High Concentrations Of Cryoprotectants Research Articles

Cryopreservation of human kidney organoids

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1’s efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.

P-116 Relationship between exposure in equilibration solution pre-vitrification for human collapsed blastocysts and miscarriage rate in ART treatments

Abstract Study question To explore the relationship between equilibration time pre-vitrification of human collapse blastocyst and clinical pregnancy and miscarriage rate in patients undergoing ART treatments Summary answer The miscarriage rate was significantly lower in the group with a short equilibration time of 7-8 minutes compared to the group of 9-10 minutes What is known already The introduction of vitrification represents one of the most important advancement in ART. However, protocols currently applied to cryopreserve human embryos still have some weak points that might be improved. A critical aspect is represented by the high concentration of cryoprotectants (CPAs) used during the vitrification and some of this CPAs might impact negatively cellular metabolism and function. Thus, this study investigated whether a shorter time in the equilibration solution before vitrification of artificially collapsed blastocysts might have an impact on survival and pregnancy outcomes, as well as live birth rate and risk of miscarriage in patients undergoing ART. Study design, size, duration This prospective study was performed at the Centre for Reproductive Medicine, Haikou Mary Hospital, China from March 2018 to May 2022. Informed consent for experimentation with human subjects was obtained before the patients start the ovarian stimulation. Female age <35 years, and causes of infertility included male factors, female infertility, and unexplained infertility. Following the fresh embryo transfer, supernumerary good quality blastocysts ≥ 2, according to Gardner’s score were vitrified and allocated as discussed below Participants/materials, setting, methods The study included a total of 831 expanded blastocysts, which were divided into two groups according to the equilibration time before vitrification: group (A) 7-8 minutes (413); and group (B) 9-10 minutes (418). Expanded blastocysts (grade 3 or more) were artificially shrunk by applying one or two laser pulses. Patients were included in the study only when at least two blastocysts were available for vitrification, in order to be allocated one in each group Main results and the role of chance Results: A total of 831 vitrified-warmed blastocysts were analysed in this study, of which 825 survived at the warming step (99.3%: 825/831). All surviving blastocysts were transferred in 585 embryo transfers. Overall, the clinical pregnancy rate per transfer was CPR: 68.5% (401/585), and live birth and miscarriage rates: 61.0% (357/585) and 11.0% (44/401). No significant differences were observed between the two groups regarding the mean age of patients, the average number of blastocysts transferred, the basal FSH, BMI, and infertility duration. Results show the same survival rate after warming for group A (99.3%) and group B (99.3%), as well as similar CPR (A: 69.1% versus B: 68.0%).The live birth (A: 63.8% versus B: 58.3%) and multiple gestation rates (A: 20.8% versus B: 23.5%) were comparable in the two groups. When analysing the overall miscarriage rate, data displayed a statistically significant difference (P < 0.05) in favour of group A (7.6%) compared to group B (14.2%). There were no differences between the two groups concerning the prevalence of male babies (A: 57.1% and B: 55.4%), average gestational length (A: 38.67±1.37 versus B: 38.33±1.21), preterm birth rate (A: 19.2% versus B: 20.6%), and birth weight (A: 2.95±0.58 versus B: 3.05±0.63 kg). Limitations, reasons for caution This is not a randomized controlled trial, it is a prospective observational cohort study aiming to calculate the effect of a shorted ES time on the risk of miscarriage. Also, potential confounding factors due to the heterogeneous nature of the sample investigated may impair the validity of our conclusions. Wider implications of the findings Results demonstrate that a shorter equilibration time resulted in optimal survival, clinical pregnancy, and live birth rates compared to exposure to ES for 9-10. Thus, suggesting that a longer exposure to the ES is not needed, and might negatively effect cellular metabolism and function, increasing the risk of miscarriage. Trial registration number Not applicatione

Testicular Tissue Vitrification: a Promising Strategy for Male Fertility Preservation.

