Cryoprotectant Research Articles

Effects of Cryoprotectant Concentration and Exposure Time during Vitrification of Immature Pre-Pubertal Lamb Cumulus-Oocyte Complexes on Nuclear and Cytoplasmic Maturation.

Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.

Cryopreserving the intact intervertebral disc without compromising viability.

Tissue cryopreservation requires saturation of the structure with cryoprotectants (CPAs) that are also toxic to cells within a short timeframe unless frozen. The race between CPA delivery and cell death is the main barrier to realizing transplantation banks that can indefinitely preserve tissues and organs. Unrealistic cost and urgency leaves less life-threatening ailments unable to capitalize on traditional organ transplantation systems that immediately match and transport unfrozen organs. For instance, human intervertebral discs (IVD) could be transplanted to treat back pain or used as ex vivo models for studying regenerative therapies, but both face logistical hurdles in organ acquisition and transport. Here we aimed to overcome those challenges by cryopreserving intact IVDs using compressive loading and swelling to accelerate CPA delivery. CPAs were tested on bovine nucleus pulposus cells to determine the least cytotoxic solution. Capitalizing on our CPAs Computed Tomography (CT) contrast enhancement, we imaged and quantified saturation time in intact bovine IVDs under different conditions in a bioreactor. Finally, the entire protocol was tested, including 1 week of frozen storage, to confirm tissue viability in multiple IVD regions after thawing. Results showed cryopreserving medium containing dimethyl sulfoxide and ethylene glycol gave over 7.5 h before cytotoxicity. While non-loaded IVDs required over 3 days to fully saturate, a dynamic loading protocol followed by CPA addition and free-swelling decreased saturation time to <5 h. After cryopreserving IVDs for 1 week with the optimized CPA and permeation method, all IVD regions had 85% cell viability, not significantly different from fresh unfrozen controls. This study created a novel solution to a roadblock in IVD research and development. Using post-compression swelling CPA can be delivered to an intact IVD over 20× more quickly than previous methods, enabling cryopreservation of the IVD with no detectable loss in cell viability.

Trehalose, Glycerol and Dimethyl Sulfoxide Supplementation for Cryopreservation Media of Honey Bee Drone Semen

The cryogenic storage of honey bee drone (Apis mellifera) semen is a rapidly developing practice, driven by the need to preserve bee colonies and the decline in gamete quality following cryopreservation. Advances in cryobiology, coupled with procedural refinements and the development of new methods for cryopreservation of male gametes at different temperatures, provide promising results for improving drone semen preservation. Innovative strategies involve the incorporation of additives within preservation media to enhance important spermatozoa parameters. Our study aimed to assess the effectiveness of cryoprotectants (CPAs), namely trehalose, glycerol, and dimethyl sulfoxide (DMSO), on the motion characteristics of honey bee drone sperm cells during cryopreservation and subsequent thawing. Using Sperm Class Analyzer (SCA) and Computer-Assisted Sperm Analysis (CASA), we evaluated the impact of these supplements on sperm motility and kinetic parameters. Our findings indicated significant differences in sperm motility (non-progressiveness and progressiveness) and kinetics (curvilinear velocity and linearity) across treatments with trehalose, DMSO, and glycerol. Trehalose supplementation retained significantly higher percentages of sperm motility and kinetic parameters post-cryopreservation compared to DMSO and glycerol. Nevertheless, despite the utilization of new protocols, media, and supplementation, the results obtained are often unsatisfactory. The research provides valuable information for enhancing the efficacy of cryopreservation media to optimize the successful outcome of honey bee drone spermatozoa storage.

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 &amp;lt;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 &amp;lt; 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

Multifunctional Laser-Induced Graphene-Based Microfluidic Chip for High-Performance Oocyte Cryopreservation with Low Concentration of Cryoprotectants.

Oocyte cryopreservation is essential in the field of assisted reproduction, but due to the large size and poor environmental tolerance of oocytes, cell freezing technology needs further improvement. Here, a Y-shaped microfluidic chip based on 3D graphene is ingeniously devised by combining laser-induced graphene (LIG) technology and fiber etching technology. The prepared LIG/PDMS microfluidic chip can effectively suppress ice crystal size and delay ice crystal freezing time by adjusting surface hydrophobicity. In addition, LIG endows the microfluidic chip with an outstanding photothermal effect, which allows to sharply increase its surface temperature from 25 to 71.8°C with 10s of low-power 808nm laser irradiation (0.4Wcm-2). Notably, the LIG/PDMS microfluidic chip not only replaces the traditional cryopreservation carriers, but also effectively reduces the dosage of cryoprotectants (CPAs) needed in mouse oocyte cryopreservation. Even when the concentration of CPAs is cut in half (final concentration of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO)), the survival rate of oocytes is still as high as 92.4%, significantly higher than the control group's 85.8%. Therefore, this work provides a novel design strategy to construct multifunctional microfluidic chips for high-performance oocytes cryopreservation.

