L. cv. Diaoganxing).

The combination of pretreatment and vacuum freeze-drying (VFD) technology is an effective technique for extending the shelf life of apricots, reducing costs and energy consumption. However, the impact of pretreatment on the freeze-drying and quality characteristics of apricots is still unclear. The effects of ultrasound (US), freeze-thaw (FT), and their combination (FT-US) on water migration and quality characteristics of apricot slices on VFD were studied. LR-NMR and SEM showed that pretreatment significantly reduced the time (19.05%-33.33%) and energy consumption (17.67%-35.66%) of the VFD process. Compared with the control group, the US, FT, and FT-US improved the color, texture, rehydration ability, and flavor of apricot slices. Among them, FT-US retained the most biologically active substances and antioxidant capacity, with the highest sensory score. Overall, FT-US pretreatment induced changes in the microstructure and chemistry of apricots, which contributed to the production of high-quality VFD apricot slices.

Tailoring Hydrogel Structures: Investigating the Effects of Multistep Cellulose Defibrillation on Polyvinyl Alcohol Composites.

Defibrillating cellulose through various grinding steps and incorporating it into hydrogels introduces unique properties that warrant thorough exploration. This study investigates cellulose defibrillation at different steps (15-120) using an ultra-fine friction grinder, blended with high-molecular-weight polyvinyl alcohol (PVA), and crosslinked via freeze-thawing. A critical discovery is the influence of defibrillation on the hydrogel structure, as evidenced by reduced crystallinity, thermal degradation, and the enhanced swelling of PVA chains. Despite an increased elastic modulus of up to 120 steps, the synthesized material maintains remarkable strength under hydrated conditions, holding significant promise in biomaterial applications.

Vanillin/fungal-derived carboxy methyl chitosan/polyvinyl alcohol hydrogels prepared by freeze-thawing for wound dressing applications.

In this study, we developed hydrogels using polyvinyl alcohol (PVA), vanillin (V), and a fungus-derived carboxymethyl chitosan (FC) using a freeze-thaw-based method. These hydrogels were strengthened by bonding, including Schiff's base bonding between V and FC and hydrogen bonding between PVA, FC, and V. The physiological properties of these PFCV hydrogels were characterized by FTIR, TGA, compressive mechanical testing, and rheology and water contact angle measurements. FTIR spectra confirmed the effective integration of FC and V into the PVA network. TGA results showed that FC and V enhanced the thermal stability of PFCV hydrogels. Mechanical tests showed increasing the amount of V reduced mechanical properties but did not alter the elastic character of hydrogels. SEM images displayed a well-interconnected porous structure with excellent swelling capacity. In addition, we examined biological properties using cell-based in vitro studies and performed antibacterial assessments to assess suitability for potential wound dressing applications. Prestoblue™ and live/dead cell analysis strongly supported skin fibroblast attachment and viability, DPPH assays indicated substantial antioxidant activity, and PFCV hydrogels showed enhanced antibacterial effects against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). In summary, incorporating V and FC into PVA hydrogels appears to be attractive for wound dressing applications.

Improvement in the quality of frozen dough for fried flour products by electrostatic field-assisted freezing.

BACKGROUND: With the development of the food industry, frozen dough technology has gradually become an indispensable part of dough processing but its quality is often reduced due to freezing during the production process. Electrostatic field-assisted freezing (EF) technology, a key research project in recent years, reduces the physical damage to food materials by reducing or changing the size of ice crystals in frozen products. RESULTS: In this study, different intensities of electrostatic fields were used to assist in the repeated freezing and thawing of dough. The effects of electrostatic fields on the freezing nucleation process were evaluated by measuring dough freezing curves, low field nuclear magnetic resonance, and melting enthalpy. It was found that the freezing time of frozen dough added with electrostatic field-assisted freezing processing was shortened, the rate at which hardness, viscosity, and elasticity decreased was reduced, and the indicators of water distribution and protein secondary structure components were closer to those of fresh dough. CONCLUSION: This experiment used electrostatic field-assisted freezing to reduce the damage to the dough structure during the freezing process, improve the quality of frozen dough and fried products, and improve the freezing efficiency of frozen dough. It provides a new idea for the study of frozen dough. © 2023 Society of Chemical Industry.

Strategies for Engineering of Extracellular Vesicles.

