Cryoablation with KCl Solution Enhances Necrosis and Apoptosis of HepG2 Liver Cancer Cells.

Cryoablation has become a valuable treatment modality for the management of liver cancer. However, one of the major challenges in cryosurgery is the incomplete cryodestruction near the edge of the iceball. This issue can be addressed by optimizing cryoablation parameters and administering thermotropic drugs prior to the procedure. These drugs help enhance tumor response, thereby strengthening the destruction of the incomplete frozen zone in liver cance. In the present study, the feasibility and effectiveness of a thermophysical agent, KCl solution, were investigated to enhance the cryodestruction of HepG2 human liver cancer cells. All cryoablation parameters were simultaneously optimized in order to significantly improve the effect of cryoablation, resulting in an increase in the lethal temperature from - 25 °C to - 17 °C. Subsequently, it was found that the application of KCl solution prior to freezing significantly decreased cell viability post-thaw compared to cryoablation treatment alone. This effect was attributed to the eutectic effect of KCl solution. Importantly, it was found that the combination of KCl solution and freezing was less effective when applied to LO2 human liver normal cells. The data revealed that the ratio of mRNA levels of Bcl-2 and bax decreased significantly more in HepG2 cells than in LO2 cells when cryoablation was used with KCl solution. In conclusion, the results of this study demonstrate the effectiveness of KCl solution in promoting cryoablation and describe a novel therapeutic model for the treatment of liver cancer that may distinguish between cancer and normal cells.

Quantitative phosphoproteomics explain cryopreservation-induced reductions in ram sperm motility.

Sperm cryopreservation decreases motility, probably due to changes in protein phosphorylation. Our objective was to use quantitative phosphoproteomics for systematic comparative analyses of fresh versus frozen-thawed sperm to identify factors causing cryo-injury. Ejaculates were collected (artificial vagina) from six Dorper rams, pooled, extended, and frozen over liquid nitrogen. Overall, 915, 3382, and 6875 phosphorylated proteins, phosphorylated peptides, and phosphorylation sites, respectively, were identified. At least two modified sites were present in 57.94% of the 6875 phosphosites identified, of which AKAP4 protein contained up to 331 modified sites. There were 732 phosphorylated peptides significantly up-regulated and 909 significantly down-regulated in frozen-thawed versus fresh sperm. Moreover, the conserved motif [RxxS] was significantly down-regulated in frozen-thawed sperm. Phosphorylation of sperm-specific proteins, e.g., AKAP3/4, CABYR, FSIP2, GSK3A/B, GPI, and ODF1/2 make them potential biomarkers to assess the quality of frozen-thawed ram sperm. Furthermore, these differentially phosphorylated proteins and modification sites were implicated in cryopreservation-induced changes in sperm energy production, fiber sheath composition, and various biological processes. We concluded that abnormal protein phosphorylation modifications are key regulators of reduced sperm motility. These novel findings implicated specific protein phosphorylation modifications in sperm cryo-injury. SIGNIFICANCE: This study used phosphorylated TMT quantitative proteomics to explore regulation of epigenetic modifications in frozen-thawed ram sperm. This experiment demonstrated that ram sperm freezing affects phosphorylation site modifications of proteins, especially those related to functions such as sperm motility and energy production. Furthermore, it is important to link functions of phosphorylated proteins with changes in sperm quality after freezing and thawing, and to clarify intrinsic reasons for sperm quality changes, which is of great importance for elucidating mechanisms of sperm freezing damage. Based on these protein markers and combined with cryoprotectant design theory, it provides a theoretical basis and data reference to study sperm cryoprotectants.

Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers.

