Cryobiology最新文献

Cryopreservation of fish gonadal tissue is an important technique for preserving genetic variability. However, this technique involves the use of cryotubes, plastic containers with low degradability that are expensive and difficult to obtain in certain parts of the world. Therefore, this study aimed to evaluate the efficiency of gelatin and hypromellose hard capsules as a sustainable and accessible alternative container to the cryotube for vitrification of zebrafish (Danio rerio) gonadal tissue. The gonadal tissues (testicular or ovarian) were vitrified in cryotubes, hard-gelatin, and hard-hypromellose capsules. Gelatin capsules exhibited comparable efficacy to cryotubes in preserving spermatogonia viability (33.03 ± 10.03 % and 37.96 ± 8.35 %, respectively), whereas hypromellose capsules showed decreased viability (18.38 ± 2.09 %). Immature oocyte viability remained unaffected by the capsule materials, with no difference compared to cryotubes at all oocyte stages (Primary Growth: p < 0.0001; Cortical Alveolar: p < 0.0001; Vitellogenic: p < 0.0001). Mitochondrial activity and lipid peroxidation demonstrated no difference among cryotubes and capsules for both gonadal tissues. However, antioxidant activity was notably higher in gelatin capsules (Testes: 147.2 ± 32.32 μg; Ovary: 87.98 ± 10.91 μg) than in cryotubes (Testes: 81.04 ± 26.05 μg; Ovary: 54.35 ± 11.23 μg) and hypromellose capsules (Testes: 62.36 ± 17.10 μg; Ovary: 63.96 ± 7.51 μg), likely due to the inherent antioxidant properties of gelatin. The results obtained in this study demonstrate that the cryotube can be replaced by gelatin capsules for vitrification of both gonadal tissues of zebrafish, being a sustainable and accessible alternative as it is a low-cost and environmentally friendly container.

The paper discusses the impact of cell size on cytotoxicity and expansion lysis during the osmotic excursions resulting from the contact of hMSCs from UCB with Me2SO. It builds upon the mathematical model recently presented by the authors, which pertains to a population of cells with uniform size. The objective is to enhance the model's relevance by incorporating the more realistic scenario of cell size distribution, utilizing a Population Balance Equations approach. The study compares the capability of the multiple-sized model to the single-sized one to describe system behavior experimentally measured through cytofluorimetry and Coulter counter when, first, suspending hMSCs in hypertonic solutions of Me2SO (at varying osmolality, system temperature, and contact times), and then (at room temperature) pelleting by centrifugation before suspending the cells back to isotonic conditions. Simulations demonstrate that expansion lysis and cytotoxic effect are not affected by cell size for the specific system hMSCs/Me2SO, thus confirming what was found so far by the authors through a single-size model. On the other hand, simulations show that, when varying the adjustable parameters of the model that are expected to change from cell to cell lineages, expansion lysis is sensitive to cell size, while cytotoxicity is not, being mainly influenced by external CPA concentration and contact duration. More specifically, it is found that smaller cells suffer expansion lysis more than larger ones. The findings suggest that different cells from hMSCs may require a multiple-sized model to assess cell damage during osmotic excursions in cryopreservation.

Spermatogonia cryopreservation can be a strategy for future conservation actions. The neotropical Siluriformes Pseudopimelodus mangurus was already classified as vulnerable on the Red List of Threatened Species. P. mangurus spermatogonial cells were isolated, assessed, and cryopreserved. Fragments of the testis were enzymatically dissociated, purified using Percoll density gradient, and submitted to differential plating. Fractionated cells were evaluated by microscopy, ddx4 (vasa) relative expression, and alkaline phosphatase activity. Cryopreservation was conducted using ethylene glycol, glycerol, dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), and propanediol at 1 M, 1.5 M, and 2 M. Cell viability was evaluated and cell concentration was determined. Cell fractions from 20 % and 30 % Percoll gradient bands showed the highest concentrations of spermatogonia. The fraction mix showed 54 % purity and 93 % viability. After differential plating, 60 % purity and 92 % viability were obtained. Spermatogonial cells showed high alkaline phosphatase activity compared to spermatocytes and spermatids. The relative spermatogonial ddx4 expression from the Percoll density gradient was about twice as high as in samples from the testis and the differential plating. The increased ddx4 expression indicated the enrichment of spermatogonial cells by density gradient step and dead cells expressing ddx4 in differential plating, or ddx4 decreasing expression during cell culture. For this reason, cells from the Percoll gradient were chosen for cryopreservation. Propanediol at 1 M demonstrated the best condition for spermatogonial cell cryopreservation, presenting 98 % viability, while dimethylacetamide at 2 M represented the least favorable condition, with approximately 47 % viability. These findings are essential for P. mangurus spermatogonial cell cryopreservation, aiming to generate a spermatogonia cryobank for future conservation efforts.

