BACKGROUND: Ice seeding enhances extracellular ice formation, minimizes intracellular damage and improves cell viability. But the real-time evaluation of ice-seeding within cryovials is problematic, and it is often assumed that the solution will be sufficiently ice-seeded if an ice-seeding process is used. OBJECTIVE: To construct and evaluate a real-time sensor for monitoring the ice seeding process via the detection of the solution capacitance. MATERIALS AND METHODS: The sensor used consisted of conductive plates and an insulating sleeve, which measured the capacitance in aqueous solutions. The capacitance was recorded over a frequency of 1 kHz to 5 MHz, and temperature was recorded simultaneously to monitor the freezing dynamics. RESULTS: The constructed sensor was able to detect significant changes in capacitance before and after freezing. The optimal monitoring frequency was 1 kHz, which registered the largest capacitance changes. Real-time monitoring confirmed the crystallization process, with the rapid increase in capacitance indicating the completion of ice formation. CONCLUSION: The capacitance sensor permits the accurate real-time monitoring of ice seeding and can be used for improving the outcomes of cryopreservation and freeze-drying. The refining of the sensor and coupling with ultrasonic technology will maximize its efficiency and reliability.

This review recounts the advances in cryopreservation technologies of red blood cells (RBC). The limitations of traditional glycerol-based methods and the latest developments in cryopreservation with no or less glycerol are elaborated. Although the use of glycerol at high-concentrations effectively prevents ice crystal formation, the associated osmotic stress damages RBC. Moreover, the cumbersome and time-consuming deglycerolization process not only increases operational costs, but also poses the risks of contamination and additonal cell damage, thereby limiting the application of frozen blood for emergency situations. To overcome these challenges, novel strategies have focused on rapid freezing with no or less glycerol, new permeable cryoprotectants and the synergistic use of non-permeable macromolecular/saccharide protectants (such as trehalose and hydroxyethyl starch). These approaches aim to achieve more efficient and safer preservation through multi-faceted protective mechanisms via the reduction of ice crystal damage, osmotic stress and oxidative damage. New strategies demonstrate significant advantages in streamlining processes (eliminating or simplifying deglycerolization steps), improving cell recovery and quality (low hemolysis) and enhancing in vivo efficacy. However, challenges such as long-term storage stability, scale-up production costs, standardization and regulatory approval remain critical issues to be addressed before clinical translation can be realized. Future research needs to focus on optimizing cryoprotectant formulations, elucidating molecular mechanisms, establishing a standardized quality control system and clinical validation, thereby revolutionizing blood inventory management.

BACKGROUND: Sperm cryopreservation is essential for assisted reproductive technologies, yet current methods face challenges regarding cryoprotectant toxicity and protocol standardization. OBJECTIVE: To develop an optimized cryopreservation system by investigating cryoprotective mechanisms and establishing standardized freezing protocols without using any animal-derived material. MATERIALS AND METHODS: Thermodynamic analysis was performed to evaluate the cryoprotectant efficacy of glycerol (i.e., its ice crystal inhibition properties). An optimized formulation combining 10% glycerol with recombinant human serum albumin (rHSA) was developed to replace traditional egg yolk components. A novel freezing protocol incorporating gradient cooling and precise nucleation at-8.5 degrees C was implemented using a custom-designed automated cryopreservation device (+/- 1 degrees C precision). RESULTS: The optimized formulation significantly reduced the melting enthalpy while it maintained post-thaw sperm motility of 75.4 +/- 3.2% and DNA fragmentation index <15%. The freezing protocol achieved 82.6 +/- 4.1% recovery rate and reduced ice crystal formation by 62.3%. Preclinical study demonstrated significant improvements versus conventional methods (increased motility by 27.8% and enhanced DNA integrity by 34.6%, both p < 0.01). CONCLUSION: The study established a standardized, pathogen-free cryopreservation system with demonstrated efficacy in preserving sperm quality. The findings provide both technical solutions for clinical practice and fundamental insights into cryoprotective mechanisms.