The cryopreservation of endothelial cell monolayers is an important step that bridges the cryopreservation of cells in suspension to that of tissues. Previous studies have identified clear distinctions in freezing mechanisms between cells in suspension and in monolayers, as well as developed novel protocols for monolayer cryopreservation. Recently, our group has shown that human umbilical vein endothelial cell (HUVEC) and porcine corneal endothelial cell (PCEC) monolayers grown on Rinzl plastic substrate can be cryopreserved in 5% dimethyl sulfoxide, 6% hydroxyethyl starch, and 2% chondroitin sulfate, following a slow-cooling protocol (-1 °C/min) with rapid plunge into liquid nitrogen from -40 °C. However, membrane integrity assessments were done immediately post thaw, which may result in an overestimation of cell viability due to possible delayed injury responses. Here, we show that for the optimal protocol condition of plunge at the -40 °C interrupt temperature, HUVEC and PCEC monolayers exhibited no significant immediate post-thaw injuries nor delayed injury responses during the 24-h post-thaw overnight culture period. HUVEC monolayers experienced no significant impact to their natural growth rate during the post-thaw culture, while PCEC monolayers experienced significantly higher growth than the unfrozen controls. The difference in the low-temperature responses between HUVEC and PCEC monolayers was further shown under high temperature plunge conditions. At these suboptimal plunge temperatures, HUVEC monolayers exhibited moderate immediate membrane injury but a pronounced delayed injury response during the 24-h post-thaw culture, while PCEC monolayers showed significant immediate membrane injury but no additional delayed injury response during the same period. Therefore, we provide further validation of our group's previously designed endothelial monolayer cryopreservation protocol for HUVEC and PCEC monolayers, and we identify several cell-type-specific responses to the freezing process.

An updated review on the application of proteomics to explore sperm cryoinjury mechanisms in livestock animals.

This comprehensive review critically examines the application of proteomics in understanding sperm cryoinjury mechanisms in livestock animals, in the context of the widespread use of semen cryopreservation for genetic conservation. Despite its global adoption, cryopreservation often detrimentally affects sperm quality and fertility due to cryoinjuries. These injuries primarily arise from ice crystal formation, osmotic shifts, oxidative stress, and the reorganization of membrane proteins and lipids during freezing and thawing, leading to premature capacitation-like changes. Moreover, the cryopreservation process induces proteome remodeling in mammalian sperm. Although there have been technological advances in semen cryopreservation, the precise mechanisms of mammalian sperm cryoinjury remain elusive. This review offers an in-depth exploration of how recent advancements in proteomic technologies have enabled a detailed investigation into these molecular disruptions. It presents an analysis of protein-level alterations post-thaw and their impact on sperm viability and functionality. Additionally, it discusses the role of proteomics in refining cryopreservation techniques to mitigate cryoinjury and enhance reproductive outcomes in livestock. This work synthesizes current knowledge, highlights gaps, and suggests directions for future research in animal reproductive science and biotechnology.

Time-Sequential Monitoring of the Early Mesothelial Reaction in the Pleura after Cryoinjury.

(1) Background: An early mesothelial reaction of the pleura, leading to fibrosis, has been reported in animals after chemical or heavy metal exposure. However, the visual monitoring of early time-sequential mesothelial reaction-associated cryoinjury has not been fully investigated. Therefore, this study aimed to evaluate and visualize the early mesothelial reactions seen following cryoinjury using rabbit pleura. (2) Methods: We monitored the early mesothelial reaction in rabbit pleurae after cryoinjury using optical coherence tomography (OCT), in real-time, which was then compared with pathological images. Due to the penetration limit of OCT, we made a thoracic window to image the parietal and visceral pleurae in vivo. We also used an innovative technique for capturing the microstructure in vivo, employing a computer-controlled intermittent iso-pressure breath hold to reduce respiratory motion, increasing the resolution of OCT. We organized three sample groups: the normal group, the sham group with just a thoracic window, and the experimental group with a thoracic window and cryotherapy. In the experimental group, localized cryoinjury was performed. The mesothelial cells at the level of pleura of the cryotherapy-injured site were visualized by OCT within the first 30 min and then again after 2 days at the same site. (3) Results: In the experimental group, focal thickening of the parietal pleura was observed at the site of cryoinjury using OCT after the first injury, and it was then confirmed pathologically as focal mesothelial cell proliferation. Two days after cryoinjury, diffuse mesothelial cell proliferation in the parietal pleura was noted on the reverse side around the cryoinjured site in the same rabbit. In the sham group, no pleural reaction was found. The OCT and pathological examinations revealed different patterns of mesothelial cell reactions between the parietal and visceral pleurae: the focal proliferation of mesothelial cells was found in the parietal pleura, while only a morphological change from flat cells to cuboidal cells and a thickened monolayer without proliferation of mesothelial cells were found in the visceral pleural. (4) Conclusions: An early mesothelial reaction occurs following cryoinjury to the parietal and visceral pleurae.

