Multiscale transport and 4D time-lapse imaging in precision-cut liver slices (PCLS).

Background: Monitoring cellular processes across different levels of complexity, from the cellular to the tissue scale, is important for understanding tissue structure and function. However, it is challenging to monitor and estimate these structural and dynamic interactions within three-dimensional (3D) tissue models. Objective: The aim of this study was to design a method for imaging, tracking, and quantifying 3D changes in cell morphology (shape and size) within liver tissue, specifically a precision-cut liver slice (PCLS). A PCLS is a 3D model of the liver that allows the study of the structure and function of liver cells in their native microenvironment. Methods: Here, we present a method for imaging liver tissue during anisosmotic exposure in a multispectral four-dimensional manner. Three metrics of tissue morphology were measured to quantify the effects of osmotic stress on liver tissue. We estimated the changes in the volume of whole precision cut liver slices, quantified the changes in nuclei position, and calculated the changes in volumetric responses of tissue-embedded cells. Results: During equilibration with cell-membrane-permeating and non-permeating solutes, the whole tissue experiences shrinkage and expansion. As nuclei showed a change in position and directional displacement under osmotic stress, we demonstrate that nuclei could be used as a probe to measure local osmotic and mechanical stress. Moreover, we demonstrate that cells change their volume within tissue slices as a result of osmotic perturbation and that this change in volume is dependent on the position of the cell within the tissue and the duration of the exposure. Conclusion: The results of this study have implications for a better understanding of multiscale transport, mechanobiology, and triggered biological responses within complex biological structures.

Comparison of liquid nitrogen-free slow freezing protocols toward enabling a practical option for centralized cryobanking of ovarian tissue.

Geographically distributed ovarian tissue cryobanks remain limited due to the high facility and staff costs, and cold transportation to centers is associated with ischemia-induced tissue damage that increases with transport distance. It is ideal to perform the cryopreservation procedure at a tissue removal site or local hospital before shipment to cost-effective centralized cryobanks. However, conventional liquid nitrogen-based freezers are not portable and require expensive infrastructure. To study the possibility of an ovarian tissue cryopreservation network not dependent on liquid nitrogen, we cryopreserved bovine ovarian tissue using three cooling techniques: a controlled rate freezer using liquid nitrogen, a liquid nitrogen-free controlled rate freezer, and liquid nitrogen-free passive cooling. Upon thawing, we evaluated a panel of viability metrics in frozen and fresh groups to examine the potency of the portable liquid nitrogen-free controlled and uncontrolled rate freezers in preserving the ovarian tissue compared to the non-portable conventional controlled rate freezer. We found similar outcomes for reactive oxygen species (ROS), total antioxidant capacity (TAC), follicular morphology, tissue viability, and fibrosis in the controlled rate freezer groups. However, passive slow cooling was associated with the lowest tissue viability, follicle morphology, and TAC, and the highest tissue fibrosis and ROS levels compared to all other groups. A stronger correlation was found between follicle morphology, ovarian tissue viability, and fibrosis with the TAC/ROS ratio compared to ROS and TAC alone. The current study undergirds the possibility of centralized cryobanks using a controlled rate liquid nitrogen-free freezer to prevent ischemia-induced damage during ovarian tissue shipment.

Diversity and ice nucleation activity of Pseudomonas syringae in drone-based water samples from eight lakes in Austria.