Irreversible electroporation (IRE) is an emerging tumor ablation modality that employs high-intensity pulsed electric fields to disrupt cell membranes. However, achieving complete tumor destruction while preserving adjacent critical structures remains a major challenge. In this study, we investigated whether high-aspect-ratio silver nanowires (AgNWs) could enhance IRE efficacy in a three-dimensional hydrogel-based cell model. We systematically evaluated cell viability, ablation area, conductivity, and temperature under various pulse parameters and AgNW concentrations. Increased electric field strength, longer pulse durations, and higher pulse numbers all potentiated the cytotoxic effect. Under identical pulsing conditions, the addition of 70 μg/mL AgNWs expanded the ablation zone by 42.2% ± 7.5%, allowing lower-energy pulse protocols to achieve ablation comparable to that of higher-energy settings. This enhancement was not attributable to changes in bulk conductivity; rather, the nanowires appeared to amplify the local electric field in the vicinity of cells, thereby boosting treatment sensitivity without introducing significant thermal effects. Collectively, these in vitro findings suggest that AgNWs can potentiate IRE ablation at reduced energy levels, offering a promising adjuvant strategy. Nevertheless, further validation through in vivo studies and comprehensive long-term safety assessments is essential prior to any clinical translation.

Cervical cancer remains a prominent cause of cancer-related mortality among women worldwide because of chronic infection with high-risk human papillomavirus (HPV) and disparate access to prevention and treatment. The current research evaluates the anticancer activity of Gypenoside XVII, a bioactive saponin of Gynostemma pentaphyllum, in HeLa cells as a model of cervical cancer. MTT, Annexin V-PI, and Hoechst 33342 assays showed dose-dependent growth inhibition with typical apoptotic morphology. Flow cytometry revealed G0/G1 cell-cycle arrest, while pathway interrogation revealed participation of mitochondrial and death-receptor cascades, in agreement with caspase-9 and caspase-8 activation, respectively. Collectively, these findings position Gypenoside XVII as a natural-product bioactive with potential both as an anticancer lead and as a functional-food ingredient, deserving of further preclinical development.

Emerging evidence demonstrates that ubiquitination consistently affects the freezing tolerance of fully hydrated seeds. In this study, PN (parthenolide), the ubiquitination inhibitor, was applied in seed imbibition of Brassica oleracea followed by slow cooling (−3 °C h⁻1). Transcriptomic analysis (RNA-seq) identified the protein processing in the endoplasmic reticulum (ER) pathway as the most enriched pathway in PN-treated samples, with pronounced alterations in ER-associated degradation (ERAD) functionality. Real-time PCR further showed that the mRNA level of heat shock proteins (HSPs), critical components of ERAD, was induced during slow cooling in the control treatment but was systemically decreased by PN treatment. Given that murine double minute2 (MDM2) is a known target of PN in Homo sapiens, we identified three MDM2-like homologs in Brassica oleracea through protein sequence homology searches using human MDM2 as the query. Real-time PCR demonstrated that BoMDM2 like-2 (named as BoHNR, Brassica oleracea HSP-Negative Regulation) was significantly increased under PN imbibition in slow cooling treatment. Knockdown of BoHNR by RNAi was associated with increased mRNA of BoHSPs and higher freezing tolerance of hydrated seeds. Altogether, modifying the function of BoHNR can change the level of HSPs and freezing tolerance of hydrated Brassica seeds.

Vitrification (ice-free) is considered more effective than slow freezing for oocyte cryopreservation. However, high concentrations of toxic cryoprotective agents (CPAs) are typically required, followed by multi-step washing to remove CPAs, risking osmotic shock and impairing oocytes development. This study aimed to establish a hydrogel encapsulation strategy using a microfluidic system to improve vitrification outcomes. Three different sizes of oocyte-loaded sodium alginate hydrogel microspheres (OHMs) ( 262 µm, 193 µm, and 156 µm) were prepared using an optimized three-channel microfluidic system. The effects of particle size, vitrification solutions (VS1, VS2, VS3, and VS4), CPA loading durations (4, 8, and 12 min), and warming/dilution procedures (one-step, two-step, and three-step) on oocyte vitrification were systematically evaluated. OHMs with a particle size of 262 µm, loaded with VS3 (containing 10

This research proposes a novel combination therapy that utilizes ultrasound-assisted cryoablation to enhance the lethality of cryoablation in Hepatocellular Carcinoma (HepG2) cells. Ultrasound is used to induce ice nucleation, followed by thawing at a set temperature. The efficacy of this approach was evaluated by assessing cell survival rates in hepatocellular carcinoma cells using Acridine Orange/ Propidium Iodide (AO/PI) staining and the Cell Counting Kit-8 (CCK-8), comparing ultrasound-assisted cryoablation with cryoablation alone. At a cooling rate of 10 degrees C/min and lower nucleation temperatures, a significant reduction in cell survival rate was observed (15.60 +/- 2.60 %). Cryomicroscopic observations revealed that enhanced intracellular and extracellular ice formation led to increased cellular damage and higher cryoablation lethality. This innovative therapy provides a promising approach for localized treatment and highlights the potential of cryoablation devices incorporating ultrasound- induced nucleation.

