Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation.

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.

Immediate versus delayed single blastocyst transfer following the first stimulated IVF cycle in the freeze-all strategy: a study protocol for a randomised controlled trial.

INTRODUCTION: In recent years, the use of frozen embryo transfers (FET) has rapidly increased following the freeze-all strategy due to the advantages of increased maternal safety, improved pregnancy rates, lower ectopic pregnancy rates and better obstetric and neonatal outcomes. Currently, there is still no good scientific evidence to support when to perform FET following a stimulated in vitro fertilisation (IVF) cycle in the freeze-all strategy. METHODS/ANALYSIS: This will be a randomised controlled trial. A total of 828 women undergoing their first FET following their first stimulated IVF cycle in the freeze-all strategy will be enrolled and randomised into one of the following groups according to a computer-generated randomisation list: (1) the immediate group, in which FET will be performed in the first menstrual cycle following the stimulated IVF cycle; or (2) the delayed group, in which FET will be performed at least in the second menstrual cycle following the stimulated IVF cycle. The primary outcome will be live birth, which is defined as the delivery of any infants at ≥22 gestational weeks with heartbeat and breath. ETHICS/DISSEMINATION: Ethical approval was granted by the Ethics Committee of Assisted Reproductive Medicine at the Shanghai JiAi Genetics & IVF Institute (JIAI E2019-15). Written informed consent will be obtained from each woman before any study procedure is performed, according to good clinical practice. The results of this trial will be disseminated in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04371783.

Effect of post-vitrification cryopreservation duration on singleton birth-weight in frozen-thawed blastocysts transfer cycles.

Introduction: This study aimed to explore the effect of cryopreservation duration after blastocyst vitrification on the singleton birth-weight of newborns to assess the safety of long-term preservation of frozen-thawed blastocyst transfer (FBT) cycles. Methods: This was a retrospective observational study conducted at the Gynecological Endocrinology and Assisted Reproduction Center of the Peking Union Medical College Hospital. Patients who gave birth to singletons between January 2006 and December 2021 after undergoing FBT cycles were included. Five groups were formed according to the duration of cryopreservation of embryos at FBT: Group I included 274 patients with a storage time < 3 months. Group II included 607 patients with a storage time of 3-6 months. Group III included 322 patients with a storage time of 6-12 months. Group IV included 190 patients with a storage time of 12-24 months. Group V included 118 patients with a storage time of > 24 months. Neonatal outcomes were compared among the groups. Multivariate linear regression analysis was performed to evaluate birth-weights and other birth-related outcomes. Results: A total of 1,511 patients were included in the analysis. The longest cryopreservation period was 12 years. The birth-weights of neonates in the five groups were 3344.1 ± 529.3, 3326.1 ± 565.7, 3260.3 ± 584.1, 3349.9 ± 582.7, and 3296.7 ± 491.9 g, respectively (P > 0.05). The incidences of preterm birth, very preterm birth, low birth-weight, and very low birth-weight were similar in all groups (P > 0.05). The large-for-gestational-age and small-for-gestational-age rates did not differ significantly among the groups (P > 0.05). After adjusting for confounding factors that may affect neonatal outcomes, a trend for an increased risk of low birth-weight with prolonged cryopreservation was observed. However, cryopreservation duration and neonatal birth-weight were not significantly correlated (P > 0.05). Conclusion: The duration of cryopreservation after blastocyst vitrification with an open device for more than 2 years had no significant effect on the birth-weight of FBT singletons; however, attention should be paid to a possible increase in the risk of low birth-weight.

Effect of fumigation height and time on cryopreservation of ram semen.

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I-IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

In situ monitoring of loading and elution processes of cryoprotectants at the single-cell level with Raman spectroscopy.

BACKGROUND: The analysis of cell membrane permeability plays a crucial role in improving the procedures of cell cryopreservation, which will affect the specific parameter settings in loading, removal and cooling processes. However, existing studies have mostly focused on deriving permeability parameters through osmotic theoretical models and cell volume response analysis, and there is still a lack of the direct experimental evidence and analysis at the single-cell level regarding the migration of cryoprotectants. RESULTS: In this work, a side perfusion microfluidics chips combined with Raman spectroscopy system was built to monitor in situ the Raman spectroscopy of extracellular and intracellular solution during loading and elution process with different cryoprotectant solution systems (single and dual component). And it was found that loading a high concentration cryoprotectant solution system through a single elution cycle may result in significant residual protective agent, which can be mitigated by employing a multi-component formula but multiple elution operations are still necessary. Furthermore, the collected spectral signals were marked and analyzed to was perform preliminary relative quantitative analysis. The results showed that the intracellular concentration changes can be accurately quantified by the Raman spectrum and are closely related to the extracellular solution concentration changes. SIGNIFICANCE AND NOVELTY: By using the method of small flow perfusion (≤20 µL/min) in the side microfluidic chip after the gravity sedimentation of cells, the continuous loading and elution process of different cryoprotectants on chip and the spectral acquisition can be realized. The intracellular and extracellular concentrations can be quantified in situ based on the ratio of spectral peak intensities. These results indicate that spectroscopic analysis can be used to effectively monitor intracellular cryoprotectant residues.