Destruction of spermatogonial stem cells in juvenile men survivors of pediatric cancers leads to infertility as a side effect of gonadotoxic therapies. Sperm freezing before cancer treatment is commonly used in the clinic for fertility preservation, but this method is not applicable for prepubertal boys due to the lack of mature sperm. In these cases, cryopreservation of testicular tissues is the only option for fertility preservation. Although controlled slow freezing (CSF) is the most common procedure for testicular tissue cryopreservation, vitrification can be used as an alternative method. Controlled vitrification has prevented cell damage and formation of ice crystals. Procedures were done easily and quickly with a brief exposure time to high concentration of cryoprotectants without expensive equipment. Different studies used vitrification of testicular tissues and they assessed the morphology of seminiferous tubules, apoptosis, and viability of spermatogonial cells. Transplantation of vitrified testicular tissue into infertile recipient mice as well as in vitro culture of vitrified tissues was done in previous studies and their findings showed complete spermatogenesis and production of mature sperm. Review articles usually have compared controlled slow freezing with vitrification. In this review, we focused only on the vitrification method and its results. Despite promising results, many studies have been done for finding an optimal cryopreservation protocol in order to successfully preserve fertility in prepubertal boys.

The Effect of Cryopreservation on the Survival of Nocardia farcinica and Yersinia pestis vaccine strains.

Pathogenic microorganisms are valuable biological resources, closely related to biosecurity, human health, environmental protection, and renewable energy. It is very important to properly preserve the microbial resources by methods to maintain the purity, viability, and integrity, and to avoid prolonged degradation. The present work aims to explore the cryopreservation technology of Nocardia farcinica (Gram-positive bacteria) and Yersinia pestis vaccine strains (Gram-negative bacteria). The effects of cryoprotectants (CPAs), freezing temperature, and freeze-thaw cycles on the two bacteria in the cryopreservation process were studied. The results showed that the addition of CPAs (glycerol, propylene glycol, sucrose, glucose, l-carnitine, l-proline, and skim milk) significantly enhanced the survival rates of the N. farcinica and Y. pestis vaccine strains. However, high concentrations of CPAs can produce biochemical toxicity in the two pathogens. The utilization of composite CPAs not only reduced the toxicity but also improved the survival rates of samples during cryopreservation. The optimal composite CPA for N. farcinica is 0.292 M sucrose, 0.62 M l-carnitine, and 2.82 M glycerol. The optimal composite CPA for Y. pestis is 0.62 M l-carnitine, 8.46 M glycerin, and 0.292 M sucrose. The results showed that the quality of the strains stored at -80°C and -196°C was better. For the case of freeze-thaw cycles, the two pathogens have different degrees of reduction, and the survival rate of Y. pestis decreased more than that of N. farcinica. The uniform distribution of bacteria in CPAs can form uniform nucleation sites in the solution system, which is beneficial to the cryopreservation of strains, as can be seen from the experimental results from a differential scanning calorimeter. This study may provide a reference for better preservation of precious natural biological resources of pathogenic microorganisms.

Cryoprotective Metabolites Are Sourced from Both External Diet and Internal Macromolecular Reserves during Metabolic Reprogramming for Freeze Tolerance in Drosophilid Fly, Chymomyza costata.

Many cold-acclimated insects accumulate high concentrations of low molecular weight cryoprotectants (CPs) in order to tolerate low subzero temperatures or internal freezing. The sources from which carbon skeletons for CP biosynthesis are driven, and the metabolic reprogramming linked to cold acclimation, are not sufficiently understood. Here we aim to resolve the metabolism of putative CPs by mapping relative changes in concentration of 56 metabolites and expression of 95 relevant genes as larvae of the drosophilid fly, Chymomyza costata transition from a freeze sensitive to a freeze tolerant phenotype during gradual cold acclimation. We found that C. costata larvae may directly assimilate amino acids proline and glutamate from diet to acquire at least half of their large proline stocks (up to 55 µg per average 2 mg larva). Metabolic conversion of internal glutamine reserves that build up in early diapause may explain the second half of proline accumulation, while the metabolic conversion of ornithine and the degradation of larval collagens and other proteins might be two additional minor sources. Next, we confirm that glycogen reserves represent the major source of glucose units for trehalose synthesis and accumulation (up to 27 µg per larva), while the diet may serve as an additional source. Finally, we suggest that interconversions of phospholipids may release accumulated glycero-phosphocholine (GPC) and -ethanolamine (GPE). Choline is a source of accumulated methylamines: glycine-betaine and sarcosine. The sum of methylamines together with GPE and GPC represents approximately 2 µg per larva. In conclusion, we found that food ingestion may be an important source of carbon skeletons for direct assimilation of, and/or metabolic conversions to, CPs in a diapausing and cold-acclimated insect. So far, the cold-acclimation- linked accumulation of CPs in insects was considered to be sourced mainly from internal macromolecular reserves.

