Search Results (109)

Global amphibian populations are declining rapidly and the development of effective cryopreservation protocols for germ cells has become a critical tool in ex situ conservation programs. Post-thaw membrane-integrity viability in four endangered Korean amphibians (Dryophytes suweonensis, Pelophylax chosenicus, Kaloula borealis, and Hynobius yangi) were evaluated. Sperm were cryopreserved using dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) at 10–30% (v/v) in combination with 0.6 M sucrose, and were frozen at two suspension heights (5 cm vs. 10 cm) above liquid nitrogen. Post-thaw membrane-integrity viability was assessed using a SYBR-14/propidium iodide membrane-integrity assay (LIVE/DEAD kit). Low concentrations of permeating cryoprotectants (CPs) improved membrane-integrity viability, whereas high concentrations led to high toxicity, particularly with DMSO. Across species, DMF produced the highest membrane-integrity viability and the most consistent performance. The cooling rate influenced membrane-integrity viability, with samples frozen at 10 cm exhibiting greater viability, reflecting the balance between intracellular ice formation during rapid cooling and solution effects during slow cooling. Optimal conditions for D. suweonensis were 15% DMSO at 10 cm (86.5% membrane-integrity viability); for P. chosenicus, 10% DMF at 10 cm (75.5%); and for K. borealis, 10% DMSO at 5 cm (81.6% membrane-integrity viability). Hynobius yangi showed modest improvement under 15% DMF at 5 cm (19.7%), although overall membrane-integrity viability was low. ED₅₀ modeling indicated species-specific thresholds requiring low CP concentrations. Sperm cryopreservation outcomes in amphibians are strongly influenced by CP type, concentration, cooling regime, and species physiology. GLM and ED₅₀ modeling provide a methodological framework for refining cryopreservation strategies for non-model, endangered species. Full article

To enhance the scope of therapeutic interventions using star polymeric nanoparticles of desired concentrations, an understanding of the effect of converting aqueous formulations into stable redispersible dry powders by freeze drying on their physicochemical and biological properties is essential. We demonstrate that parameters such as the choice of the cryoprotectant, its molecular weight, and concentration play an important role during lyophilization and reconstitution processes. We hypothesized that utilizing cryoprotectants akin to shell-forming polymers may be ideal in protection against aggregation and keeping the nanostructures intact during lyophilization and reconstitution, as well as retaining the overall biological efficacy of their cargo. Through an evaluation of miktoarm polymer-based nanoparticles, we demonstrate that PEG_2k at 1% w/v concentration provides the optimized cryoprotection, and the resulting solid formulations upon redispersion in an aqueous medium preserve the desired nanoparticle and curcumin properties. PEG_2k at 1% w/v is more efficient than PEG_5k and saccharides including glucose, sucrose, trehalose, and mannitol in enhancing the integrity of micelles during lyophilization and reconstitution. Addition of PEG_2k 1% w/v (with or without lyophilization and redispersion) enhances drug release in PBS buffer, while it has no impact in the cell culture media. Nanoformulations protect endothelial cells from cytotoxicity of curcumin, and addition of cryoprotectant or the lyophilization/redispersion processes did not impair anti-inflammatory efficacy of curcumin. Full article

Cryopreservation is a well-established method for extending platelet shelf-life and addressing supply shortages. Traditionally, this involves dimethyl sulfoxide (DMSO) as a cryoprotective agent (CPA), but recent studies suggest that using controlled rate freezing (CRF) with only NaCl may offer a less toxic alternative. To explore further optimization, this study assessed whether adding 10% choline chloride–glycerol, a deep eutectic solvent (DES), could enhance platelet quality in CRF/NaCl cryopreservation. Ten double-dose buffy coat platelet units were divided into test (DES-treated) and control (NaCl-only) groups. After DES exposure (10% for 20 min), all units were prepared using the NaCl protocol and frozen at −80 °C with CRF equipment, then stored for over 90 days. Upon thawing and reconstitution in AB plasma, no significant differences were observed in platelet content post-thaw between control and test units (255 ± 43 vs. 257 ± 41 × 10⁹/unit), post-thaw recovery (>85%): respectively, Δψ (JC-1% pos 63 ± 15 vs. 68 ± 17), LDH (% of total 10 ± 6 vs. 9 ± 6), (CD63% 77 ± 9 vs. 82 ± 7), (CD62P % 72 ± 15 vs. 76 ± 11), (CD42b % 78 ± 9 vs. 80 ± 9), (CD61% 79 ± 9 vs. 78 ± 9), (CD41% 81 ± 11 vs. 83 ± 7), (PAC-1% 33 ± 10 vs. 32 ± 8), (Pecam-1% 78 ± 11 vs. 80 ± 8), (GPIV % 72 ± 10 vs. 74 ± 11), (LAMP-1% 26 ± 14 vs. 11 ± 9), (MPCD61+ % 41 ± 11 vs. 46 ± 10), (ROTEM CT 56 ± 7 vs. 55 ± 6), (ROTEM CFT 110 ± 70 vs. 106 ± 67) and (ROTEM MCF 35 ± 6 vs. 36 ± 6). These findings support the feasibility of CPA-free CRF-based platelet cryopreservation while maintaining functional integrity. Full article