Bacteria from the Pseudomonas syringae complex (comprised of at least 15 recognized species and more than 60 different pathovars of P. syringae sensu stricto) have been cultured from clouds, rain, snow, streams, rivers, and lakes. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as ice+) that catalyzes the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in the U.S. and France, little is known about the genetic diversity and ice nucleation activity of P. syringae in other parts of the world. We investigated the haplotype diversity and ice nucleation activity at -8 °C (ice+) of strains of P. syringae from water samples collected with drones in eight freshwater lakes in Austria. A phylogenetic analysis of citrate synthase (cts) sequences from 271 strains of bacteria isolated from a semi-selective medium for Pseudomonas revealed that 69% (188/271) belonged to the P. syringae complex and represented 32 haplotypes in phylogroups 1, 2, 7, 9, 10, 13, 14 and 15. Strains within the P. syringae complex were identified in all eight lakes, and seven lakes contained ice+ strains. Partial 16S rDNA sequences were analyzed from a total of 492 pure cultures of bacteria isolated from non-selective medium. Nearly half (43.5%; 214/492) were associated with the genus Pseudomonas. Five of the lakes (ALT, GRU, GOS, GOL, and WOR) were all distinguished by high levels of Pseudomanas (p ≤ 0.001). HIN, the highest elevation lake, had the highest percentage of ice+ strains. Our work highlights the potential for uncovering new haplotypes of P. syringae in aquatic habitats, and the use of robotic technologies to sample and characterize microbial life in remote settings.

Comparative effects of a calcium chelator (BAPTA-AM) and melatonin on cryopreservation-induced oxidative stress and damage in ovarian tissue.

Oncology treatments cause infertility, and ovarian tissue cryopreservation and transplantation (OTCT) is the only option for fertility preservation in prepubertal girls with cancer. However, OTCT is associated with massive follicle loss. Here, we aimed to determine the effect of supplementation of slow freezing and vitrification media with BAPTA-AM and melatonin alone and in combination on ovarian tissue viability, reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), and follicular morphology and viability. Our results indicated that BAPTA-AM and melatonin can significantly improve ovarian tissue viability and the TAC/ROS ratio and reduce ROS generation in frozen-thawed ovarian tissues in slow freezing and vitrification procedures. BAPTA-AM was also found to be less effective on TAC compared to melatonin in vitrified ovarian tissue. While supplementation of slow freezing and vitrification media with BAPTA-AM and/or melatonin could increase the percentage of morphologically intact follicles in cryopreserved ovarian tissues, the differences were not significant. In conclusion, supplementation of cryopreservation media with BAPTA-AM or melatonin improved the outcome of ovarian tissue cryopreservation in both vitrification and slow freezing methods. Our data provide some insight into the importance of modulating redox balance and intracellular Ca2+ levels during ovarian tissue cryopreservation to optimize the current cryopreservation methods.

Rational synthesis of total damage during cryoprotectant equilibration: modelling and experimental validation of osmomechanical, temperature, and cytotoxic damage in sea urchin (Paracentrotus lividus) oocytes.

Sea urchins (e.g., Paracentrotus lividus) are important for both aquaculture and as model species. Despite their importance, biobanking of urchin oocytes by cryopreservation is currently not possible. Optimized cryoprotectant loading may enable novel vitrification methods and thus successful cryopreservation of oocytes. One method for determining an optimized loading protocol uses membrane characteristics and models of damage, namely osmomechanical damage, temperature damage (e.g., chill injury) and cytotoxicity. Here we present and experimentally evaluate existing and novel models of these damage modalities as a function of time and temperature. In osmomechanical damage experiments, oocytes were exposed for 2 to 30 minutes in hypertonic NaCl or sucrose supplemented seawater or in hypotonic diluted seawater. In temperature damage experiments, oocytes were exposed to 1.7 °C, 10 °C, or 20 °C for 2 to 90 minutes. Cytotoxicity was investigated by exposing oocytes to solutions of Me2SO for 2 to 30 minutes. We identified a time-dependent osmotic damage model, a temperature-dependent damage model, and a temperature and time-dependent cytotoxicity model. We combined these models to estimate total damage during a cryoprotectant loading protocol and determined the optimal loading protocol for any given goal intracellular cryoprotectant concentration. Given our fitted models, we find sea urchin oocytes can only be loaded to 13% Me2SO v/v with about 50% survival. This synthesis of multiple damage modalities is the first of its kind and enables a novel approach to modelling cryoprotectant equilibration survival for cells in general.

Meta-analysis of the Boyle van 't Hoff relation: Turgor and leak models explain non-ideal volume equilibrium.