Granulocyte-Macrophage Colony-Stimulating Factor Cytokine Addition After the Freeze-Thawing Process Improves Human Sperm Motility and Vitality in Asthenoteratozoospermia Patients.

The cryopreservation-thawing process of spermatozoa cells has negative impacts on their structure, function, and fertility parameters, which are known as cryoinjury. Asthenozoospermia patients are more susceptible to cryoinjury. Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases sperm glucose uptake via the induction of glucose transporters, resulting in increased sperm motility. This study aimed to investigate the efficiency of GM-CSF supplementation of the cryopreservation media for semen samples of asthenoteratozoospermia patients. The study was carried out on 20 semen samples from infertile men referred to diagnosing semen analysis. To avoid subjective bias, two main sperm motility parameters, including velocity along the curvilinear path and velocity along the straight-line path were considered by the computer-assisted sperm analysis system. Afterward, each semen sample was divided into three equal aliquots and randomly assigned to one of the following groups: group I (control, freezing media only), group II (+GM-CSF, freezing medium supplemented with 2 µL/mL GM-CSF), or group III (GM-CSF added after thawing and washing). Following semen thawing, standard parameters, mitochondrial membrane potential (MMP), and the DNA Fragmentation Index were analyzed. Total sperm motility (progressive and non-progressive) improved significantly in group III samples after a 30-minute incubation with GM-CSF compared with the control group (26.5% ± 3.1% vs. 17.51% ± 2.59%). However, no differences in progressive motility or sperm morphology were found among the three thawed samples. The percentage of vitality was significantly higher in group III compared with the other two groups (28.38% ± 3.4% vs. 22.4% ± 3.08% and 22.14% ± 2.77%, respectively) (p < 0.05). JC-1 levels (a marker of MMP) were not significantly different between the examined groups (44.95% ± 8.26% vs. 36.61% ± 6.95% vs. 46.67% ± 7.7%, for control, group II, and group III, respectively) (p > 0.05). GM-CSF may be advantageous as an additive after freezing, improving total motility and viability after 30 minutes of post-thaw incubation; however, when supplied to the freezing media before cryopreservation, it is unable to protect against cryoinjury.

New strategies for germ cell cryopreservation: Cryoinjury modulation.

Cryopreservation is an option for the preservation of pre- or post-pubertal female or male fertility. This technique not only is beneficial for human clinical applications, but also plays a crucial role in the breeding of livestock and endangered species. Unfortunately, frozen germ cells, including oocytes, sperm, embryos, and spermatogonial stem cells, are subject to cryoinjury. As a result, various cryoprotective agents and freezing techniques have been developed to mitigate this damage. Despite extensive research aimed at reducing apoptotic cell death during freezing, a low survival rate and impaired cell function are still observed after freeze-thawing. In recent decades, several cell death pathways other than apoptosis have been identified. However, the relationship between these pathways and cryoinjury is not yet fully understood, although necroptosis and autophagy appear to be linked to cryoinjury. Therefore, gaining a deeper understanding of the molecular mechanisms of cryoinjury could aid in the development of new strategies to enhance the effectiveness of the freezing of reproductive tissues. In this review, we focus on the pathways through which cryoinjury leads to cell death and propose novel approaches to enhance freezing efficacy based on signaling molecules.

Engineered heart tissue maturation inhibits cardiomyocyte proliferative response to cryoinjury.