Cell spheroids are widely studied for their potential applications in tissue engineering and regenerative medicine. The present work investigated the effects of cryopreservation on spheroids derived from ovine fibroblasts, depending on spheroid size (140 or 220 µm). Specifically, it explored how cryopreservation impacted several biological and physical parameters including cell damage, viability, metabolism, adhesion, proliferation, and spheroid mass density, weight, and diameter at three time points after thawing. A Live/Dead assay provided a visual assessment of cell damage, cell viability and metabolic activity were assessed by an Alamar Blue assay, and a replating assay evaluated cell adhesion and proliferation capabilities. Spheroid mass density, weight, and diameter were quantified by the W8 Biophysical Analyzer, creating accurate biophysical profiles. Real-time PCR (RT-PCR) analysis was employed to uncover gene expression changes following cryopreservation. Our findings indicate that spheroids measuring 140 µm in diameter largely maintained their biophysical features and cell viability post-cryopreservation, whereas those at 220 µm exhibited a decline in both vitality and mass density. The reduced vitality of 220 µm spheroids likely reflects size-related limitations in cryoprotectant diffusion and stress within the core. Overall, this study provides a comprehensive understanding of how cryopreservation affects ovine fibroblast spheroid biophysics and cellular integrity, laying the groundwork for improved preservation techniques for cell spheroids. Full article

Additives such as surfactants (Tween-20) and cryoprotectants (glycerol and trehalose) are often used in enzymatic assays to improve the quality and long-term stabilization of proteins. However, these additives can affect the enzymatic activity and the enzyme’s affinity for active compounds, such as inhibitors, and must be considered during assay design since a slight shift in enzyme behavior may compromise the reliability of the results. In this study, the effects of Tween-20, glycerol, and trehalose on hyaluronidase (Hyal) were systematically evaluated by assessing their influence both directly—through microscale thermophoresis (MST) signals of the labeled enzyme (Hyal*)—and indirectly, by monitoring the formation of the final product of the degradation of hyaluronic acid, tetrasaccharide (Tet), using capillary electrophoresis (CE/UV). Hyal was labeled for the first time with ATTO-647 NHS ester, a commercial dye compatible with MST. Efficient labeling was achieved in a phosphate-based buffer without loss of catalytic activity. Tween-20 showed no impact on MST signals nor on enzymatic performance when used between 0.005 and 0.05% (v/v). Glycerol also did not interfere with MST measurements; however, it significantly reduced catalytic activity at concentrations above 2% (v/v). Trehalose affected Hyal* fluorescence in a concentration-dependent manner and enhanced catalytic activity even at 0.02% (v/v). Full article

The present research aimed to evaluate the effectiveness of new organo-mineral biostimulants in an apple orchard, including their relevance to spring frosts and to enhancing yield. The study evaluated the effects of foliar sprays with organo-mineral fertilizers on apple yield, comparing three treatments: 1—control (no treatment); 2—foliar spray with a 1% blend of “WPU” Antifreeze and 1% “WP Drip Ca + Mg”; 3—foliar application using a 3% solution of both “WPU” Antifreeze and “WP Drip Ca + Mg”. The NPC “White Pearl” foliar sprays exhibited cryoprotective properties to spring frosts through multiple mechanisms, i.e., prevention of cellular dehydration via elevated bound water content and accumulation of osmoprotective compounds including proline and soluble sugars. This research shows that the applied treatments improved carbohydrate metabolism by enhancing the biosynthesis of glucose and starch, as well as changing the donor–acceptor relationships between the leaf apparatus and the fruit toward the forming apple, promoting a better outflow of assimilates into ripening fruits. The 1% solution treatment enhanced apple yield by 70% (1.7-fold) relative to the untreated control. These findings indicate that the “White Pearl” organo-mineral fertilizer NPC (especially at 1% concentration) could serve as an effective supplement to conventional apple farming practices, boosting overall productivity. Full article