The use of sodium caseinate in the freezing of sheep semen.

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.

Evaluation of DNAmAge in paired fresh, frozen, and formalin-fixed paraffin-embedded heart tissues.

The continued development in methylome analysis has enabled a more precise assessment of DNA methylation, but treatment of target tissue prior to analysis may affect DNA analysis. Prediction of age based on methylation levels in the genome (DNAmAge) has gained much interest in disease predisposition (biological age estimation), but also in chronological donor age estimation in crime case samples. Various epigenetic clocks were designed to predict the age. However, it remains unknown how the storage of the tissues affects the DNAmAge estimation. In this study, we investigated the storage method impact of DNAmAge by the comparing the DNAmAge of the two commonly used storage methods, freezing and formalin-fixation and paraffin-embedding (FFPE) to DNAmAge of fresh tissue. This was carried out by comparing paired heart tissue samples of fresh tissue, samples stored by freezing and FFPE to chronological age and whole blood samples from the same individuals. Illumina EPIC beadchip array was used for methylation analysis and the DNAmAge was evaluated with the following epigenetic clocks: Horvath, Hannum, Levine, Horvath skin+blood clock (Horvath2), PedBE, Wu, BLUP, EN, and TL. We observed differences in DNAmAge among the storage conditions. FFPE samples showed a lower DNAmAge compared to that of frozen and fresh samples. Additionally, the DNAmAge of the heart tissue was lower than that of the whole blood and the chronological age. This highlights caution when evaluating DNAmAge for FFPE samples as the results were underestimated compared with fresh and frozen tissue samples. Furthermore, the study also emphasizes the need for a DNAmAge model based on heart tissue samples for an accurate age estimation.

In vitro studies on the effects of cryopreserved platelet-rich plasma on cells related to wound healing.

Platelet-rich plasma (PRP) holds promise as a therapeutic modality for wound healing; however, immediate utilization encounters challenges related to volume, concentration, and consistency. Cryopreservation emerges as a viable solution, preserving PRP's bioactive components and extending its shelf life. This study explores the practicality and efficacy of cryopreserved platelet-rich plasma (cPRP) in wound healing, scrutinizing both cellular mechanisms and clinical implications. Fresh PRP and cPRP post freeze-thaw underwent assessment in macrophage, fibroblast, and endothelial cell cultures. The impact of cPRP on active component release and cell behavior pertinent to wound healing was evaluated. Varied concentrations of cPRP (1%, 5%, 10%) were examined for their influence on cell polarization, migration, and proliferation. The results showed minimal changes in cPRP's IL-1ß levels, a slight decrease in PDGF-BB, and superior effects on macrophage M2 polarization and fibroblast migration, while no statistical significance was observed in endothelial cell angiogenesis and proliferation. Remarkably, 5% PRP exhibited the most significant stimulation among all cPRP concentrations, notably impacting cell proliferation, angiogenesis, and migration. The discussion underscores that cPRP maintains platelet phenotype and function over extended periods, with 5% cPRP offering the most favorable outcomes, providing a pragmatic approach for cold storage to extend post-thaw viability and amplify therapeutic effects.

What is the context? Platelet-rich plasma (PRP) is a potential bioactive material for wound healing, but using it immediately faces issues like volume, concentration, and consistency.Low-temperature freezing is a method employed to preserve PRP. However, the current understanding of the effects of the freezing-thawing process on the components of PRP and its impact on cells relevant to wound healing remains unclear.What is new? This study explores the feasibility and effectiveness of using cryopreserved PRP at −80°C for promoting wound healing. This research stands out for its focus on cellular responses and practical implications in therapeutic contexts.To understand their distinct impact on different cell types relevant to wound healing, the study meticulously examined various final concentrations of cPRP (1%, 5%, 10%).The study identified the superior effects of 5% cPRP on crucial cellular activities, notably in cell polarization, proliferation, angiogenesis, and migration.What is the impact? Low-temperature freezing can be considered an effective method for PRP preservation.Some bioactive components in cPRP exhibit subtle changes; however, these changes result in better effects on certain cell types related to healing.The study illustrates that all concentrations of cPRP effectively enhance cell proliferation, migration, and differentiation, emphasizing the comparable efficacy of cryopreserved PRP to non-cryopreserved PRP.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.