There has been much recent attention paid to the interaction of cell volume, its regulation, and the molecular biology of the cell. Cells are generally assumed to behave as linear osmometers, with their water volume linearly proportionate to the inverse of osmotic pressure as described by the Boyle van 't Hoff (BvH) relation. This study evaluates the generality of this and other long-standing assumptions about cell responses to anisotonic conditions. We present alternative models that account for osmoregulation including mechanical resistance to volumetric expansion (the turgor model) and ion-osmolyte leakage (the leak model). To evaluate the generality of the BvH relation and determine the suitability of alternative models, we performed a comprehensive survey of the literature and a careful analysis of the resulting data, and then we used these data to compare among models. We identified 137 articles published from 1964 to 2019 spanning 14 animal species and 26 cell types and determined the BvH relation is not an appropriate general model but is adequate when restricted to the hypertonic region. In contrast, models that account for either mechanical resistance or ion-osmolyte leakage fit well to almost all collected data. The leak model has fitted parameters that are in the same range as the current literature estimate, while the turgor model typically requires an elastic modulus value of one or multiple orders of magnitude larger than literature values. However, confirmation of the underlying mechanism of osmotic regulation is required at the cell-specific level and cannot be assumed a priori.

Major Histocompatibility Complex-B haplotype and ovarian graft response.

Gonadal tissue transfer is considered one of the best methods to preserve genetic variability. Poultry hosts can receive a gonad from a donor of a different genetic background, sustain the growth of this graft, and produce gametes from it. Unfortunately, the host's strong immune response may significantly reduce the gonadal graft's ability to reach maturity. Our study aimed to evaluate the influence of MHC-B alleles in rejecting a gonadal graft of similar or different genetic backgrounds. In the first experiment, ovarian tissue was transplanted to chicks of similar genetic backgrounds, either Lohmann White (LW) with variable MHC-B or Barred Rock (BR) with fixed MHC-B. The sustained growth of donor ovarian tissues occurred in (4/7 hosts) BR (MHC-B matched) hosts only-one of these graft-positive-BR hens produced eggs derived from the donor ovary. No grafts were recovered when the host and the donor had an LW background (0/9; MHC-B mismatched). In the second experiment, ovarian transplantation was done between chicks of either similar or different genetic backgrounds (Brown Leghorn [BL], BR, and BL/BR F1). The 2 pure lines contained only one MHC-B allele, whereas the F1 heterozygotes had both. All host birds were given a daily dose of an immunosuppressant (mycophenolate mofetil) until maturity. The success rate was assessed by microsatellite genotype confirmation of donor-derived ovaries plus physiological and histological analyses of ovarian grafts. In this second experiment, 11 out of 43 ovarian hosts laid eggs. However, all fertilized eggs from these hens were derived from the remnant host ovarian tissue, not from the donor ovaries. A necropsy assessment was done on all 43 host birds. Ten donor grafts were recovered from hosts having matched (6 hosts) and mismatched (4 hosts) MHC-B, and none were functional. Interestingly, 6 of them were enclosed by a serous membrane capsule filled with fluid and had various tissue growth. In addition, clusters of immune cells were observed in all recovered donor grafts. Our results demonstrated that genetic background could greatly influence the success of gonadal transfer in chickens.

Silymarin mediated osmotic responses and damage in HepG2 cell suspensions and monolayers.

Maintenance of cells within a volume range compatible with their functional integrity is a critical determinant of cell survival after cryopreservation, and quantifying this osmotically induced damage is a part of the rational design of improved cryopreservation protocols. The degree that cells tolerate osmotic stress significantly impacts applicable cryoprotocols, but there has been little research on the time dependence of this osmotic stress. Additionally, the flavonoid silymarin has been shown to be hepatoprotective. Therefore, here we test the hypotheses that osmotic damage is time-dependent and that flavonoid inclusion reduces osmotic damage. In our first experiment, cells were exposed to a series of anisosmotic solutions of graded hypo- and hypertonicity for 10-40 min, resulting in a conclusion that osmotically induced damage is time dependent. In the next experiment, adherent cells preincubated with silymarin at the concentration of 10-4 mol/L and 10-5 mol/L showed a significant increase in cell proliferation and metabolic activity after osmotic stress compared to untreated matched controls. For instance, when adherent cells preincubated with 10-5 mol/L silymarin were tested, resistance to osmotic damage and a significant increase (15%) in membrane integrity was observed in hypo-osmotic media and a 22% increase in hyperosmotic conditions. Similarly, significant protection from osmotic damage was observed in suspended HepG2 cells in the presence of silymarin. Our study concludes that osmotic damage is time dependent, and the addition of silymarin leads to elevated resistance to osmotic stress and a potential increase in the cryosurvival of HepG2 cells.