Our objective was to investigate the predictive value of monocyte-related inflammatory factors, including monocyte to high-density lipoprotein cholesterol ratio (MHR) and monocyte to lymphocyte ratio (MLR), for the recurrence of atrial fibrillation (AF) after cryoablation in AF patients. The 570 patients who underwent cryoablation were divided into AF recurrence group and non-recurrence group based on follow-up results. The multivariable logistic regression analysis was used to evaluate the effect of MHR and MLR on AF patients. The AF-free survival status of patients was tested by Kaplan-Meier method. ROC analysis was performed to assess the predictive value of MHR and MLR for post-ablation recurrence of AF. A total of 113 (19.8 %) patients relapsed, while 457 patients (80.2 %) had no AF recurrence during follow up. Patients with AF recurrence had higher MHR values (0.37 ± 0.14 vs. 0.33 ± 0.14; P = 0.004) and higher MLR values (0.49 ± 0.32 vs. 0.18 ± 0.07; P < 0.001) compared to those without AF recurrence. MHR≥0.34 combined with MLR≥0.24 (HR = 9.979, 95 % CI: 6.070–16.407, P < 0.001) was an independent factor for predicting AF recurrence after cryoablation in patients by logistic regression analysis. The ROC analysis showed that the AUC for the combination of the MHR and MLR variables was 0.974 (95 % CI: 0.962–0.985) and had the highest diagnostic sensitivity (97.4 %). Elevated baseline values of the monocyte-related inflammatory factors, MHR and MLR, have a certain predictive value for increased AF recurrence after cryoablation.

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to −4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.

Cryoprotective agents play a critical role in minimizing cell damage caused by ice formation during cryopreservation. However, high concentrations of CPAs are toxic to cells and tissues. Required concentrations of CPAs can be reduced by utilizing higher cooling and warming rates, but insight into the thermophysical properties of biological solutions in the vitrification method is necessary for the development of cryopreservation protocols. Most studies on thermophysical properties under ultra-rapid cooling conditions have been qualitatively based on visualization. Differential scanning calorimetry methods are ideal for studying the behavior of biomaterials in various freezing conditions quantitatively and accurately, though previous studies have been predominantly restricted to slower cooling rates. Here, we developed an ultra-rapid cooling method for DSC that can achieve minimal cooling rates exceeding 2000 °C/min. We investigated the thermophysical vitrification behavior of ternary solutions of phosphate buffer saline (1X), dimethyl sulfoxide or glycerol and ice blocking polymers (X-1000 or Z-1000). We quantified the impact of solute concentration on ice crystal formation during rapid cooling. Our findings support the expectation that increasing the solute concentration reduces the amount of ice formation, including devitrification. Devitrification increases from 0 % to 40 % (v/v) Me₂SO and then reduces significantly. The relative amounts of devitrification to the total ice formation are 0 %, 60 %, 0 % in 20 %, 40 %, 60 % (v/v) Me₂SO, and 2 %, 48 %, 49 % in 20 %, 40 %, 60 % (v/v) glycerol, respectively. The results suggest that at low concentrations, such as below 20 % (v/v) for Me₂SO or glycerol, increasing the warming rate after ultra-rapid freezing is not essential to eliminate devitrification. Furthermore, ice blocking polymers do not reduce ice formation substantially and cannot eliminate devitrification under ultra-rapid cooling conditions. In conclusion, our results provide insights into the impact of solute concentration on ice formation and devitrification during rapid cooling, which can be practical for optimizing cryopreservation protocols.

It is thought that surface melting and puffing of freeze-dried amorphous materials are related to the difference between the surface temperature (T_sur) and freeze-concentrated glass transition temperature (T_g’) of the materials. Although T_g’ is a material-specific parameter, T_sur is affected by the type and amount of solute and freeze-drying conditions. Therefore, it will be practically useful for preventing surface melting and puffing if T_sur can be calculated using only the minimum necessary parameters. This study aimed to establish a predictive model for the surface melting and puffing of freeze-dried amorphous materials according to the calculated T_sur. First, a T_sur-predictive model was proposed under the thermodynamic equilibrium assumptions. Second, solutions with various solute mass fractions of sucrose, maltodextrin, and sucrose-maltodextrin mixture were prepared, and three material-specific parameters (T_g’, unfrozen water content, and true density) were experimentally determined. According to the proposed model with the parameters, the T_sur of the samples was calculated at chamber pressures of 13, 38, and 103 Pa. The samples were freeze-dried at the chamber pressures, and their appearance was observed. As expected, surface melting and puffing occurred at calculated T_sur > T_g’ with some exceptions. The water activity (a_w) of the freeze-dried samples increased as the T_sur − T_g’ increased. This will have resulted from surface melting and puffing, which created a covering film, thereby preventing subsequent dehydration. The observations suggest that the proposed model is also useful for predetermining the drying efficiency and storage stability of freeze-dried amorphous materials.