The cellular and molecular mechanisms that are responsible for the poor regenerative capacity of the adult heart after myocardial infarction (MI) are still unclear and their understanding is crucial to develop novel regenerative therapies. Considering the lack of reliable in vitro tissue-like models to evaluate the molecular mechanisms of cardiac regeneration, we used cryoinjury on rat Engineered Heart Tissues (rEHTs) as a new model which recapitulates in part the in vivo response after myocardial injury of neonatal and adult heart. When we subjected to cryoinjury immature and mature rEHTs, we observed a significant increase in cardiomyocyte (CM) DNA synthesis when compared to the controls. As expected, the number of mitotic CMs significantly increases in immature rEHTs when compared to mature rEHTs, suggesting that the extent of CM maturation plays a crucial role in their proliferative response after cryoinjury. Moreover, we show that cryoinjury induces a temporary activation of fibroblast response in mature EHTs, similar to the early response after MI, that is however incomplete in immature EHTs. Our results support the hypothesis that the endogenous maturation program in cardiac myocytes plays a major role in determining the proliferative response to injury. Therefore, we propose rEHTs as a robust, novel tool to in vitro investigate critical aspects of cardiac regeneration in a tissue-like asset free from confounding factors in response to injury, such as the immune system response or circulating inflammatory cytokines.

Bax/Bcl-2 Expression Ratio Analysis of Rat Ovary Vitrified with Date Juice Concentrate as a Natural Extracellular Cryoprotectant.

Background: The use of extremely low temperatures in vitrification is known to cause cryoinjury so that it can trigger the activation of the intrinsic apoptotic pathway, which can damage the structural integrity of the pre-antral follicle. Based on that, it is necessary to use an appropriate cryoprotectant to protect the preserved cell. Aims: This study aimed to identify the potential use of date juice concentrate (DJC) as a natural extracellular cryoprotectant to suppress the rate of apoptosis after vitrification. Settings and Design: This experimental research uses 24 samples of ovarian rats. Rats were fed and drank an ad libitum. Materials and Methods: Ovaries were isolated in the proestrus phase, then processed into slides for immunohistochemistry (IHC) staining using anti-Bax and anti-Bcl-2 antibodies. IHC results were evaluated for the brown colour using ImageJ IHC Profiler. The results were analysed as an optical density and displayed in the Bax/Bcl-2 ratio. Statistical Analysis Used: All data were statistically analysed with either parametric (analysis of various) or non-parametric (Kruskal-Wallis) tests. Results: The combination of EG 7.5% + DJC 15% (KP2) showed the lowest Bax/Bcl-2 ratio in primordial and primary follicles. Meanwhile, the lowest Bax/Bcl-2 ratio in secondary follicles is found in KP4 (EG 15% + DJC 15%). The DJC is known to contain a dominant amount of glucose. The DJC shows antioxidant activity and contains antioxidant compounds, phenols and flavonoids. Conclusion: The sugar content and antioxidant compounds of DJC can protect against follicle membrane damage, so the rate of intrinsic apoptosis pathway is also suppressed initially with Bax protein suppression in the mitochondrial membrane.

Comparative iTRAQ proteomics identified proteins in fresh and frozen thawed yak spermatozoa.

The changes in semen and cryodamage after the cryopreservation process negatively affect sperm function and motility. However, possible proteomic alterations of yak semen during cryopreservation have not yet been achieved. In this study, we compared proteomes of fresh and frozen thawed yak sperm using iTRAQ combined with LC-MS/MS proteome approach. Totally, 2064 proteins were quantitatively identified, including 161 in fresh sperm that showed significant differences compared to frozen thawed sperm. According to the Gene ontology (GO) enrichment analysis, differentially expressed proteins (DEPs) are predominantly associated with spermatogenesis, tricarboxylic acid cycle, ATP synthesis, and differentiation biological process. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEPs were mainly involved in metabolic pathways related to pyruvate metabolism, carbon metabolism, glycolysis/gluconeogenesis, together with the citrate (TCA) cycle. In the analysis of the protein-protein interaction (PPI) network, 15 potential proteins (PDHB, DLAT, PDHA2, PGK1, TP5C1, etc.) that could be related to the sperm quality of the yaks were obtained. Furthermore, 6 DEPs were validated by parallel reaction monitoring (PRM), confirming that the iTRAQ data were reliable. These results indicate that cryopreservation alters the proteome of yak sperm, which is possibly related to cryodamage and fertilization ability.