Cold denaturation of gluten proteins during prolonged frozen storage or repeated freeze–thaw cycles can severely affect the quality of frozen cereal products. While both processes have been studied individually, their combined effects and underlying mechanisms remain unclear. This study systematically evaluated the hydration properties and conformational changes in gluten proteins stored at −73 °C and −23 °C, with or without freeze–thaw cycling. Compared to continuous storage, freeze–thaw cycles reduced water-holding capacity by 9.1–12.2% and increased oil-holding capacity by 5.3–10.3%, indicating aggravated structural damage. Ultra-low temperature storage (−73 °C) suppressed ice crystal growth, preserved hydration, and limited hydrophobic residue exposure. Spectroscopic analyses revealed a temperature-dependent shift from α-helices to β-sheets and β-turns, which was accelerated by freeze–thaw cycles. Enhanced hydrophobic interactions and tryptophan exposure further indicated destabilization. Molecular dynamics simulations showed that increased hydrogen bonding between proteins and water contributed to unfolding at low temperatures, while temperature fluctuations intensified denaturation through repeated hydrogen bond breakage and reformation. These results underscore the critical role of thermal instability in cold denaturation and offer mechanistic insights for improving cryoprotection strategies in frozen food systems. Full article

(1) Background: Cryopreservation of epididymal spermatozoa in dogs is challenging due to their lower cryotolerance compared to ejaculated spermatozoa. Given the limited sperm volume obtained per individual, efficient recovery and preservation techniques are essential. (2) Methods: This study assessed sperm collection and cryopreservation methods from the cauda epididymis of ten dogs undergoing routine elective castration. After dissection and mincing, the cauda epididymidis tissue was incubated in 0.9% saline at 38 °C for either 10- or 30-min. Samples were analyzed for concentration and motility using AndroVision^® software (CASA; AndroVision™; Minitüb GmbH) (Tiefenbach, Germany). Additional evaluations included histological examination, hypoosmotic swelling test, live/dead staining, and morphological assessments. Three extenders, custom-made Tris-Fructose-Citrate (Tris), custom-made Uppsala, and commercial Optixcell^®, were used for cryopreservation and compared for post-thaw sperm quality. (3) Results: No significant differences were found between the 10- and 30-min incubation groups regarding sperm motility, viability, or histological integrity. The total sperm counts were 292 × 10⁶ ± 175 × 10⁶ for the 10 min group and 233 × 10⁶ ± 162 × 10⁶ for the 30 min group (p = 0.56). Histological sections revealed no significant difference in residual intraluminal spermatozoa between groups, indicating that 10 min of incubation is sufficient for effective sperm migration. Post-thaw sperm motility was significantly higher with Uppsala (17.2 ± 12.2%) and Optixcell^® (11.7 ± 6.5%) compared to Tris (4.7 ± 4.8%). Morphological abnormalities were lowest in samples preserved with Optixcell^® (37.5 ± 10.1%, p = 0.005). (4) Conclusion: A 10 min incubation period is adequate for efficient recovery of epididymal sperm in dogs. Among the tested extenders, Uppsala and Optixcell^® demonstrated superior cryoprotective effects, resulting in better post-thaw motility and reduced morphological abnormalities compared to Tris. Full article

Lipofilling has far more applications than cosmetic surgery alone. Due to its high content of stromal vascular fraction (SVF) cells, lipoaspirate can also be used to treat wounds, as its cellular components may accelerate wound healing. Using our CELT^PLUS protocol, we can increase the number of SVF cells per volume. Unfortunately, some patients require more than one treatment to achieve an optimal outcome, but would unnecessarily suffer from repeated liposuction. Therefore, our objective was to test whether cryopreserving CELT^PLUS fat could offer a solution, potentially avoiding the need for repeated liposuction procedures. DMSO was used as a cryoprotective agent for proof-of-principle testing, although other non-toxic cryoprotective agents should be considered in the future. The rest of our freezing protocol is a clinically friendly attempt to facilitate the translation into clinical practice. We tested the cryopreserved tissue using histological evaluation, metabolism measurement, SVF cell yield estimation, PCRs from both whole tissue and from cultured SVF cells, and Oil Red “O” staining. We found that freezing CELT^PLUS fat with DMSO yields better results than without cryoprotection in all evaluated methods. Until non-toxic cryoprotective agents are tested on CELT^PLUS fat, we do not recommend initiating animal or human testing. Full article