General tissue mass transfer model for cryopreservation applications

Successful cryopreservation of complex specimens, such as tissues and organs, would greatly benefit both the medical and scientific research fields. Vitrification is one of the most promising techniques for complex specimen cryopreservation, but toxicity remains a major challenge because of the high concentration of cryoprotectants (CPAs) needed to vitrify. Our group has approached this problem using mathematical optimization to design less toxic CPA equilibration methods for cells. To extend this approach to tissues, an appropriate mass transfer model is required. Fick’s law is commonly used, but this simple modeling framework does not account for the complexity of mass transfer in tissues, such as the effects of fixed charges, tissue size changes, and the interplay between cell membrane transport and transport through the extracellular fluid. Here, we propose a general model for mass transfer in tissues that accounts for all of these phenomena. To create this model, we augmented a previously published acellular model of mass transfer in articular cartilage to account for the effects of cells. We show that the model can accurately predict changes in CPA concentration and tissue size for both articular cartilage and pancreatic islets, tissue types with vastly different properties.

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for Me2SO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification.

Sand-mediated ice seeding enables serum-free low-cryoprotectant cryopreservation of human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and/or introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above −10 °C, which enables cryopreservation of hiPSCs with no serum, much-reduced cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by their pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.

Mass transfer into biological matter using isochoric freezing

This paper is a theoretical study of a protocol for transport of high concentrations of cryoprotectants into biological matter, using isochoric freezing. Unlike isobaric freezing, where the entire system freezes at temperatures lower than the freezing temperature, in isochoric freezing a substantial portion of the system remains unfrozen at temperatures below freezing. In isochoric freezing cryopreservation, the system is designed in such a way that the biological matter remains unfrozen and surrounded by an unfrozen solution. The protocol in this study involves the freezing of an isochoric systems along the “liquidus line” at which water and ice are in thermodynamic equilibrium. Rejection of solutes by ice increases the concentration of the solutes in the unfrozen solution surrounding the unfrozen biological matter, leading, thereby, to transport of increasingly higher concentrations of cryoprotectants into the biological matter, as the temperature of the system is lowered and the toxicity of the cryoprotectants is reduced.

Effect of vitrification on global gene expression dynamics of bovine elongating embryos.

Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming in the cryopreservation process. Many of these factors can potentially affect gene expression. In this study, invitro-produced bovine embryos at the blastocyst stage were subjected to vitrification. Four recipients each were used for transferring non-vitrified (n=80) and vitrified (n=80) embryos. A total of 12 non-vitrified and 9 vitrified viable day-14 (D14) embryos were recovered by uterine flushing. RNA-seq analysis of the whole embryo or isolated trophectoderm (TE) from vitrified and fresh recovered D14 embryos revealed a total of 927 and 4376 genes with changed expression in embryos and TE isolates, respectively, as a result of vitrification. In addition, we found 671 and 61 genes commonly up- or downregulated in both vitrified whole embryos and TE. Commonly upregulated pathways by vitrification included epithelial adherens junctions, sirtuin signalling, germ cell-sertoli cell junction, ATM signalling, NER and protein ubiquitination pathways. The commonly downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling and mTOR signalling pathways. Our analysis identified specific pathways and implicated specific gene expression patterns affecting embryo developmental competence that are important to cryopreservation.