Prediction of water status in post-harvested agriculture products enduring drying is critical to maintain storage conditions. This study focused on the efficiency of multispectral imaging a novel nondestructive analytical tool by combining various machine-learning models such as Feedforward Neural Network (FNN), Decision Tree Regression, Support Vector Regression, and k-nearest neighbors in the prediction of water fractions during freeze-drying of mushrooms. Spectra from multispectral imaging of the Vis-NIR (405-970 nm) region were combined with machine learning models for the quantification of free water (FW), immobilized water (IM), bound water (BW) and total water (TW) during freeze-drying (FD) of shiitake mushrooms. Water distribution tests through low-field nuclear magnetic resonance demonstrated that 36 h of drying sublimates 90.55% freezable water. The modeling approach performed well, and FNN was found to be the best compared to the others. Its prediction efficiency was 97.77% and 95.95% in BW and TW, respectively. In terms of root mean square error, this model obtained the lowest prediction errors compared to the rest of the models for all water fractions. However, the FNN model prediction deviation is determined with the best bias value of 0.1312 for FW. This study provides an excellent platform in predicting the water status and food quality with a rapid and nondestructive multispectral Vis-NIR spectroscopic approach during drying. The techniques successfully handled the complex spectral data when combined with chemometrics and could be useful in the future for the detection of the chemical composition of agricultural products.

BACKGROUND: Cryopreservation faces challenges from intracellular ice formation (IIF) and solution damage, influenced by cooling rates. Ice seeding mitigates supercooling risks, yet traditional methods like contact nucleation are impractical. Ultrasonic ice seeding emerges as a promising alternative, leveraging cavitation to induce nucleation without disrupting temperature stability. OBJECTIVE: To evaluate ultrasonic ice seeding’s efficacy in reducing IIF and enhancing post-thaw survival of Hep-G2 liver cancer cells. MATERIALS AND METHODS: A cryogenic microscopy platform and ultrasonic device (40 kHz, 500W) were constructed. Hep‐G2 cells were cryopreserved using three methods including 10% DMSO as cryoprotectant: cell freezing container (10%‐CFC), programmable controlled-rate freezing (10%‐PC), and programmable freezing with ultrasonic ice seeding (10%‐PC+UIC). Survival rates were assessed via AOPI staining, while intracellular ice formation and cell morphology were analyzed microscopically. RESULTS: The 10%‐PC+UIC group achieved the highest survival rate (93.29 ± 1.20%), surpassing 10%‐PC (90.32 ± 1.60%) and 10%‐CFC (80.33 ± 3.36%). Ultrasonic ice seeding reduced intracellular ice occurrence to 6.43% versus 20.71% in spontaneous crystallization. Microscopy revealed smaller, uniform ice crystals and controlled cell dehydration/rehydration dynamics, minimizing membrane damage. CONCLUSIONS: Ultrasonic ice seeding significantly improves HepG2 cell survival by reducing IIF through controlled nucleation. Its non‐contact, contamination‐free operation offers practical advantages for large-scale cryopreservation, outperforming traditional methods. This technique holds potential for broader applications in biological and medical storage protocols.

Peripheral blood mononuclear cells (PBMCs) are important immune cells. However, traditional slow-freezing methods delay the proliferation of PBMCs and damage T-cell subsets. Therefore, there is an urgent need to develop an alternative cooling procedure that can effectively preserve the viability and function of PBMCs. In this study, we optimized the cryopreservation of PBMCs using ultrasonic ice seeding and analyzed post-cryopreservation T-cell subtypes using flow cytometry. An ultrasonic ice-seeding apparatus was constructed to achieve contactless ice seeding by combining an ultrasonic generating device and a controlled-rate freezer. The results showed that the cooling procedure involving ultrasonic ice seeding exhibited superior efficacy compared to the conventional slow-freezing approach. Following cryopreservation, the viability and cumulative proliferation of PBMCs were 94.97% and 204.47%, respectively. The proportion of naive T cells (Tn) after cryopreservation and thawing accounted for up to 18.35%. By incorporating ultrasonic ice seeding, the optimized cryopreservation procedure enhanced the post-thaw viability, cumulative proliferative capacity, and proportion of T-cell subtypes in PBMCs, providing a novel and effective approach for PBMC cryopreservation.