Extracellular vesicles (EVs) are membrane vesicles released by cells into the extracellular space. EVs mediate cell-to-cell communication through local and systemic transportation of biomolecules such as DNA, RNA, transcription factors, cytokines, chemokines, enzymes, lipids, and organelles within the human body. EVs gained a particular interest from cancer biology scientists because of their role in the modulation of the tumor microenvironment through delivering bioactive molecules. In this respect, EVs represent an attractive therapeutic target and a means for drug delivery. The advantages of EVs include their biocompatibility, small size, and low immunogenicity. However, there are several limitations that restrict the widespread use of EVs in therapy, namely, their low specificity and payload capacity. Thus, in order to enhance the therapeutic efficacy and delivery specificity, the surface and composition of extracellular vesicles should be modified accordingly. In this review, we describe various approaches to engineering EVs, and further discuss their advantages and disadvantages to promote the application of EVs in clinical practice.

Investigation and Comparison of Active and Passive Encapsulation Methods for Loading Proteins into Liposomes.

In this work, four different active encapsulation methods, microfluidic (MF), sonication (SC), freeze-thawing (FT), and electroporation (EP), were investigated to load a model protein (bovine serum albumin-BSA) into neutral liposomes made from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (Chol) and charged liposomes made from DSPC:Chol:Dioleoyl-3-trimethylammonium propane (DOTAP), DSPC:Chol:1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and DSPC:Chol:phosphatidylethanolamine (PE). The aim was to increase the protein encapsulation efficiency (EE%) by keeping the liposome size below 200 nm and the PDI value below 0.7, which warrants a nearly monodisperse preparation. Electroporation (100 V) yielded the best results in terms of EE%, with a dramatic increase in liposome size (>600 nm). The FT active-loading method, either applied to neutral or charged liposomes, allowed for obtaining suitable EE%, keeping the liposome size range below 200 nm with a suitable PDI index. Cationic liposomes (DSPC:Chol:DOTAP) loaded with the FT active method showed the best results in terms of EE% (7.2 ± 0.8%) and size (131.2 ± 11.4 nm, 0.140 PDI). In vitro release of BSA from AM neutral and charged liposomes resulted slower compared to PM liposomes and was affected by incubation temperature (37 °C, 4 °C). The empty charged liposomes tested for cell viability on Human Normal Dermal Fibroblast (HNDF) confirmed their cytocompatibility also at high concentrations (1010 particles/mL) and cellular uptake at 4 °C and 37 °C. It can be concluded that even if both microfluidic passive and active methods are more easily transferable to an industrial scale, the FT active-loading method turned out to be the best in terms of BSA encapsulation efficiencies, keeping liposome size below 200 nm.

Specific antioxidant enzymes are involved in the freeze-thawing response of industrial baker's yeasts.

In this study, the biochemical basis of resistance to slow freezing and thawing (F-T) stress was explored in two baker yeast industrial strains of Saccharomyces cerevisiae that presented differential tolerance to freezing in order to be in the frozen bakery industry. Strain Y8, used commercially in sweet baking doughs, exhibited greater stress tolerance than Y9, a strain employed in regular doughs. Survival of Y8 was higher than that of Y9 (30% vs 12%) after F-T or other reactive oxygen species (ROS) inducing stresses compared to their non-stressed controls. The superior F-T tolerance of Y8 was related to its lower ROS accumulation capacity, determined by fluorometry in cell-free extracts and in vivo, by fluorescence microscopy upon F-T, being Y8 ROS accumulation 2-fold lower than that of Y9. That, in turn, could be positively associated with Y8's higher constitutive activities of cytosolic catalase (CAT) and superoxide dismutase by a significant activation (25%) of Y8 CAT after F-T. That would complement the protective effects of other protectant molecules like trehalose, present at high concentration in this strain.

Design, development and in vitro quantification of novel electrosprayed everolimus-loaded Soluplus®/Polyvinyl alcohol nanoparticles via stability-indicating HPLC method in cancer therapy.

Everolimus (RAD001) a mammalian target of rapamycin has been hampered by poor solubility, affecting its dissolution rate, a relationship that extends to low bioavailability. Nanoparticles (NP) based on Soluplus (SOL®) and Polyvinyl alcohol (PVA) was fabricated by electrospraying (ES) for the delivery of RAD001 to improve anti-tumour efficacy. Electrospraying with established experimental conditions produced PVA-SOL®-RAD001 NP with 71 nm mean diameter, smaller particle size distribution and >90 % encapsulation efficiency. Various polymer-drug concentrations exposed to various freeze-thaw (F/T) cycles were studied for NP optimisation and to enhance its mechanical properties. The optimised NP formulation demonstrated complete encapsulation as well as a sustained and pH dependent drug release profile for in vitro release test. In addition, to specifically study the degradation profile of RAD001 and to quantify RAD001 in the fabricated NP, a new HPLC method was developed and validated. The purpose and novelty of the HPLC method was also to ensure that RAD001 can be detected at low amounts where other conventional characterisation methods are unable to detect. The developed HPLC method was accurate, precise, robust and sensitive with LOD and LOQ values of 4.149 and 12.575 µg/mL. In conclusion, the novel developed HPLC system can be applied for the quantification of different chemotherapeutic agents and the novel electrosprayed hydrogel NP is a potential drug delivery system to increase solubility and bioavailability of RAD001 in cancer therapy.