Ovarian tissue cryopreservation and transplantation: a review on reactive oxygen species generation and antioxidant therapy.

Cancer is the leading cause of death worldwide. Fortunately, the survival rate of cancer continues to rise, owing to advances in cancer treatments. However, these treatments are gonadotoxic and cause infertility. Ovarian tissue cryopreservation and transplantation (OTCT) is the most flexible option to preserve fertility in women and children with cancer. However, OTCT is associated with significant follicle loss and an accompanying short lifespan of the grafts. There has been a decade of research in cryopreservation-induced oxidative stress in single cells with significant successes in mitigating this major source of loss of viability. However, despite its success elsewhere and beyond a few promising experiments, little attention has been paid to this key aspect of OTCT-induced damage. As more and more clinical practices adopt OTCT for fertility preservation, it is a critical time to review oxidative stress as a cause of damage and to outline potential ameliorative interventions. Here we give an overview of the application of OTCT for female fertility preservation and existing challenges; clarify the potential contribution of oxidative stress in ovarian follicle loss; and highlight potential ability of antioxidant treatments to mitigate the OTCT-induced injuries that might be of interest to cryobiologists and reproductive clinicians.

An impedance based microfluidic sensor for evaluation of individual red blood cell solute permeability.

-In this paper, we investigate a microfluidic based sensing device for cell membrane permeability measurements in real time with applications in rapid assessment of red blood cell (RBC) quality at the individual cell level. The microfluidic chip was designed with unique abilities to line up the RBCs in the centerline of the microchannel using positive dielectrophoresis (p-DEP) forces, rapid mixing of RBCs with various media (e.g. containing permeating or nonpermeating solutes) injected from different inlets to achieve high mixing efficiency. The chip detects the impedance values of the RBCs within 0.19 s from the start of mixing with other media, at ten electrodes along the length of the channel and enables time series measurements of volume change of individual cell caused by cell osmosis in anisosmotic fluids over a 0.8 s postmixing timespan. This technique enables estimating water permeability of individual cell accurately. Here we first present confirmation of a linear voltage-diameter relationship in polystyrene bead standards. Next, we show that under equilibrium conditions, the voltage-volume relationship in rat red blood cells (RBCs) is linear, corresponding to previously published Boyle van 't Hoff plots. Using rat cells as a model for human, we present the first measurement of water permeability in individual red blood cells and confirm that these data align with previously published population level values for human RBC. Finally, we present preliminary evidence for possible application of our device to identify individual RBCs infected with Plasmodium falciparum malaria parasites. Future developments using this device will address the use of whole blood with non-homogenous cell populations, a task currently performed by clinical Coulter counters.

Technologies for Vitrification Based Cryopreservation.

Cryopreservation is a unique and practical method to facilitate extended access to biological materials. Because of this, cryopreservation of cells, tissues, and organs is essential to modern medical science, including cancer cell therapy, tissue engineering, transplantation, reproductive technologies, and bio-banking. Among diverse cryopreservation methods, significant focus has been placed on vitrification due to low cost and reduced protocol time. However, several factors, including the intracellular ice formation that is suppressed in the conventional cryopreservation method, restrict the achievement of this method. To enhance the viability and functionality of biological samples after storage, a large number of cryoprotocols and cryodevices have been developed and studied. Recently, new technologies have been investigated by considering the physical and thermodynamic aspects of cryopreservation in heat and mass transfer. In this review, we first present an overview of the physiochemical aspects of freezing in cryopreservation. Secondly, we present and catalog classical and novel approaches that seek to capitalize on these physicochemical effects. We conclude with the perspective that interdisciplinary studies provide pieces of the cryopreservation puzzle to achieve sustainability in the biospecimen supply chain.