Step-by-step protocol for alternative injury models in newt cardiac regeneration.

Although the heart is one of the most important organs for animal survival, its regenerative capacity varies among animal species. Notably, adult mammals cannot regenerate their hearts after damage such as acute myocardial infarction. In contrast, some vertebrate animals can regenerate the heart throughout their lives. Cross-species comparative studies are important to understand the full picture of cardiac regeneration in vertebrates. Among the animal species able to regenerate the heart, some urodele amphibians, such as newts, possess a remarkable capacity for this process. Standardized methods of inducing cardiac regeneration in the newt are needed as a platform for studies comparing newts and other animal models. The procedures presented here describe amputation and cryo-injury techniques for the induction of cardiac regeneration in Pleurodeles waltl, an emerging model newt species. Both procedures consist of simplified steps that require no special equipment. We additionally show some examples of the regenerative process obtained using these procedures. This protocol has been developed for P. waltl. However, these methods are also expected to be applicable to other newt and salamander species, facilitating comparative research with other model animals.

Cryopreservation: A Comprehensive Overview, Challenges, and Future Perspectives.

Cryopreservation is the most prevalent method of long-term cell preservation. Effective cell cryopreservation depends on freezing, adequate storage, and correct thawing techniques. Recent advances in cryopreservation techniques minimize the cellular damage which occurs while processing samples. This article focuses on the fundamentals of cryopreservation techniques and how they can be implemented in a variety of clinical settings. The article presents a brief description of each of the standard cryopreservation procedures, such as slow freezing and vitrification. Alongside that, the membrane permeating and nonpermeating cryoprotectants are briefly discussed, along with current advancements in the field of cryopreservation and variables influencing the cryopreservation process. The diminution of cryoinjury incurred by the cell via the resuscitation process will also be highlighted. In the end application of cryopreservation techniques in many fields, with a special emphasis on stem cell preservation techniques and current advancements presented. Furthermore, the challenges while implementing cryopreservation and the futuristic scope of the fields are illustrated herein. The content of this review sheds light on various ways to enhance the output of the cell preservation process and minimize cryoinjury while improving cell revival.

Optimized addition of nitric oxide compounds in semen extender improves post-thaw seminal attributes of Murrah buffaloes.

Semen dilution and cryopreservation alter the homogeneity of seminal plasma, resulting in a non-physiological redox milieu and consequently poor sperm functionality. Considering the concentration-specific bimodal action of nitric oxide (NO) in the regulation of sperm functions, cryopreservation media supplemented with optimized concentrations can improve the semen attributes. The present study aimed to evaluate the effect of adding an optimized concentration of sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME) in an extender on in vitro semen quality. An aliquot of semen samples (n = 32) from Murrah buffalo bulls (n = 8) was divided into control (C) and treatment (T-I: SNP in extender at 1 µmol/L; T-II: L-NAME in extender at 10 µmol/L). Fresh semen quality parameters showed no significant difference at 0 h except for the structural integrity in the T-II group. Post-thaw semen quality parameters and sperm kinematics using computer-aided sperm analysis (CASA) revealed significantly higher (p < 0.05) cryoresistance in the treatment groups. Viability, acrosome integrity, and membrane integrity were significantly higher (p < 0.05) in both treatment groups; however, the results were pervasive in T-II. Lower abnormal spermatozoa were observed in both T-I and T-II. SNP supplementation led to a significant rise (p < 0.05) in NO, whereas L-NAME reduced the NO concentration in post-thawed samples, which was directly correlated with different sperm functionality and associated biomarkers viz. total antioxidant capacity (TAC) and thiobarbituric acid reactive substance (TBARS). It was concluded that the cryopreservation media supplemented with SNP and L-NAME at 1 µmol/L and 10 µmol/L, respectively, lower the cryo-damage and improve post-thaw seminal attributes.

Nerve Protection During Prostate Cryosurgery.