Astragalus sinicus, a key leguminous green manure widely cultivated in Southern China’s rice-based cropping systems, plays a pivotal role in sustainable agriculture by enhancing soil organic matter sequestration, improving rice yield, and elevating grain quality. The symbiotic nitrogen-fixing association between A. sinicus and its matching rhizobia is fundamental to its agronomic value; however, suboptimal inoculant efficiency and field application methodologies constrain its full potential. To address these limitations, we conducted a multi-phase study involving (1) rhizobial strain screening under controlled greenhouse conditions, (2) an optimized lyophilization protocol evaluating cryoprotectant (trehalose, skimmed milk powder and others), and (3) seed pelleting trails with rhizobial viability and nodulation assessments over different storage periods. Our results demonstrate that Mesorhizobium huakuii CCBAU 33470 exhibits a superior nitrogen-fixing efficacy, significantly enhancing key traits in A. sinicus, including leaf chlorophyll content, tiller number, and aboveground biomass. Lyophilized inoculants prepared with cryoprotectants (20% trehalose or 20% skimmed milk powder) maintained >90% bacterial viability for 60 days and markedly improved nodulation capacity relative to unprotected formulations. The optimized seed pellets sustained high rhizobial loads (5.5 × 10³ cells/seed) with an undiminished viability after 15 days of storage and nodulation ability after 40 days of storage. This integrated approach of rhizobial selection, inoculant formulation, and seed coating overcomes cultivation bottlenecks, boosting symbiotic nitrogen fixation for A. sinicus cultivation. Full article

This study investigated quality changes in seaweed–yak patties before and after freeze–thaw by varying seaweed addition levels (10–70%). Macroscopically, the effects on water-holding capacity, textural properties, and oxidative indices of restructured yak patties were evaluated. Microscopically, the impact of seaweed-derived bioactive ingredients on patty microstructure and myofibrillar protein characteristics was examined. LF-NMR and MRI showed that 40% seaweed addition most effectively restricted water migration, reduced thawing loss, and preserved immobilized water content. Texture profile analysis (TPA) revealed that moderate seaweed levels (30–40%) enhanced springiness and minimized post-thaw hardness increases. SEM confirmed that algal polysaccharides formed a denser protective network around the muscle fibers. Lipid oxidation (MDA), free-radical measurements, and non-targeted metabolomics revealed a significant reduction in oxidative damage at 40% seaweed addition, correlating with increased total phenolic content. Protein analyses (particle size, zeta potential, hydrophobicity, and SDS-PAGE) demonstrated a cryoprotective effect of seaweed on myofibrillar proteins, reducing aggregation and denaturation. These findings suggest that approximately 40% seaweed addition can improve the physicochemical stability and antioxidant capacity of frozen seaweed–yak meat products. This work thus identifies the optimal seaweed addition level for enhancing freeze–thaw stability and functional quality, offering practical guidance for the development of healthier, high-value restructured meat products. Full article

The cryopreservation of three-dimensional (3D) biofabricated constructs is a key enabler for their clinical application in regenerative medicine. Unlike two-dimensional (2D) cultures, 3D systems such as encapsulated cell spheroids, molded hydrogels, and bioprinted tissues present specific challenges related to cryoprotectant (CPA) diffusion, thermal gradients, and ice formation during freezing and thawing. This review examines the current strategies for preserving 3D constructs, focusing on the role of biomaterials as cryoprotective matrices. Natural polymers (e.g., hyaluronic acid, alginate, chitosan), protein-based scaffolds (e.g., silk fibroin, sericin), and synthetic polymers (e.g., polyethylene glycol (PEG), polyvinyl alcohol (PVA)) are evaluated for their ability to support cell viability, structural integrity, and CPA transport. Special attention is given to cryoprotectant systems that are free of dimethyl sulfoxide (DMSO), and to the influence of hydrogel architecture on freezing outcomes. We have compared the efficacy and limitations of slow freezing and vitrification protocols and review innovative approaches such as temperature-controlled cryoprinting, nano-warming, and hybrid scaffolds with improved cryocompatibility. Additionally, we address the regulatory and manufacturing challenges associated with developing Good Manufacturing Practice (GMP)-compliant cryopreservation workflows. Overall, this review provides an integrated perspective on material-based strategies for 3D cryopreservation and identifies future directions to enable the long-term storage and clinical translation of engineered tissues. Full article