Optimizing Bull Semen Cryopreservation Media Using Multivariate Statistics Approaches.

Cryo-injury reduces post-thaw semen quality. Extender components play a protective role, but existing experimental approaches do not elucidate interactions among extender components, semen samples, and post-thaw quality. To identify optimal concentrations for 12 extender ingredients, we ran 122 experiments with an I-optimal completely random design using a large dataset from our previous study. We obtained a maximum predicted total motility of 70.56% from an I-optimal design and 73.75% from a Monte Carlo simulation. Individual bull variations were significant and interacted with extenders independently. 67% of bulls reliably preferred extender formulations to reach maximum motility. Multifactor analysis suggests that some antioxidants may offer superior protection over others. Partial least squares path modeling (PLS-PM) found the highest positive loadings for glutathione in the antioxidant class, glycerol in the CPA class, and fructose in the basic compounds class. The optimal ranges for milk, water, and ethylene glycol were extremely narrow. Egg yolk, cholesterol-loaded cyclodextrin, and nerve growth factor had medium-loading impacts. PLS-PM showed that CPA, osmoregulators, and basic components were the most efficient contributors to motility, while the antioxidant and extracellular protectant classes had less efficiency. Thus, ingredients, concentrations, and interactions of extender compounds are critical to extender formulation, especially when using multiple compounds with the same function.

Permeation of individual cryoprotectants and their different combinations into mouse liver tissue.

Development of successful tissue cryopreservation methods requires specific knowledge regarding tissue permeation of individual cryoprotective agents (CPAs) and their combinations. The present study assessed the permeation of dimethyl sulfoxide, ethylene glycol, and propylene glycol into liver tissue, and addressed whether the diffusion coefficient of individual CPAs changes when combining CPAs. To do this, mouse liver slices were exposed at room temperature to 3.5 mol/L concentrations of CPAs individually or in combination for 15, 30, 45, and 60 min. Subsequently, tissue CPA concentrations were determined using a gas chromatography/mass spectrometry (GC/MS) method. Our results show that (1) the GC/MS method allows measurement of multiple CPA concentrations in a single small tissue sample, (2) dimethyl sulfoxide has a higher diffusion coefficient than ethylene glycol and propylene glycol, and (3) the CPA diffusivity appears to decrease in mixtures with multiple CPAs. These findings may help the development of effective tissue cryopreservation methods.

Machine learning and hypothesis driven optimization of bull semen cryopreservation media.

Cryopreservation provides a critical tool for dairy herd genetics management. Due to widely varying inter- and within-bull post thaw fertility, recent research on cryoprotectant extender medium has not dramatically improved suboptimal post-thaw recovery in industry. This progress is stymied by the interactions between samples and the many components of extender media and is often compounded by industry irrelevant sample sizes. To address these challenges, here we demonstrate blank-slate optimization of bull sperm cryopreservation media by supervised machine learning. We considered two supervised learning models: artificial neural networks and Gaussian process regression (GPR). Eleven media components and initial concentrations were identified from publications in bull semen cryopreservation, and an initial 200 extender-post-thaw motility pairs were used to train and 32 extender-post-thaw motility pairs to test the machine learning algorithms. The median post-thaw motility after coupling differential evolution with GPR the increased from 52.6 ± 6.9% to 68.3 ± 6.0% at generations 7 and 17 respectively, with several media performing dramatically better than control media counterparts. This is the first study in which machine learning was used to determine the best combination of constituents to optimize bull sperm cryopreservation media, and provides a template for optimization in other cell types.

Cryopreservation of Semen in Domestic Animals: A Review of Current Challenges, Applications, and Prospective Strategies.