Storage conditions affect the composition of the lyophilized secretome of multipotent mesenchymal stromal cells.

The widespread use of multipotent mesenchymal stromal cell-derived secretome (MSC-sec) requires optimal preservation methods. Lyophilization offers benefits like concentrating the secretome, reducing the storage volume, and making storage conditions more flexible. This study evaluated the influence of storage duration and temperature on lyophilized MSC-sec. The conditioned medium from Wharton's jelly MSCs was stored at - 80 °C or lyophilized with or without trehalose. Lyophilized formulations were kept at - 80 °C, - 20 °C, 4 °C, or room temperature (RT) for 3 and 30 months. After storage and reconstitution, the levels of growth factors and cytokines were assessed using multiplex assay. The storage of lyophilized MSC-sec at - 80 °C ensured biomolecule preservation for 3 and 30 months. Following 3 month storage at 4 °C and RT, a notable decrease occurred in BDNF, bNGF, and sVCAM-1 levels. Prolonged 30 month storage at the same temperatures significantly reduced BDNF, bNGF, VEGF-A, IL-6, and sVCAM-1, while storage at - 20 °C decreased BDNF, bNGF, and VEGF- A levels. Trehalose supplementation of MSC-sec improved the outcome during storage at 4 °C and RT. Proper storage conditions were crucial for the preservation of lyophilized MSC-sec composition. Short-term storage at various temperatures maintained over 60% of the studied growth factors and cytokines; long-term preservation was only adequate at -80 °C.

Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa.

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

Laboratory based correlative cryo-soft X-ray tomography and cryo-fluorescence microscopy.

Cryo-soft X-ray tomography is the unique technology that can image whole intact cells in 3D under normal and pathological conditions without labelling or fixation, at high throughput and spatial resolution. The sample preparation is relatively straightforward; requiring just fast freezing of the specimen before transfer to the microscope for imaging. It is also possible to image chemically fixed samples where necessary. The technique can be correlated with cryo fluorescence microscopy to localize fluorescent proteins to organelles within the whole cell volume. Cryo-correlated light and soft X-ray tomography is particularly useful for the study of gross morphological changes brought about by disease or drugs. For example, viral fluorescent tags can be co-localized to sites of viral replication in the soft X-ray volume. In general this approach is extremely useful in the study of complex 3D organelle structure, nanoparticle uptake or in the detection of rare events in the context of whole cell structure. The main challenge of soft X-ray tomography is that the soft X-ray illumination required for imaging has heretofore only been available at a small number of synchrotron labs worldwide. Recently, a compact device with a footprint small enough to fit in a standard laboratory setting has been deployed ("the SXT-100") and is routinely imaging cryo prepared samples addressing a variety of disease and drug research applications. The SXT-100 facilitates greater access to this powerful technique and greatly increases the scope and throughput of potential research projects. Furthermore, the availability of cryo-soft X-ray tomography in the laboratory will accelerate the development of novel correlative and multimodal workflows by integration with light and electron microscope based approaches. It also allows for co-location of this powerful imaging modality at BSL3 labs or other facilities where safety or intellectual property considerations are paramount. Here we describe the compact SXT-100 microscope along with its novel integrated cryo-fluorescence imaging capability.

The cold chain delivery of organs for transplantation: from research laboratories and individual enthusiasts to pan- global networks in 50 years.

It is some 50 years since the first published reports appeared of ex vivo preservation of organs for transplantation. Over the intervening decades, organ preservation strategies have become one essential component of world-wide clinical transplant services. In the formative years, translational research in organ hypothermic preservation was grappling with the questions about whether static or dynamic storage was preferable, and the practical implications of those choices. Those studies were also informing the newly expanding clinical transplant services. During the middle years, both preservation modalities were practiced by individual group choices. By the 2000s, the shift in donor demographics demanded a re-evaluation of organ preservation strategies, and now a new era of research and development is promoting adoption of new technologies. In this review we outline many important academic studies which have contributed to this successful history, and give profile to the increasing innovative approaches which are being evaluated for the future. Doi.org/10.54680/fr24310110112.