Freezing Hu ram semen: influence of different penetrating cryoprotectants and egg yolk level on the post-thaw quality of sperm.

The Hu sheep is a renowned breed known for its high reproductive rate. It is in estrus all year round, and its breeding population is gradually expanding. However, the current techniques for cryopreserving semen have limited effectiveness, which hinders the continuous development of this species. The purpose of this study is to explore the effects of different penetrating cryoprotectants (CPAs) and egg yolk (EY) concentrations on the cryopreservation of Hu ram semen to determine the most effective combination. In this study, the effects of glycerol (GLY), ethylene glycol (EG), dimethylacetamide, dimethyl sulfoxide, different proportions of GLY and EG, EY on sperm quality after thawing were investigated by detecting sperm total motility (TM), progressive motility (PM), straight-line velocity, curvilinear velocity, average path velocity, amplitude of lateral head displacement, wobble movement coefficient, average motion degree, functional integrity (plasma membrane integrity, acrosome integrity) and reactive oxygen species (ROS) level. When GLY and EG were added together, compared to other concentration groups, 6% GLY significantly (p<0.05) increased TM, PM, plasma membrane integrity, and acrosome integrity of thawed sperm. Additionally, it significantly (p<0.05) decreased the ROS level of sperm. In this study, the TM, PM, and membrane integrity of the 6% EG were significantly (p<0.05) higher than those of the control, 1% GLY+5% EG and 6% GLY+6% EG groups. Compared to other concentration groups, 20% EY significantly (p<0.05) improved the TM, PM, and plasma membrane integrity of thawed sperm. However, the integrity of the acrosome increased with the higher concentration of EY. In conclusion, the post-thawed Hu ram semen diluted with a diluent containing 6% GLY and 20% EY exhibited higher quality compared to the other groups.

Exploring the Cryopreservation Mechanism and Direct Removal Strategy of TAPS in Red Blood Cell Cryopreservation.

Cryopreservation of red blood cells (RBCs) plays an indispensable role in modern clinical transfusion therapy. Researchers are dedicated to finding cryoprotectants (CPAs) with high efficiency and low toxicity to prevent RBCs from cryopreservation injury. This study presents, for the first time, the feasibility and underlying mechanisms of a novel CPA called tris(hydroxymethyl)aminomethane-3-propanesulfonic acid (TAPS) in RBCs cryopreservation. The results demonstrated that the addition of TAPS achieved a post-thaw recovery of RBCs at 79.12 ± 0.67%, accompanied by excellent biocompatibility (above 97%). Subsequently, the mechanism for preventing RBCs from cryopreservation injury was elucidated. On one hand, TAPS exhibits a significant amount of bound water and effectively inhibits ice recrystallization, thereby reducing mechanical damage. On the other hand, TAPS demonstrates high capacity to scavenge reactive oxygen species and strong endogenous antioxidant enzyme activity, providing effective protection against oxidative damage. Above all, TAPS can be readily removed through direct washing, and the RBCs after washing showed no significant differences in various physiological parameters (SEM, RBC hemolysis, ESR, ATPase activity, and Hb content) compared to fresh RBCs. Finally, the presented mathematical modeling analysis indicates the good benefits of TAPS. In summary, TAPS holds potential for both research and practical in the field of cryobiology, offering innovative insights for the improvement of RBCs cryopreservation in transfusion medicine.

The crystallization properties of antifreeze GelMA hydrogel and its application in cryopreservation of tissue-engineered skin constructs.

Gelatin methacrylate (GelMA) hydrogels are expected to be ideal skin tissue engineering dressings for a wide range of clinical treatments. Herein, we report the preparation of GelMA or antifreeze GelMA hydrogel sheets with different GelMA concentrations, crosslinking times, and cryoprotectant (CPA) concentrations. The crystallization properties of GelMA or antifreeze GelMA hydrogel sheets were studied by cryomicroscopy and differential scanning calorimetry (DSC). It was found that the growth of ice crystals was slower when GelMA hydrogel concentration was more than 7%. The 10% DMSO-7% GelMA hydrogel sheets crosslinked for 60 min showed no ice crystal formation and growth during cooling and warming. The DSC results showed that the vitrification temperature of the 10% DMSO-7% GelMA hydrogel sheet was -111°C. Furthermore, slow freezing and rapid freezing of fibroblast-laden GelMA or antifreeze GelMA hydrogel sheets, and tissue-engineered skin constructs were studied. The results showed no significant difference in cell survival between slow (88.8% ± 1.51) and rapid (89.2% ± 3.00) freezing of fibroblast-loaded 10% DMSO-7% GelMA hydrogel sheets, and significantly higher than that of 7% GelMA hydrogel sheets (33.4% ± 5.46). The cell viability was higher in tissue-engineered skin constructs after slow freezing (86.34% ± 1.45) than rapid freezing (72.74% ± 1.34). We believe that the combination of antifreeze hydrogels and tissue engineering will facilitate the cryopreservation of tissue engineering constructs.