Equilibrium vitrification of mouse embryos using low concentrations of cryoprotectants

Previously, we developed a method for vitrification of mouse embryos in a near-equilibrium state using EFS35c, PB1 medium containing 35% (v/v) ethylene glycol, and 0.98 M sucrose. This method has advantages in both slow freezing and vitrification. However, since the vitrification solution in this method contains high concentrations of cryoprotectants and thus has high osmolality, the solution would injure oocytes and embryos with high sensitivity to chemical toxicity and high osmolality. In this study, we examined whether embryos could be vitrified in a near-equilibrium state using a solution containing low concentrations of cryoprotectants and thus with low osmolality. To investigate whether embryos were vitrified in a near-equilibrium state, 2-cell mouse embryos were vitrified with EDFS10/10a, PB1 medium containing 10% (v/v) ethylene glycol, 10% (v/v) DMSO, and 0.4 M sucrose, in liquid nitrogen, stored at −80 °C for 4–28 days, and warmed in water at 25 °C. The viability of the embryos was evaluated by the appearance of embryos after warming and developmental ability. When embryos were vitrified in liquid nitrogen using EDFS10/10a, the survival and developmental ability into blastocysts after storage at −80 °C for 7 days were high, indicating that embryos were vitrified in a near-equilibrium state. A high proportion of embryos vitrified with EDFS10/10a developed to term after transportation with dry ice, re-cooling in liquid nitrogen, and transfer to recipients. Therefore, new equilibrium vitrification developed in this study may be useful for oocytes and embryos that are highly sensitive to the toxicity of cryoprotectants and high osmolality.

Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.

Assessment of Survivability of Vitrified Embryos Depending on the Stage of Development

Freezing embryos of excellent and good quality allows you to maintain reproductive potential for patientsin the future and achieve pregnancy using one stimulation cycle. Cryopreservation of embryos can becarried out at any stage of development. Vitrification as a method of cryopreservation combines the use ofhighly concentrated solutions of cryoprotectants and rapid (almost instantaneous) cooling by immersingsamples directly in liquid nitrogen. Due to this, the main goal of vitrification is achieved - the absence of theformation of ice crystals, which can damage the cell structure. The advantages of vitrification are as follows:a high concentration of cryoprotectants and a high cooling/thawing rate eliminate the damage caused by theformation of intracellular ice, as well as the undesirable osmotic effects of dehydration and rehydration dueto extracellular crystal formation.

The recent state of cryopreservation techniques for ex-situ gene conservation and breeding purposes in small ruminants: A review

The viewpoint of the recent cryopreservation techniques (CT) suggests the use of a reduced volume of cryopreservation solution, high concentration of cryoprotectants and ultra-rapid cooling and warming rates help to reduce cryo-injury and maximize the viability of the preserved animal genetic resources (AnGR). The CT had now become widely accepted as one of the best methods of choice for the ex-situ conservation of AnGR due to its high success rate recorded and no-invasive nature as compared to the conventional slow rate freezing (CSRF). Rapid advances and wide acceptability of the use of assisted reproductive technologies (ART’s) particularly artificial insemination (AI) in animal breeding had resulted in a greater loss of a large number of good quality genes in virtually almost all the native breeds of animals across the globe. Small ruminant (SR) animals are not an exception in such present predicaments situation of erosion and dilution of the valuable AnGR among the native breeds. As a result of this, 148 and 16 breeds of sheep and goats respectively have already become extinct in Europe and the Caucasus. In view of the aforementioned situation, the present review aimed at exploring some of the current states of development, roles played and potentials of CT in the conservation of SR genes and genome for the immediate and future breeding purposes for sustainable development. It basically covers; animal genetic resource, the need to conserve AnGR, tools for ex situ in vitro conservation of AnGR and recent developments in breeding and cryopreservation of SR AnGR.
 Cryopreservation is playing a pivotal role in ex-situ gene conservation of AnGR. Decline in genetic diversity among SR breed population was high in Europe and the Caucasus. There is therefore, need for improvent on current stringent measures on conservation of AnGR in this region of the world.