Cryosurgery is a minimally invasive approach to the treatment of focal prostate cancer (PCa). A major complication is the cryoinjury to the cavernous nerve in the neurovascular bundle (NVB). This nerve cryoinjury halts conduction of action potentials (APs) and can eventually result in erectile dysfunction and therefore diminished quality of life for the patient. Here, we propose the application of cryoprotective agents (CPA) to the regions of the nerves in the NVB, prior to prostate cryosurgery, to minimize non-recoverable loss of AP conduction. We modeled a cryosurgical procedure based on data taken during a clinical case and applied ex-vivo porcine phrenic nerves and rat sciatic nerve with temperature profile of NVB. The APs were measured before and after the CPA exposures and during 3 h of recovery. Comparisons of AP amplitude recovery with various CPA compositions reveal that certain CPAs (e.g., 5% DMSO + 7.5% Trehalose and 5% M22 for porcine and rat nerves, respectively) showed little or no toxicity and effective cryoprotection from freezing (on average 48% and 30% of recovered AP, respectively). In summary, we demonstrate that neural conduction can be preserved after exposure to freezing conditions if CPAs are properly selected and deployed onto the nerve.

Seed morphoanatomy of the genus Passiflora L. (Passifloraceae) by scanning electron microscopy and light microscopy.

Morphoanatomical analysis of seeds contributes to knowledge of the development of seedlings and identification of species, as well as supporting conservation studies. The conservation of the species belonging to the Passiflora genus is crucial due to of the threats to the genetic resources of these species. Thus, the objective of this study was to morphoanatomically characterize Passiflora seeds, verify possible injuries to the tissues after cryopreservation and thus contribute to the conservation strategies of the species of this genus. Initially, seeds of Passiflora coccinea, P. edulis, P. gibertii, P. maliformis, P. morifolia, P. setacea, P. suberosa, and P. tenuifila collected from the Passion Fruit Active Germplasm Bank of the Embrapa Cassava and Fruits research unit (Embrapa Mandioca e Fruticultura) were analyzed. Then, their length, width and thickness, shape of the base and tip, and ornamentations present on the body and edge of the seeds were evaluated. The seeds of the species were placed in cryotubes and immersed in liquid nitrogen to assess possible cryoinjuries. The tegument and tissues of the seeds were examined by scanning electron microscopy. The seeds had varied biometric data, with average values of 4.63 mm for length, 3.28 mm for width, and 1.51 mm for thickness. Six ornamentation types were observed: reticulate for the species P. coccinea; finely reticulate for P. edulis; foveolate reticulate for P. gibertii and P. setacea; alveolate reticulate for P. maliformis and P. tenuifila; coarsely reticulate for P. morifolia; and falsifoveolate reticulate for P. suberosa. Some seeds suffered tegument cracks due to the freezing in liquid nitrogen, but without physiological damages to the embryo and endosperm. The cryopreservation of the seeds in the presence of the tegument significantly reduced the cryoinjuries caused to the embryo. Cryopreservation can be promising for long-term conservation of passion fruit seeds.

Repeated freeze-thaw cycles in freeze-tolerant treefrogs: novel interindividual variation of integrative biochemical, cellular, and organismal responses.

The freeze-tolerant anuran Dryophytes chrysoscelis, Cope's gray treefrog, mobilizes a complex cryoprotectant system that includes glycerol, glucose, and urea to minimize damage induced by freezing and thawing of up to 65% of body water. In this species' eastern Northern American temperate habitat, oscillations of temperature above and below freezing are common; however, the effects of repeated freezing and thawing in this species are unstudied. The biochemical and physiological effects of repeated freeze-thaw cycles were therefore evaluated and compared with cold acclimation and single freeze-thaw episodes. Glycerol was elevated in plasma, liver, and skeletal muscle of both singly and repeatedly frozen and thawed animals compared with cold-acclimated frogs. In contrast, urea was unchanged by freezing and thawing, whereas glucose was elevated in singly frozen and thawed animals but was reduced toward cold acclimation levels after repeated bouts of freezing. Overall, the cryoprotectant system was maintained, but not further elevated, in all tissues assayed in repeatedly frozen and thawed animals. For repeated freeze-thaw only, hepatic glycogen was depleted and plasma hemoglobin, indicative of erythrocyte hemolysis, increased. Postfreeze recovery of locomotor function, including limb and whole body movement, was delayed with repeated freeze-thaw and was associated with glycerol accumulation and glycogen depletion. Individuals that resumed locomotor function more quickly also accumulated greater cryoinjury. Integrated analyses of cryoprotectant and cryoinjury accumulation suggest that winter survival of D. chrysoscelis may be vulnerable to climate change, limited by carbohydrate stores, cellular repair mechanisms, and plasticity of the cryoprotectant system.