Cryopreservation of bovine semen using extract of Cinnamomum zeylanicum.

BACKGROUND: Antioxidants minimise oxidative stress and enhance sperm quality in the process of cryopreservation. OBJECTIVE: To assess the impact of Cinnamomum zeylanicum extract as an additive during the post-dilution and post-thaw stages of Murrah buffalo semen cryopreservation. MATERIALS AND METHODS: The semen sample was diluted using Tris-Egg-Yolk-Citric-Acid-Fructose-Glycerol extender and subsequently divided into three groups: Group 1, TEYCAFG without any additives or controls (C); Group 2, TEYCAFG fortified with a 50 ug/mL aqueous extract of cinnamon (T1); and Group 3, TEYCAFG fortified with a 50 ug/mL ethanolic extract of cinnamon (T2). The evaluation included an assessment of progressive motility, live spermatozoa, sperm abnormalities, HOST, CMPT, and enzyme leakage (GOT and GPT) at both the post-dilution and post-thaw stages. RESULTS: The groups that received cinnamon supplementation demonstrated statistically significant improvements (p<0.05) in various parameters, including an increase in the progressive motility, live spermatozoa, and HOS-positive spermatozoa, as well as greater distance traveled by vanguard spermatozoa compared to the control group. Furthermore, the cinnamon-added groups exhibited a significant decrease (p<0.05) in the percentage of sperm abnormalities and lower enzyme leakage (GOT and GPT) in post-thawed semen. CONCLUSION: Aqueous extract of C. zeylanicum at a concentration of 50 µg/mL provides superior protection of sperm structures and functions as compared to both the ethanolic extract of C. zeylanicum at the same concentration and the control group. Doi.org/10.54680/fr24310110712.

Liquid nitrogen improves the decellularization effectiveness of whole-ovary.

BACKGROUND: Ovarian tissue cryopreservation for fertility preservation carries a risk of malignant cell re-seeding. Artificial ovary is a promising method to solve such a problem. However, ovary decellularization protocols are limited. Hence, further studies are necessary to get better ovarian decellularization techniques for the construction of artificial ovary scaffolds. OBJECTIVE: To establish an innovative decellularization technique for whole porcine ovaries by integrating liquid nitrogen with chemical agents to reduce the contact time between the scaffolds and chemical reagents. MATERIALS AND METHODS: Porcine ovaries were randomly assigned to three groups: novel decellularized group, conventional decellularized group and fresh group. The ovaries in the novel decellularized group underwent three cycles of freezing by liquid nitrogen and thawing at temperatures around 37 degree C before decellularization. The efficiency of the decellularization procedure was assessed through histological staining and DNA content analysis. The maintenance of ovarian decellularized extracellular matrix(ODECM) constituents was determined by analyzing the content of matrix proteins. Additionally, we evaluated the biocompatibility of the decellularized extracellular matrix(dECM) by observing the growth of granulosa cells on the ODECM scaffold in vitro. RESULTS: Hematoxylin and eosin staining, DAPI staining and DNA quantification techniques collectively confirm the success of the novel decellularization methods in removing cellular and nuclear components from ovarian tissue. Moreover, quantitative assessments of ODECM contents revealed that the novel decellularization technique preserved more collagen and glycosaminoglycan compared to the conventional decellularized group (P<0.05). Additionally, the novel decellularized scaffold exhibited a significantly higher number of granulosa cells than the conventional scaffold during in vitro co-culture (P<0.05). CONCLUSION: The novel decellularized method demonstrated high efficacy in eliminating DNA and cellular structures while effectively preserving the extracellular matrix. As a result, the novel decellularized method holds significant promise as a viable technique for ovarian decellularization in forthcoming studies. Doi.org/10.54680/fr24310110212.

Visualization of intracellular ice formation and growth in mouse oocytes.