29 Time-lapse analysis of bovine embryos derived after invitro fertilization from vitrified and fresh oocytes

Oocyte vitrification enables the long-term conservation of female genetic resources. However, obtaining viable bovine embryos from vitrified oocytes has proven to be difficult. Embryo development is a dynamic process, and critical stages can go unnoticed with the use of traditional morphologic assessments. Therefore, the aim was to evaluate morphokinetic parameters in embryos derived from fresh versus vitrified oocytes. After 22h of IVM, oocytes were divided into (1) oocytes surrounded by multiple layers of cumulus cells (COCs; n=275) and (2) oocytes partially denuded, leaving only corona radiata (CR; n=178). Then, two-thirds of the CR oocytes were subjected to one of two vitrification protocols as follows: high concentration of cryoprotectants (CR-H; n=171; Ortiz-Escribano et al. 2016 Theriogenology 86, 635-641) and low concentration of cryoprotectants (CR-L; n=163; Ishii et al. 2018 J. Reprod. Develop. 6). After warming, vitrified oocytes were incubated ~2h in maturation medium. Invitro fertilization and culture were performed simultaneously for all groups. Four to eight zygotes from each group were assigned randomly to time lapse (Primo Vision; Vitrolife), and group culture was performed in leftovers as a control. Differences between groups in survival (intact zygotes after IVF), cleavage, and blastocyst rates were evaluated by logistic regression. Morphokinetic data (time to reach first (1-2 cells), second (3-4 cells), third (5-8), fourth (9-16 cells), fifth (>16 cells), cleavage, and blastocyst stage, and time in lag phase) were investigated. Survival rates in COCs (98%) and CR (96%) groups were not different; CR-H (87%) showed lower survival than COCs but similar survival to that of CR. Group CR-L (82%) had a lower survival rate than the rest of the groups (P<0.05). Higher cleavage rate (83%) was found in COCs compared with the rest of the groups. The CR and CR-H groups showed similar cleavage rate (65 and 55%, respectively), whereas CR-L had a lower cleavage rate (42%) than other groups (P<0.05). As expected, both vitrified groups showed lower blastocyst rates (4% for CR-H and 10% for CR-L) than fresh COCs (44%; P<0.05). Morphokinetics measures were affected by the treatments: time to reach first cleavage was similar for COCs (35.3h), CR (38.4h), and CR-L (34.8h), whereas CR-H was slower (42.4h). However, the fourth division was reached earlier by CR-H (51.7h), which was significantly faster than for CR-L (88.8 h; P<0.05) but similar for COCs (71.3h) and CR (60.7h). In conclusion, more morphokinetic data are needed to compare different vitrification methods and confirm whether time-lapse analysis can be used to predict blastocyst outcome after oocyte vitrification.