Freeze-drying (FD) of foods significantly alters nutritional composition which are strongly related to water status. This study aims to compare the prediction, stability and robustness of more advanced machine learning models such as genetic algorithms (GA) and whale optimization (WO) to overcome, enhance the stability and error limitations of partial least square (PLS), back propagation neural network (BPNN), and support vector machine (SVM) with complex spectra. Multispectral imaging spectra of visible-near infrared (Vis-NIR) (405-970 nm) of water fractions of Lentinus edodes coupled GA and WO models. Low-field nuclear magnetic resonance (LF-NMR) showed that FD removed 90.55 % of total water (TW) within 36 h of drying. However, a rapid sublimation rate was observed during first 12 h (74.20 %), while only 16.35 % of water evaporated in next 24 h due to the presence of less amount of free water (FW). Advancement of models improved the stability and prediction accuracy of water status. Developed models showed more precise prediction and robustness in terms of ratio of prediction to deviation (RPD). For TW, GA and WO of BPNN found the most improved and stable model R2p = 0.9799 and 0.9556, respectively. GA-SVM has shown excellent results in IW with R2p = 0.9432 and lower RMSEP = 6.376. Additionally, GA-BPNN obtained excellent RPD of 5.0545, confirming robustness and stability with complex spectral data. In conclusion, research provides an excellent opportunity for integrating advanced optimization approached and identify the complex Vis-NIR spectroscopic date with potential expansion in future work in assessment of water status during processing.

Developing bioartificial liver and hepatocyte transplantation technology causes increasing hepatocyte cell demand. Effective long-term hepatocyte cell preservation methods are necessary to promote. Progressive cooling is a key preservation technology for cell banks. However, the cell solution needs to be supercooled in a slow freezing process. The high degree of supercooling possibly induces uncontrollable intracellular ice formation. This work designs an ultrasonic ice-seeding system for L-02 hepatocyte cell preservation, reducing supercooling and improving cell survival rate. The effect of ultrasonic intensities on the hepatocyte’s survival rate was investigated and optimized. The results prove the calorimetric method can efficiently measure the ultrasonic intensity dissipated in the hepatocyte cell preservation solution. When the ultrasonic intensity is 0.0329 W/cm2 ∼ 0.4316 W/cm2, the hepatocyte survival rate is over 90%. There is no significant difference between experiment groups (p < 0.05) when the ultrasonic intensity is larger than 0.4316 W/cm2. The hepatocyte cell survival rate reduced significantly with the increase of ultrasonic intensity. The 7-day hepatic function indicator experiment results indicate that the ultrasonic ice seeding has the weakest impact on hepatocyte cells in the four groups. The secretion of urea, albumin and glucose proved that ultrasonic ice seeding technology does not affect cell secretion and has an enormous advantage in cryopreservation. It can be widely applied to cell freezing fields.

Oocyte vitrification has a wide range of applications in assisted reproduction and fertility preservation. It requires precise cryoprotectant agents (CPAs) loading and removal sequences to alleviate osmotic shock, which requires manual manipulation by an embryologist. In this study, a microfluidic system was developed to facilitate the precise adjustment of the CPA concentration around the oocyte by linear loading and removal of CPA. In addition, the microfluidic-based automated vitrification (MAV) device combines CPA loading/removal process, with vitrification process, thereby achieving automated oocyte vitrification. Oocytes were vitrified by Cryotop/QC manual method and MAV method. The results showed that the survival, cleavage, and blastocyst rates of oocytes were 80.44, 54.17, and 32.95

The freeze-drying is a technology that preserves biological samples in a dry state, which is beneficial for storage, transportation, and cost saving. In this study, the bovine pericardium was treated with a freeze-drying protectant composed of polyethylene glycol (PEG) and trehalose (Tre), and then freeze-dried. The results demonstrated that the mechanical properties of the pericardium treated with PEG + 10% w/v Tre were superior to those of the pericardium fixed with glutaraldehyde (GA). The wet state water content of the rehydrated pericardium, determined using the Karl Fischer method, was (74.81 ± 1.44)%, which was comparable to that of the GA-fixed pericardium. The dry state water content was significantly reduced to (8.64 ± 1.52)%, indicating effective dehydration during the freeze-drying process. Differential scanning calorimetry (DSC) testing revealed that the thermal shrinkage temperature of the pericardium was (84.96 ± 0.49) ℃, higher than that of the GA-fixed pericardium (83.14 ± 0.11) ℃, indicating greater thermal stability. Fourier transform infrared spectroscopy (FTIR) results showed no damage to the protein structure during freeze-drying. Hematoxylin and eosin (HE) staining demonstrated that the freeze-drying process reduced pore formation, prevented ice crystal growth, and resulted in a tighter arrangement of tissue fibers. The frozen-dried bovine pericardium was subjected to tests for cell viability and hemolysis rate. The results revealed a cell proliferation rate of (77.87 ± 0.49)%, corresponding to a toxicity grade of 1. Additionally, the hemolysis rate was (0.17 ± 0.02)%, which is below the standard of 5%. These findings indicated that the frozen-dried bovine pericardium exhibited satisfactory performance in terms of cytotoxicity and hemolysis, thus meeting the relevant standards. In summary, the performance of the bovine pericardium treated with PEG + 10% w/v Tre and subjected to freeze-drying could meet the required standards.