BACKGROUND: Characterization of intracellular ice formation (IIF) in oocytes during the freezing and thawing processes will contribute to optimizing their cryopreservation. However, the observation of the ice formation process in oocytes is limited by the spatiotemporal resolution of the cryomicroscope systems. OBJECTIVE: To observe the intracellular icing of oocytes during cooling and rewarming, and to study the mechanism of formation and growth of intracellular ice in oocytes. MATERIALS AND METHODS: Mouse oocytes were frozen at different cooling rates to induce intracellular ice formation using a cryomicroscopy system consisting of a microscope equipped with a cryogenic cold stage, an automatic cooling system, a temperature control system, and a high-speed camera. The growth patterns of intracellular ice in oocytes were analyzed from the images recorded. Finally, the growth rate of intracellular ice formation in oocytes was calculated using an automatic intracellular ice tracking method. RESULTS: The IIF temperature decreased gradually with the increase in cooling rate. Initiation sites of IIF could be classified into three categories: marginal type, internal type and coexisting type. There was a strong predominance for ice crystal initiation site in the oocytes, with up to 80% of the initiation sites located in the marginal region. The intracellular ice growth modes of darkening and twitching cells were characterized by "spreading" and "clustering", respectively. In addition, twitching cells started to recrystallize during rewarming, while darkening cells did not. The instantaneous maximal growth rate of ice crystals in twitching cells was about 10 times higher than that in darkening cells. CONCLUSION: By visualising the growth of ice crystals in mouse oocytes during cooling and rewarming, we obtained valuable information on the kinetics of ice formation and melting in these cells. This information can help us understand how ice formation and melting affect the viability and quality of oocytes after cryopreservation. Doi.org/10.54680/fr24310110412.

Cryo-storage of porcine hides at the industrial scale for tissue engineering and regenerative medicine application.

BACKGROUND: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process. OBJECTIVE: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine. MATERIALS AND METHODS: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants. RESULTS: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage. CONCLUSION: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.

Aquaporin expression in cryopreserved human sperm: exploring the capabilities of natural deep eutectic solvents (NADEs).

BACKGROUND: Aquaporins (AQPs) are essential proteins that facilitate the rapid movement of water and cryoprotective agents (CPAs) during the cryopreservation process, and ensure the cryo-tolerance of sperm cells. OBJECTIVE: This study evaluated the preservation of aquaporin levels in human sperm after undergoing freezing using natural deep eutectic solvents (NADES) as CPAs for cryoprotection. MATERIALS AND METHODS: From June 2021 to October 2022, 35 semen samples with normal sperm parameters were acquired from the Mehr Infertility Treatment Institute in Rasht, Iran. The samples were divided into several groups for analysis: control group (not frozen), group frozen with SpermFreeze Solution, and groups frozen with different NADESs, including ChS, ChX, ChU, ChG, GlyP, and EtP. After thawing, various aspects for each group were assessed, including the integrity and condensation of sperm chromatin, viability, motility, integrity of acrosome, and the expression of AQP1, AQP3, AQP7, AQP8, and AQP9 genes. RESULTS: The analysis of gene expression revealed that freezing with ChS and GlyP preserved the expression of the AQP1 and AQP3 genes compared to the control group. Regarding AQP7 and AQP8, significant differences were not observed in expression levels between certain NADES groups (e.g., ChS, ChU, and GlyP) and the control group. Additionally, samples frozen with specific NADESs, such as ChS, ChG, EtP, and GlyP, exhibited preserved levels of AQP9 expression when compared to the control group. CONCLUSION: These findings emphasize the importance of NADES in preserving the expression of aquaporins in cryopreserved human sperm and their important fertility parameters. Doi.org/10.54680/fr24310110512.

A brief review of the methodology and cryoprotectants in selected fish and mammalian species.

Cryopreservation is a valuable technique used to assist in the genetic improvement of cultured stocks and provide a continuous supply of good-quality semen for artificial insemination. Conserving semen by cryopreservation serves several purposes (e.g. artificial reproductive technologies and species conservation) and is also used in the clinical treatment of human infertility. However, the lifespan of cryopreserved semen is influenced by a range of factors, including storage temperature, cooling rate, chemical composition of the extender, the concentration of cryoprotectant, reactive oxygen species, seminal plasma composition and hygienic control. The choice of cryoprotectant is a vital factor underlying the success of animal semen cryopreservation. In this regard, extensive research has been carried out on various cryoprotectants, such as egg yolk, dimethyl sulfoxide, methanol, ethylene glycol and dimethylacetamide. Recent studies have also described the use of a range of new cryoprotectants for cryopreservation, including compounds of plant origin (soy), amino acids, antifreeze proteins, carbohydrates and cyclodextrins. Moreover, semen cryopreservation and storage require the use of liquid nitrogen or ultralow refrigeration methods for both long- and short-term storage. This review summarizes the general methods used for freezing semen and discusses the use of traditional and newly emerging cryoprotectants (permeable and non-permeable) for the cryopreservation of semen in selected fish and mammalian species.