Closed system processing variables affect post-thaw quality characteristics of cryopreserved red cell concentrates.

BACKGROUND: Differences in manufacturing conditions using the Haemonetics ACP 215 cell processor result in cryopreserved red cell concentrates (RCCs) of varying quality. This work studied the effect of processing method, additive solution, and storage duration on RCC quality to identify an optimal protocol for the manufacture of cryopreserved RCCs. MATERIALS AND METHODS: RCCs were pooled-and-split and stored for 7, 14, or 21 days before cryopreservation. Units were glycerolized with the ACP 215 using a single or double centrifugation method. After thawing, the RCCs were deglycerolized, suspended in AS-3, SAGM, ESOL, or SOLX/AS-7, and stored for 0, 3, 7, 14, or 21 days before quality testing. Quality assessments included hemoglobin content, hematocrit, hemolysis, adenosine triphosphate (ATP), supernatant potassium, and mean cell volume. RESULTS: Both glycerolization methods produced RCCs that met regulatory standards for blood quality. Dual centrifugation resulted in higher hemoglobin content, fewer processing alerts, and a shorter deglycerolization time than single centrifugation processing. Units processed with AS-3 and ESOL met regulatory standards when stored for up to 21 days pre-cryopreservation and 21 days post-deglycerolization. However, ESOL demonstrated superior maintenance of ATP over RBCs in AS-3. Some RCCs suspended in SAGM and SOLX exceeded acceptable hemolysis values after 7 days of post-deglycerolization storage regardless of pre-processing storage length. CONCLUSIONS: When manufacturing cryopreserved RCCs using the ACP 215, dual centrifugation processing with AS-3 or ESOL additive solutions is preferred, with storage periods of up to 21 days both pre-processing and post-deglycerolization.

A Cryoprotectant-Gel Composite Designed to Preserve Articular Cartilage during Frozen Osteoarticular Autograft Reconstruction for Malignant Bone Tumors: An Animal-Based Study.

OBJECTIVE: We designed a highly adhesive cryoprotectant-gel composite (CGC), based on regular liquid-form cryoprotectant base (CB), aiming to protect cartilage tissue during frozen osteoarticular autograft reconstruction for high-grade sarcoma around the joint. This study aimed to evaluate its effectiveness in rat and porcine distal femur models. DESIGN: Fresh articular cartilage samples harvested from distal rat and porcine femurs were divided into 4 test groups: untreated control group, liquid nitrogen (LN) freezing group, LN freezing group pretreated with CB (CB group), and LN freezing group pretreated with CGC (CGC group). Microscopic and macroscopic evaluation of cartilage condition, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, and apoptotic protein analysis of chondrocytes were performed to confirm our results. RESULTS: In the rat model, CGC could prevent articular cartilage from roughness and preserve more proteoglycans when compared with the LN freezing and CB groups. Western blot analysis showed CGC could prevent cartilage from LN-induced apoptosis supported by caspase-3/8 apoptotic signaling cascade. Macroscopically, we observed CGC could reduce both articular clefting and loss of articular luminance after freezing in the porcine model. In both models, CGC could reduce articular chondrocytes from degeneration. Fewer TUNEL-positive apoptotic and more viable chondrocytes in cartilage tissue were observed in the CGC group in our animal models. CONCLUSION: Our study proved that CGC could effectively prevent cartilage surface and chondrocytes from cryoinjury after LN freezing. Freezing articular cartilage surrounded with high concentration of CGC can be a better alternative to preserve articular cartilage during limb salvage surgery for malignant bone tumor.