31 Effect of vitrification on global gene expression dynamics of bovine elongating embryos

The ability to cryopreserve gametes and embryos has been a valuable tool for reproductive management in all mammalian species, especially livestock. Embryo vitrification involves exposure to high concentrations of cryoprotectants and osmotic stress during cooling and warming. These factors have to affect gene expression. The elongating embryo is a stage of embryo development that can be recovered noninvasively in the cow on day (D) 14 and represents a critical stage of development when many embryos die. In this study, we aimed to evaluate the effect of vitrification on the transcriptome dynamics of D14 embryos by RNA sequencing (RNA-seq). Invitro blastocyst-stage embryos were vitrified by exposure to dimethyl sulfoxide and ethylene glycol solution, followed by placing on Cryo Loks and plunging in liquid nitrogen. After warming, embryos were loaded into straws and transferred into eight synchronized recipients, four cows received nonvitrified embryos and four cows received vitrified embryos (20 embryos per cow). Embryo flushing yielded 12 nonvitrified and 9 vitrified viable D14 embryos. Whole embryos (six nonvitrified and two vitrified embryos) or isolated trophectoderm (TE; four nonvitrified and seven vitrified) were processed for RNA-seq. The Smart-sEqn 2 protocol was followed to prepare RNA-seq libraries. Sequencing reads were prefiltered and aligned to the bovine genome, and gene expression values were calculated as fragments per kilobase of transcript per million mapped reads. Genes were deemed differentially expressed between treatments if they showed a false discovery rate P-value<0.05 and fold-change >2. Ingenuity pathway analysis was used to reveal gene ontology and pathways. Expression of 927 genes was changed in D14 embryos as a result of vitrification, with 782 and 145 genes upregulated and downregulated, respectively. In TE, vitrification resulted in 4096 and 280 upregulated or downregulated genes, respectively. Several pathways were upregulated by vitrification in both whole embryos and TE, including epithelial adherens junctions, sirtuin signalling, germ cell-Sertoli cell junction, ATM signalling, nucleotide excision repair, and protein ubiquitination pathways. Downregulated pathways included EIF2 signalling, oxidative phosphorylation, mitochondrial dysfunction, regulation of eIF4 and p70S6K signalling, mammalian target of rapamycin signalling, sirtuin singling, and nucleotide excision repair pathways. In addition, we found 671 and 61 genes upregulated and downregulated in both vitrified whole embryos and TE. Mitochondrial dysfunction and oxidative phosphorylation signalling were upregulated, whereas epithelial adherens junction and sirtuin signalling were downregulated, suggesting mitochondrial function and energy production were impaired in TE after vitrification. Our analysis identified specific pathways and implicated specific genes affected by cryopreservation and potentially affecting embryo developmental competence.

Stepped vitrification technique for human ovarian tissue cryopreservation

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants.

Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture.

Thermodynamic Theory and Experimental Validation of a Multiphase Isochoric Freezing Process.

Freezing of the aqueous solutions that comprise biological materials, such as isotonic physiological saline, results in the formation of ice crystals and the generation of a hypertonic solution, both of which prove deleterious to biological matter. The field of modern cryopreservation, or preservation of biological matter at subfreezing temperatures, emerged from the 1948 discovery that certain chemical additives such as glycerol, known as cryoprotectants, can protect cells from freeze-related damage by depressing the freezing point of water in solution. This gave rise to a slew of important medical applications, from the preservation of sperm and blood cells to the recent preservation of an entire liver, and current cryopreservation protocols thus rely heavily on the use of additive cryoprotectants. However, high concentrations of cryoprotectants themselves prove toxic to cells, and thus there is an ongoing effort to minimize cryoprotectant usage while maintaining protection from ice-related damage. Herein, we conceive from first principles a new, purely thermodynamic method to eliminate ice formation and hypertonicity during the freezing of a physiological solution: multiphase isochoric freezing. We develop a comprehensive thermodynamic model to predict the equilibrium behaviors of multiphase isochoric systems of arbitrary composition and validate these concepts experimentally in a simple device with no moving parts, providing a baseline from which to design tailored cryopreservation protocols using the multiphase isochoric technique.

The movement of water and cryoprotectants in mammalian oocytes and embryos: Membrane permeability and aquaporins

INTRODUCTION During the cryopreservation of cells, intracellular ice formation is the most common cause of cell death. To prevent this in oocytes/embryos, cytoplasm must be vitried using cell-permeating cryoprotectants, which can cause other major types of cell injury; for example, chemical toxicity of cryoprotectant during loading and osmotic swelling during removal of permeated cryoprotectant.1 Especially in vitrication, the potential toxicity of the nal vitrication solution is high, because of the high concentration of cryoprotectants needed to achieve the vitried state. erefore, the exposure time of oocytes/embryos to the vitrication solution has to be limited. However, short exposure times can cause insuf-cient permeation by the cryoprotectant and insucient dehydration/concentration of the cytoplasm, resulting in intracellular ice formation. To prevent these major injuries during vitrication, rapid movement of water and cryoprotectants is preferable.