Cryopreservation is a way to preserve germplasm with applications in agriculture, biotechnology, and conservation of endangered animals. Cryopreservation has been available for over a century, yet, using current methods, only around 50% of spermatozoa retain their viability after cryopreservation. This loss is associated with damage to different sperm components including the plasma membrane, nucleus, mitochondria, proteins, mRNAs, and microRNAs. To mitigate this damage, conventional strategies use chemical additives that include classical cryoprotectants such as glycerol, as well as antioxidants, fatty acids, sugars, amino acids, and membrane stabilizers. However, clearly current protocols do not prevent all damage. This may be due to the imperfect function of antioxidants and the probable conversion of media components to more toxic forms during cryopreservation.

Real-time computer assisted measurement of oocyte and embryo volume for assessment of transport parameters.

Cryopreservation of gametes has revolutionized both animal agriculture and human reproductive medicine. Although many new technologies have tremendously improved the cryopreservation of oocytes and embryos, osmotic stress encountered during the equilibration process can cause their loss of function. Rational cryoprotective agent (CPA) equilibration strategies can be used to minimize this stress but require trained personnel to monitor the process in individual oocytes or embryos or require the use of suboptimal average transport parameter values in mathematically guided protocols. To enable individually optimized equilibration of CPAs in individual cells, here we establish experimental and computational techniques to track the osmotic behavior of individual bovine oocytes and embryos during CPA equilibration in real time. We designed a microfluidic device to provide a controlled flow of CPA and modified standard image analysis techniques to estimate real-time cell volume changes. In particular, we used a level-set method to define a boundary within a contour plot which could automate the image analysis process. A colour based level set algorithm coupled with contour smoothing not only provided the best fit but also reduced the segmentation time to well under a second per image. The accuracy of the automated method was comparable to human segmented images for both oocytes and embryos. This technology should enable both rapid evaluation of key biophysical parameters in oocytes and embryos undergoing CPA equilibration and the development of real-time feedback-control of CPA equilibration, enabling individual oocyte- and embryo-specific optimal protocols.

Multiple cryoprotectant toxicity model for vitrification solution optimization.

Vitrification is a promising cryopreservation technique for complex specimens such as tissues and organs. However, it is challenging to identify mixtures of cryoprotectants (CPAs) that prevent ice formation without exerting excessive toxicity. In this work, we developed a multi-CPA toxicity model that predicts the toxicity kinetics of mixtures containing five of the most common CPAs used in the field (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide). The model accounts for specific toxicity, non-specific toxicity, and interactions between CPAs. The proposed model shows reasonable agreement with training data for single and binary CPA solutions, as well as ternary CPA solution validation data. Sloppy model analysis was used to examine the model parameters that were most important for predictions, providing clues about mechanisms of toxicity. This analysis revealed that the model terms for non-specific toxicity were particularly important, especially the non-specific toxicity of propylene glycol, as well as model terms for specific toxicity of formamide and interactions between formamide and glycerol. To demonstrate the potential for model-based design of vitrification methods, we paired the multi-CPA toxicity model with a published vitrification/devitrification model to identify vitrifiable CPA mixtures that are predicted to have minimal toxicity. The resulting optimized vitrification solution composition was a mixture of 7.4 molal glycerol, 1.4 molal DMSO, and 2.4 molal formamide. This demonstrates the potential for mathematical optimization of vitrification solution composition and sets the stage for future studies to optimize the complete vitrification process, including CPA mixture composition and CPA addition and removal methods.

Hydroxytyrosol and resveratrol improves kinetic and flow cytometric parameters of cryopreserved bull semen with low cryotolerance.