Frozen-Thawed Sperm Analysis of Benign Prostatic Hyperplasia Dogs Treated With Finasteride.

Benign Prostatic Hyperplasia (BPH) is a pathological condition that directly interferes with the reproductive potential of senile dogs, by leading to prostate enlargement and sperm injury, which in turn may compromise sperm freezeability. Moreover, albeit finasteride treatment reduces prostatic volume and blood supply and maintains seminal quality and testicular integrity, the effects of sperm samples submitted to cryopreservation after the finasteride treatment are still unknown. Thus, the aim of this study was to evaluate frozen-thawed semen of BPH dogs, as well as dogs subjected to BPH pharmacological treatment with finasteride. For such purpose, 20 dogs were previously selected and assigned to three experimental groups, according to BPH diagnosis and treatment with finasteride: Control (n = 9), BPH Group (n = 5) and BPH-Finasteride Group (n = 6). Semen was subjected to one-step cryopreservation protocol with tris-fructose-citric acid extender with 5% glycerol and thawed at 37°C for 30 sec. Fresh and post-thaw sperm samples were evaluated for macroscopic parameters, sperm concentration, sperm motility kinetics, sperm mitochondrial activity and potential, oxidative stress, plasmatic and acrosome membrane integrity, sperm DNA fragmentation and sperm binding test on perivitelic membrane of chicken egg yolk. Regarding fresh semen, BPH-Finasteride group had the lowest ejaculate visual aspect (opacity), higher frequency of sperm flagellar beating (BCF) and percentage of sperm with medium velocity. Control group had the highest percentage of sperm DNA integrity compared to BPH group. For the frozen-thawed semen, Control group presented the highest percentage of spermatozoa with high mitochondrial activity. However, the BPH-Finasteride group showed higher number of sperm bound to the perivitelline membrane of chicken egg yolk compared to the BPH Group. Conversely, BPH group had higher percentage of DNA damage. In conclusion, the ejaculate of BPH dogs has higher susceptibility to cryoinjury, whereas finasteride-treated dogs have increased spermatozoa functional performance, suggesting a promising use of BPH dogs as semen donors in sperm cryopreservation programs.

The response of a human haematopoietic cell line to trehalose-loaded liposomes and their effect on post-thaw membrane integrity.

Dimethyl sulphoxide (DMSO) used in haematopoietic stem cell (HSC) cryopreservation has been linked to an increased incidence of adverse reactions following transplantation. In the interest of reducing the required DMSO concentrations, we have evaluated the use of unilamellar liposomes to internalize the non-toxic, cell-impermeable disaccharide, trehalose into HSCs and characterized the cryoprotective efficacy of this strategy. A fluorescent marker, 5(6)-carboxyfluorescein (200 µmol/L), was used for trehalose internalization following a 5 h incubation at 37 °C with liposome concentrations ranging from 0.5 mM to 4 mM. Cells were frozen (1 °C/min to -80 °C) following treatment with either 3 mM or 4 mM of liposomes (5 h, 37 °C) containing 0.2 mol/L trehalose either in the presence or absence of 0.2 mol/L extracellular trehalose. Increasing the liposome concentration from 3 mM to 4 mM corresponded to a significant (p = 0.046) increase in the mean fluorescent intensity (MFI) (3 mM 512 ± 7.07; 4 mM: 916 ± 28.3). Post-thaw membrane integrity indicated that the presence of trehalose both inside and outside when internalized using a liposome concentration of 4 mM significantly improved survival relative to the sole presence of extracellular trehalose (p = 0.02). However, viability was diminished relative to a standard DMSO control (trehalose: 32.5% ± 1.7%; DMSO: 85.0% ± 4.6%). This study confirms that the protective efficacy of trehalose is enhanced when it is present on both sides of the membrane; however, it reinforces concerns surrounding the efficiency of using liposomes as a vehicle to transfer trehalose into cells.