Freeze-thaw assisted maleic acid pretreatment of eucalyptus to prepare cellulose nanocrystals and degraded lignin.

A green and effective method is proposed for the pretreatment of eucalyptus by freeze-thaw assisted maleic acid tactic, wherein the effects of freeze-thaw, maleic acid concentration, reaction time, and temperature on the fractionation were examined. Results showed that under optimal conditions (60% maleic acid, 120 °C, and 2 h), a remarkable removal of 74.5% lignin and 95.2% hemicellulose was achieved after freeze-thaw treatment. The resulting cellulose-rich solid residues were further processed with maleic acid to prepare cellulose nanocrystals, which displayed uniform sized rod-like structures and high crystallinity (62.51%). Moreover, maleic acid pretreatment resulted in lignin with low molecular weight (2110-2530) and excellent homogeneity (PDI ≤ 1.86), while maintaining a relatively intact structure. The lignin had high ß-O-4 aryl ether bond contents (≥77.5%) and abundant phenolic hydroxyl contents (2.33-3.63 mmol/g). Overall, the process exhibits notable benefits in effectively separating lignocellulose for high valorization.

Biphasic scaffolds of polyvinyl alcohol/gelatin reinforced with polycaprolactone as biomedical materials supporting for bone augmentation based on anatomical mimicking; fabrication, characterization, physical and mechanical properties, and in vitro testing.

Reinforced biphasic scaffolds were fabricated with based materials design of anatomical mimicking and evaluated to identify the certain application for maxillofacial surgery. The scaffolds created the polyvinyl alcohol (PVA) with a percentage of gelatin of 5% and were coated with polycaprolactone (PCL) that a different number of cycles 0, 1, 5, 10, and 15 cycles (PCL0, PCL1, PCL5, PCL10, and PCL15 were used to fabricate biphasic scaffolds via bubbling and freeze-thawing before reinforce with immersion coating techniques. The structure and morphology of the scaffolds were characterized and observed by a scanning electron microscope, a differential scanning calorimeter, and a thermogravimetric analyzer, respectively. The performance of the scaffolds was tested in terms of their swelling behavior, degradation, and mechanical properties. They were cultured with MC3T3E1 osteoblast cells and L929 fibroblast cells. The main biological performance of cell proliferation was analyzed, and protein synthesis, calcium synthesis, and alkaline phosphatase activity of the scaffolds were studied. Their morphology demonstrated fewer pores when coated with PCL. Mechanical strength of the modified scaffolds increased followed by the cycles of coating with PCL. The scaffolds with more cycle of PCL coating lower swelling and degradability than without PCL coating. They had more thermal stability than the scaffold without PCL coating. The scaffolds with PCL coating demonstrated better bio-functionality to activate cell response than without coating. Finally, the result exhibited that PCL10 provide a suitably reinforced biphasic scaffold with high promise for maxillofacial surgery.

Acid emulsions stabilized by soy whey concentrates and soluble soybean polysaccharides: Role of biopolymer interaction strategies on stability against environmental stresses.

This article focuses on the evaluation of different interaction strategies between soy whey concentrates (SWC) and soluble soybean polysaccharides (SSPS) at pH 3.0 on the emulsion stability against freeze-thawing and mechanical stirring. Emulsions were prepared from aqueous dispersions of both biopolymers (3.0% w/w SSPS and SWC, 1:1 mass ratio) and sunflower oil (10% w/w) by aqueous phase complexation (APC), interfacial complexation (IC) and interfacial complexation and sonication (ICS). SWC control emulsion was a poor emulsifying ability; SSPS addition, through the APC and ICS strategies, noticeably improved the SWC emulsifying properties. ICS emulsions showed the highest stability to environmental stresses, due a combination of low initial particle size, flocculation degree and steric hindrance promoted by the presence of SSPS chains at the interface. This study provides valuable information forthe utilization of whey soy proteins in acid dispersed systems stable to environmental stresses.

Versatile Assembly of Metal-Phenolic Network Foams Enabled by Tannin-Cellulose Nanofibers.