Cryopreservation of organoids: Strategies, innovation, and future prospects.

Organoid technology has demonstrated unique advantages in multidisciplinary fields such as disease research, tumor drug sensitivity, clinical immunity, drug toxicology, and regenerative medicine. It will become the most promising research tool in translational research. However, the long preparation time of organoids and the lack of high-quality cryopreservation methods limit the further application of organoids. Although the high-quality cryopreservation of small-volume biological samples such as cells and embryos has been successfully achieved, the existing cryopreservation methods for organoids still face many bottlenecks. In recent years, with the development of materials science, cryobiology, and interdisciplinary research, many new materials and methods have been applied to cryopreservation. Several new cryopreservation methods have emerged, such as cryoprotectants (CPAs) of natural origin, ice-controlled biomaterials, and rapid rewarming methods. The introduction of these technologies has expanded the research scope of cryopreservation of organoids, provided new approaches and methods for cryopreservation of organoids, and is expected to break through the current technical bottleneck of cryopreservation of organoids. This paper reviews the progress of cryopreservation of organoids in recent years from three aspects: damage factors of cryopreservation of organoids, new protective agents and loading methods, and new technologies of cryopreservation and rewarming.

Protein Cryoprotectant Ability of the Aqueous Zwitterionic Solution.

Protein cryopreservation is important for the long-term storage of unstable proteins. Recently, we found that N-acetylglucosaminyltransferase-V (GnT-V) can be cryopreserved in a deep freezer without temperature control using a dilute binary aqueous solution of 3-(1-(2-(2-methoxyethoxy)ethyl)imidazol-3-io)butane-1-carboxylate (OE2imC3C) [10 wt %, mole fraction of solute (x) = 7.75 × 10-3], an artificial zwitterion. However, it is unclear which solvent properties are required in these media to preserve unstable proteins, such as GnT-V. In this study, we investigated the melting phenomena and solution structure of dilute binary aqueous OE2imC3C solutions [x = 0-2.96 × 10-2 (0-30 wt %)] using differential scanning calorimetry (DSC) and Raman and Fourier transform infrared (FTIR) spectroscopies combined with molecular dynamics (MD) simulation to compare the cryoprotectant ability of OE2imC3C with two general cryoprotectants (CPAs), glycerol and dimethyl sulfoxide. DSC results indicated that aqueous OE2imC3C solutions can be melted at lower temperatures with less energy than the control CPA solution, with increasing x, primarily due to OE2imC3C having a higher content of unfrozen water molecules. Moreover, Raman and FTIR results showed that the high content of unfrozen water molecules in aqueous OE2imC3C solutions was due to the hydration around the ionic parts (the COO- group and imidazolium ring) and the OCH2CH2O segment. In addition, the MD simulation results showed that there were fewer structured water molecules around the OCH2CH2O segment than the hydration water molecules around the ionic parts. These solvent properties suggest that dilute aqueous OE2imC3C solutions are effective in preventing freezing, even in a deep freezer. Therefore, this medium has the potential to act as a novel cryoprotectant for proteins in biotechnology and biomedical fields.

Inulin Can Improve Red Blood Cell Cryopreservation by Promoting Vitrification, Stabilizing Cell Membranes, and Inhibiting Ice Recrystallization.

In transfusion medicine, the cryopreservation of red blood cells (RBCs) is of major importance. The organic solvent glycerol (Gly) is considered the current gold-standard cryoprotectant (CPA) for RBC cryopreservation, but the deglycerolization procedure is complex and time-consuming, resulting in severe hemolysis. Therefore, it remains a research hotspot to find biocompatible and effective novel CPAs. Herein, the natural and biocompatible inulin, a polysaccharide, was first employed as a CPA for RBC cryopreservation. The presence of inulin could improve the thawed RBC recovery from 11.83 ± 1.40 to 81.86 ± 0.37%. It was found that inulin could promote vitrification because of its relatively high viscosity and glass transition temperature (Tg'), thus reducing the damage during cryopreservation. Inulin possessed membrane stability, which also had beneficial effects on RBC recovery. Moreover, inulin could inhibit the mechanical damage induced by ice recrystallization during thawing. After cryopreservation, the RBC properties were maintained normally. Mathematical modeling analysis was adopted to compare the performance of inulin, Gly, and hydroxyethyl starch (HES) in cryopreservation, and inulin presented the best efficiency. This work provides a promising CPA for RBC cryopreservation and may be beneficial for transfusion therapy in the clinic.