Ovarian tissue cryopreservation (OTC) is currently the exclusive choice for preserving fertility in both young girls before reaching puberty and young women who require immediate chemotherapy. Ovarian tissue transplantation has proven to be effective in restoring hormonal cycles and fertility. However, in certain cancer cases, there is a potential risk of inadvertently reintroducing malignant cells when transplanting cryopreserved ovarian tissue. Therefore, the use of an artificial ovary as an innovative and complementary approach allows for the development of isolated follicles, facilitates oocyte maturation and ovulation, and can partially restore endocrine function. This paper presents a comprehensive overview of techniques used to preserve fertility in natural ovarian tissues, including slow freezing, vitrification and hydrogel encapsulation methods. Additionally, it reviews fertility preservation techniques for artificial ovarian tissues, such as strategies involving hydrogel-encapsulated follicle, scaffolding for constructing ovarian microtissues, and 3D printing engineering. Lastly, this article explores current challenges and difficulties encountered in preserving ovarian tissue fertility, while also anticipating future trends in development, making it a valuable reference for the implementation of ovarian tissue fertility preservation.

The hydrolysis of globulins is supposedly involved in freezing tolerance of hydrated lettuce seeds. Considering that globulins are constituted by two subunits with S-S bonds, lettuce seeds was imbibed in the aqueous solution of 5,5′-Dithiobis (2-nitrobenzoicacid )(DTNB), the Ellman’s reagent which can react with free sulfhydryl side chain of cysteine to form an S-S bond between the protein and thionitrobenzoic acid (TNB) residue. According to this study, DTNB improved seed freezing tolerance, as shown by the greater viability of hydrated seeds under programmed cooling (-1°C min− 1-) treatment. Calorimetric study showed delayed ice formation in DTNB treatment upon programmed cooling (-22.0°C in DTNB-treated embryos vs -19.3°C in control embryos). Two-dimensional (Two-D) electrophoresis showed that DTNB promoted the de-polymerization of seed storage globulins in cooling treatment. The globulins and their hydrolyzed polypeptides were extracted and the ice formation mode of the polypeptides with different molecular weight was detected with differential scanning calorimeter (DSC), which indicated that the smaller peptides gave higher onset temperature of ice formation and higher enthalpy of heat release. The concentration of disulfides bond and sulfhydryl groups in globulins was detected which indicated that DTNB could promote the formation of sulfhydryl groups, and reduce the amount of disulfide bonds under programmed cooling treatment. Moreover, DTNB increased the hydroxyl radical scavenging rate, which indicated that DTNB promotes the anti-oxidation of hydrated seeds.

Moisture content is the key factor that affects the freezing preservation of seeds. Previous studies have indicated that hydrolysis of seed storage globulins can enhance the freezing tolerance of hydrated seeds. Seed globulins are constituted by two subunits connected via disulfide bonds(-S-S-). As a donor for H2S, NaHS can act as a nucleophile to attack the disulfide bond of proteins and promote S-persulfidation. In this study, hydrated brassica (Brassica oleracea) seeds were treated with NaHS (5 mM) to confirm the role of the reduction the disulfide bonds of seed globulins on the freezing tolerance of hydrated seeds. The results indicated that NaHS treatment increased seed viability after slow cooling (NaHS vs. control: 55% vs. 38.3%) (P<0.05). Differential scanning calorimetry confirmed that supercooling occurred in embryos after NaHS treatment. NaHS treatment significantly increased the content of sulfhydryl groups in seed storage globulin, suggesting that NaHS promoted the reduction of disulfide bonds during slow cooling. Two-dimensional electrophoresis showed that NaHS promoted the depolymerization of seed storage globulins and accumulation of small polypeptides under slow cooling treatment. NaHS treatment also enhanced the levels of ascorbate and glutathione, which together make up an important antioxidant defense system against oxidative damage.