Metal-phenolic network (MPN) foams are prepared using colloidal suspensions of tannin-containing cellulose nanofibers (CNFs) that are ice-templated and thawed in ethanolic media in the presence of metal nitrates. The MPN facilitates the formation of solid foams by air drying, given the strength and self-supporting nature of the obtained tannin-cellulose nanohybrid structures. The porous characteristics and (dry and wet) compression strength of the foams are rationalized by the development of secondary, cohesive metal-phenolic layers combined with a hydrogen bonding network involving the CNF. The shrinkage of the MPN foams is as low as 6% for samples prepared with 2.5-10% tannic acid (or condensed tannin at 2.5%) with respect to CNF content. The strength of the MPN foams reaches a maximum at 10% tannic acid (using Fe(III) ions), equivalent to a compressive strength 70% higher than that produced with tannin-free CNF foams. Overall, a straightforward framework is introduced to synthesize MPN foams whose physical and mechanical properties are tailored by the presence of tannins as well as the metal ion species that enable the metal-phenolic networking. Depending on the metal ion, the foams are amenable to modification according to the desired application.

The migration and loss of water in emulsified surimi gels prepared with different phase states of lipids: Effect of freeze-thawing treatments.

The freeze-thawing (FT) stability generally correlates well with the economic value and acceptability of frozen surimi-based products. However, quality changes of emulsified surimi gels under FT conditions are still unclear. Therefore, the gel properties of samples with different phase states of lipids (lard, lard + soybean oil, and soybean oil) were investigated at FT conditions. Results showed that the soybean oil evidently improved the rheological behaviors of sols/gels compared to the lard group. The moisture content of samples with different lipids decreased by 2.40%-2.71% after 4 FT cycles. With increasing FT cycles, the water-holding capacity decreased accompanied by the increase of cooking loss. Spin-spin relaxation spectra and hydrogen proton density images proved the occurrence of water migration of gels during these processes. Better gel integrity was observed in samples consisting of soybean oil, where the proportion of pores was lower than those with lard regardless of FT treatments. Additionally, the intermolecular forces of gels also changed under FT treatments. There results suggested that the lipids with different phase states affected the migration and loss of water in emulsified surimi gels under FT cycles. PRACTICAL APPLICATION: The quality changes of heating-induced surimi gel products under frozen storage have been ignored, especially the emulsified surimi gels. This study discloses the changes of the gel properties in emulsified gel products with different phase states of lipids after FT treatments, which provides critical insights into the quality improvement of this novel emulsified surimi product during processing, storage, and transportation.

Effects of traditional and advanced drying techniques on the physicochemical properties of Lycium barbarum L. polysaccharides and the formation of Maillard reaction products in its dried berries.

This study explored the effect of three different industrial drying methods on the physicochemical, nutritional, and safety profile of goji berries. The hot-air (HD) and microwave drying (MD) methods yielded berries with relatively high polysaccharide content, while vacuum freeze-drying (FD) yielded dried berries with better sensory qualities but relatively less polysaccharide content. The polysaccharides obtained from the HD and MD berries had lower molecular weight, high antioxidant activity and high degrees of Maillard reaction. Further investigations revealed that all three methods, in particular HD and MD, generated high levels of intermediate Maillard reaction products (55.8-86.3 mg/kg) and advanced glycation end-products (fluorescent intensity of 26784-51712), based on significant reduction of reducing sugar and amino acids in the HD and MD berries (p < 0.05). These findings highlight the need to scrutinize the effectiveness of traditional and emerging drying technologies used to produce safe fruits.

Effects of ultrasound assisted cell wall disruption on physicochemical properties of camellia bee pollen protein isolates.

Camellia bee pollen protein isolates were extracted by cell wall disruption using ultrasonication, freeze-thawing, enzymatic hydrolysis, and their combinations. The effects of these methods on microstructure of cell wall, protein release, protein yield, physiochemical properties and structure of proteins were investigated. As compared with physical treatments (ultrasonication, freeze-thawing and their combination), the enzymatic hydrolysis significantly improved the yield of proteins, because it not only promoted the release of proteins from the inside of pollen, but also released proteins in pollen wall. The proteins extracted by enzymatic hydrolysis method also exhibited better solubility, emulsifying and gelation properties due to the partial hydrolysis of proteins by protease. In addition, when ultrasound was combined with freeze-thawing or enzymatic hydrolysis, it could further improve the yield of proteins and the functional properties of proteins, which was mainly related to the changes of protein structure induced by cavitation effect of ultrasound.