Echocardiography-guided percutaneous intramyocardial septal radiofrequency ablation (PIMSRA, Liwen procedure) is a novel treatment option for hypertrophic obstructive cardiomyopathy (HOCM). The safety and feasibility of using this procedure for cryoablation are unknown. We aimed to investigate the feasibility and safety of echocardiography-guided percutaneous intramyocardial septal cryoablation (PIMSCA) for septal thickness reduction in a canine model. Eight canines underwent PIMSCA, and had electrocardiography, echocardiography(ECG), myocardial contrast echocardiography (MCE), serological and pathological examinations during the preoperative, immediate postoperative, and 6-month follow-up. All eight canines underwent successful cryoablation and continued to be in sinus rhythm during ablation and without malignant arrhythmias. MCE showed that the ablation area had decreased myocardial perfusion after the procedure. Troponin I levels were significantly elevated [0.010 (0.005, 0.297) ng/mL vs. 3.122 (1.152, 7.990) ng/mL, p < 0.05)]. At 6-month follow-up after the procedure, all animals were alive, with thinning of the interventricular septum (7.26 ± 0.52 mm vs. 3.86 ± 0.29 mm, p < 0.05). Echocardiography showed no significant decrease in the left ventricular ejection fractions (LVEF) (54.32 ± 2.93 % vs. 54.70 ± 2.47 %, p > 0.05) or changes by pulse-wave Doppler E/A (1.17 ± 0.43 vs. 1.07 ± 0.43, p > 0.05), E/e' (8.09 ± 1.49 vs. 10.05 ± 2.68, p > 0.05). Pathological findings proved the effectiveness of cryoablation in myocardial tissues. We observed pericardial effusions and premature ventricular complexes (PVCs) associated with the procedure. Our findings provided preliminary evidence of the safety and feasibility of PIMSCA in reducing interventricular septum, which provides a potentially new treatment option for HOCM.

Cryopreservation of goat spermatozoa is challenging due to several factors, including one of the most essential, i.e., oxidative stress. It is particularly essential in goat semen due to its scanty ejaculate volume and high sperm concentration. This leaves a narrow sperm-to-seminal plasma ratio owing to marginal antioxidant support; moreover, semen extension further dilutes the antioxidant level, leading to an imbalance of oxidant-antioxidant equilibrium. The present study aimed to evaluate the effect of quercetin on curtailing oxidative stress and its reflection on the post-thaw survivability and membrane integrity of goat spermatozoa. For this study, six bucks were selected. Six ejaculates from each buck totaling 36 ejaculates were collected, which were then split into five parts; furthermore, each part was added with a semen extender having a particular concentration of additive. Group C without quercetin and T₁ containing Vitamin E at 3 mmol/mL were considered the control and positive control respectively, whereas T₂, T₃, and T₄ contain 10, 20, and 30 μmol/mL of Quercetin respectively. The final sperm concentration of each group was kept at 200 × 10⁶ spermatozoa/mL. All groups were subjected to equilibration at 4 °C for 4 h, then filled in French mini (0.25 mL) straws, followed by sealing and cryopreservation. Samples after 72 h of cryopreservation were subjected to evaluation of plasma membrane integrity and viability through staining, acrosomal integrity, and mitochondrial membrane activity through flow cytometry. Evaluation of sperm kinematics as well as the oxidant-antioxidant status of sperm (ROS and nitric oxide) and seminal plasma (SOD, CAT, GPx, FRAP, and lipid peroxidation through MDA estimation) were also carried out. Quercetin, when supplemented at 20 μmol/mL in buck semen extender, significantly (p < 0.01) improved cryopreserved sperm functions in terms of plasma membrane integrity, viability, acrosomal integrity, mitochondrial membrane activity, and sperm kinematics of buck semen. Similarly, Quercetin supplementation at 20 μmol/mL significantly reduced reactive oxygen and nitrogen species (RONS) in sperm and improved the antioxidant status of seminal plasma, which was indicated by reduced oxidative damage and improved the antioxidant status of buck semen. In conclusion, Quercetin at 20 μmol/mL reduced oxidative stress, improved semen antioxidant status, and improved sperm membranes integrity and kinematics.

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.