There is considerable interest in breeding programs to "rescue" semen with poor post-thaw fertility from bulls known as "bad freezers". We hypothesized that there may be an interaction between the post-thaw recovery of sperm and the efficacy of antioxidant addition to extenders. The current study assesses the effects of antioxidant additives hydroxytyrosol (HT) and resveratrol (RSV) on the post-thaw semen parameters in two groups of bulls classified as either high or low cryotolerant (i.e., "good" and "bad" freezers). Semen samples were collected from 40 bulls and processed in the extenders containing different concentrations of HT (experiment 1; 0, 25 and 50 µM) and RSV (experiment 2; 0.0, 0.01, 0.1 and 1 mM). In experiment 1, bulls in the low cryotolerance group had a significant improvement in post-thaw recovery at 25 µM and 50 µM (P < 0.05). These improvements were observed in motility and several cellular parameters. However, post-thaw semen quality in the high cryotolerance group was not significantly affected by the HT addition. In experiment 2, although RSV did not have any positive impact in high cryotolerance group, post-thaw recovery in the low cryotolerance bulls was significantly improved in 0.1 mM RSV. Oxidative stress markers in either high or low cryotolerance groups were not significantly changed by RSV addition (P > 0.05). It can be concluded that addition of optimized concentrations of HT and RSV to the extender could be a strategy for improving the post-thaw semen, especially in bulls with high genetic merit but low initial cryotolerance.

Exogenous Melatonin Ameliorates the Negative Effect of Osmotic Stress in Human and Bovine Ovarian Stromal Cells.

Ovarian tissue cryopreservation transplantation (OTCT) is the most flexible option to preserve fertility in women and children with cancer. However, OTCT is associated with follicle loss and an accompanying short lifespan of the grafts. Cryopreservation-induced damage could be due to cryoprotective agent (CPA) toxicity and osmotic shock. Therefore, one way to avoid this damage is to maintain the cell volume within osmotic tolerance limits (OTLs). Here, we aimed to determine, for the first time, the OTLs of ovarian stromal cells (OSCs) and their relationship with reactive oxygen species (ROS) and mitochondrial respiratory chain activity (MRCA) of OSCs. We evaluated the effect of an optimal dose of melatonin on OTLs, viability, MRCA, ROS and total antioxidant capacity (TAC) of both human and bovine OSCs in plated and suspended cells. The OTLs of OSCs were between 200 and 375 mOsm/kg in bovine and between 150 and 500 mOsm/kg in human. Melatonin expands OTLs of OSCs. Furthermore, melatonin significantly reduced ROS and improved TAC, MRCA and viability. Due to the narrow osmotic window of OSCs, it is important to optimize the current protocols of OTCT to maintain enough alive stromal cells, which are necessary for follicle development and graft longevity. The addition of melatonin is a promising strategy for improved cryopreservation media.

Sexual Violence and Nonconsensual Experiences Among Alt-Sex Communities' Members.

BACKGROUND: As behaviors, alternative sexual (alt-sex) (i.e., kink, bondage, discipline, dominance, submission, sadism, and masochism , consensual non-monogamy, swinging, leather, and fetish practices) practitioners often emphasize that consent and boundaries are key elements of alt-sex activities. Despite these emphases, individuals experience consent violations and sexual assault both prior to engaging and during their involvement in alt-sex activities. PURPOSE: This study examines alt-sex practitioners' sexual assault and nonconsensual experiences in order to highlight potential means of intervention and prevention, as well as inform clinical and legal professionals. METHODS: In collaboration with the National Coalition for Sexual Freedom, this study uses an international survey of adults in alt-sex communities (N = 2996) to examine sexual assault and nonconsensual experiences both within and outside of alt-sex contexts. RESULTS: We found a lower rate of consent violations in the alt-sex community (26%) compared to sexual assault as an adult outside of alt-sex contexts (34%) and sexual assault as a minor (40%). We found significant differences by groups in sexual assault as a minor (gender, sexual orientation, age, and live in the US or not), sexual assault outside of alt-sex contexts (gender, sexual orientation, and age), nonconsensual experiences in alt-sex contexts (gender, sexual orientation, age, and race), receiving nonconsensual touch in alt-sex contexts (gender, sexual orientation, and age), giving nonconsensual touch in alt-sex contexts (sexual orientation, age, living in the US or not, and race), and being falsely accused of nonconsensual touching in alt-sex contexts (gender, age, and living in the US or not). Within the most recent consent violation, the most common behaviors were non-kink related, except for lack of aftercare. Nearly 40% of participants reported the reasons for their most recent consent violation in alt-sex contexts were being selfish or caught up in the moment. IMPLICATIONS: Focused interventions are needed to address how different populations are experiencing assault and violations in alt-sex contexts.