The aim of this study was to evaluate the effect of the iota, kappa and lambda carrageenan fractions on the physical properties and crystal structure of a fruit sorbet prepared from frozen mango fruits. During this study, physical properties such as density, cryoscopic temperature, osmotic pressure, overrun and melting time were analyzed. In order to assess the crystal structure and its changes, microscope images were taken of each sample after 1, 30 and 90 days of storage. The stabilizers showed no significant effect on the physical properties of the ice cream mixture; however, the sample with iota carrageenan stood out for having the highest overrun (58.7%) and the sample with kappa carrageenan took the longest to melt of all tested samples (almost 21 min). This study shows a significant effect of carrageenans in reducing the initial size of ice crystals as well as reducing recrystallization during storage. The stabilizing blend using ι-carrageenan provided the most effective cryoprotective properties, with an ice crystal diameter of 9 µm. Full article

Seven wild-type lactic acid bacteria, belonging to Lactiplantibacillus plantarum and Lactococcus cremoris species, were isolated from beetroots and white mushrooms and evaluated for their safety and functional profile. Lc. cremoris isolates were sensitive to all antibiotics tested, while L. plantarum strains exhibited resistance in certain antibiotics. Among them, Lc. cremoris FBMS_5810 showed the highest cholesterol removal ability (51.89%) and adhesion capacity to Caco-2 cell lines (32.14%), while all plant origin strains exhibited strong antagonistic and inhibitory activity against foodborne pathogens, as well as high survival potential during an in vitro digestion model. Subsequently, freeze-dried immobilized Lc. cremoris FBMS_5810 cells on oat flakes were prepared with initial cell loads >8.5 log CFU/g, and the effect of trehalose as a cryoprotectant in cell viability during storage at room and refrigerated temperatures for up to 180 days was studied. A significant reduction in cell loads was observed in all cases studied. However, freeze-dried immobilized Lc. cremoris FBMS_5810 cells on oat flakes prepared using trehalose as a cryoprotectant stored at 4 °C exhibited the highest cell viability (8.75 log CFU/g) after 180 days. In the next step, functional breakfast cereals enriched with freeze-dried immobilized Lc. cremoris FBMS_5810 cells on oat flakes (produced with (MLT) or without (ML) trehalose) were developed and stored at room and refrigerated temperatures for 180 days. The initial cell levels ≥ 9.18 log CFU/g were achieved, while a significant decrease was recorded during storage in all cases. The maintenance of cell loads ≥ 7.75 log CFU/g was documented in the case of both ML and MLT samples stored at 4 °C; however, the presence of trehalose in MLT samples resulted in cell viability 7.52 log CFU/g after 180 days of storage at room temperature. Importantly, the functional breakfast cereals were accepted by the panel during the sensory evaluation. Full article

The cryopreservation of male gonadal tissue is critical to conserve genetic material and use it later via assisted reproduction. This study aimed to evaluate cryopreservation methods (slow freezing, SF; solid surface vitrification, SSV) as well as the optimal concentrations of intracellular cryoprotectants during the SSV of testicular tissue from prepubertal collared peccaries. Five pairs of testes were dissected on different days into small fragments (3 mm³) and allocated to a non-cryopreserved, a control group or one of three treatment groups: SF; SSV 3 M (1.5 M dimethyl sulfoxide [DMSO] plus 1.5 M ethylene glycol [EG]); or SSV 6 M (3 M DMSO plus 3 M EG). After one week of storage in liquid nitrogen, tissue samples were warmed and evaluated in terms of histology, viability, proliferative capacity potential, and DNA integrity. The scores for histological integrity and cellular damage for SF (2.08 ± 0.05 and 2.33 ± 0.07, respectively) were similar to the results found in SSV 6 M (1.93 ± 0.04 and 2.30 ± 0.07; p > 0.05). However, these scores were better when compared to SSV 3 M (1.87 ± 0.05 and 2.08 ± 0.06; p < 0.05). The percentage of cellular viability was around 57% after all preservation treatments (p > 0.05), which was lower than in the control group (88.8 ± 1.9%; p < 0.05). The SSV 6 M treatment was better than the other treatments regarding the proliferative capacity potential of spermatogonia cells (3.52 ± 0.03) (p < 0.05), although it was lower than in the control group (4.00 ± 0.12) (p < 0.05). Additionally, SSV 6 M led to the same DNA integrity (97.0 ± 0.7%) as in the control group (99.4 ± 0.3%). These collective findings suggest that the combination of SSV with 6 M cryoprotectants is the most efficient for the cryopreservation of testes from prepubertal collared peccaries. Full article