A microfluidic hanging droplet as a programmable platform for mammalian egg vitrification.

Egg (oocyte) vitrification is the dominant method for preserving fertility for women of reproductive age. However, the method is typically performed by hand, requiring precise (∼0.1 to 10 μL) and time-sensitive (∼1 s) liquid exchange of cryoprotectants (CPA) around eggs as well as fine handling of eggs (∼100 μm) for immersion into liquid nitrogen (LN2). Here, we developed a microfluidic platform for programmable vitrification. Our platform is based on a millimeter-sized hanging droplet inside which a given egg is suspended and subjected to liquid exchanges within seconds. After programmable exposures to CPA, the egg is extracted from the liquid-air interface of the droplet using a motorized fine-tip instrument and immersed into LN2 for vitrification. To benchmark our platform with the manual method, we vitrified over a hundred mouse eggs and found comparable percentages (∼95%) for post-vitrification survivability. In addition, our platform performs real-time microscopy of the egg thereby enabling future studies where its morphology may be linked to functional outcomes. Our study contributes to the ongoing efforts to enhance the automation of embryology techniques towards broader applications in reproductive medicine both for clinical and research purposes.

Essential dynamics of ubiquitin in water and in a natural deep eutectic solvent.

Natural deep eutectic solvents (NADESs) comprised of osmolytes are of interest as potential biomolecular (cryo)protectants. However, the way these solvents influence the structure and dynamics of biomolecules as well as the role of water remains poorly understood. We carried out principal component analysis of various secondary structure elements of ubiquitin in water and a betaine : glycerol : water (1 : 2 : ζ; ζ = 0, 1, 2, 5, 10, 20, 45) NADES, from molecular dynamics trajectories, to gain insight into the protein dynamics as it undergoes a transition from a highly viscous anhydrous to an aqueous environment. A crossover of the protein's essential dynamics at ζ ∼ 5, induced by solvent-shell coupled fluctuations, is observed, indicating that ubiquitin might (re)fold in the NADES upon water addition at ζ > ∼5. Further, in contrast to water, the anhydrous NADES preserves ubiquitin's essential modes at high temperatures explaining the protein's seemingly enhanced thermal stability.

Optimization of Deep Eutectic Solvents Enables Green and Efficient Cryopreservation.

Cryopreservation presents significant opportunities for biomedical applications including cell therapy, tissue engineering, and assisted reproduction. Dimethyl sulfoxide (DMSO), the most commonly used cryoprotectant (CPA), can be added to cells to prevent cryogenic damage. However, the toxicity of cryoprotectants restrains its further development in many areas with safety concerns such as clinical treatment. Therefore, the development of low-toxicity cryoprotectants is essential for medical research. This work reports deep eutectic solvents (DES) as naturally biocompatible osmoprotectants for green and efficient cryopreservation of human umbilical cord mesenchymal stem cells (HuMSC), which may be an ideal alternative to DMSO. The six types of DESs were explored for thermal properties, toxicity, and permeability in cells. Raman spectroscopy and viscosity studies showed that DES exhibited an improved hydrogen-bonding system as the temperature decreased. By optimizing the freezing process (cooling rate, incubation time, and loading procedure) of DES, the viability of mouse embryonic fibroblast cells (NIH-3T3) after thawing was significantly improved. The HuMSC were successfully preserved with no significant difference (p > 0.05) in cell viability (94.65%) after thawing compared with DMSO, which preserved the cell differentiation function and improved the cell proliferation rate. The mechanism of DES in cryopreservation was investigated, and it was found that DES could bind water molecules and effectively inhibit the growth of ice crystals during ice recrystallization, reducing mechanical damage to cells. This study highlights the excellent performance of DES as a low-toxicity CPA for stem cell preservation, which may be a significant advance for future clinical cell therapy.

Enhanced cryopreservation performance of PVA grafted monolayer graphite oxide with synergistic antifreezing effect and rapid rewarming

In cryopreservation of biological samples such as cells, the formation, growth and recrystallization of ice crystals can cause fatal mechanical damage to cells. Therefore, how to effectively regulate and inhibit ice crystals, reduce freezing damage, and improve cryopreservation efficiency is a critical scientific problem that needs to be addressed in the current cryopreservation field. In this paper, a series of PVA grafted single-layer graphite oxide (PVA-g-GO nanocomposites) with different degrees of polymerization and different proportions was ingeniously engineered by a one-step esterification reaction method to study their ice crystal control effect. The obtained PVA17-g-GO nanocomposite could effectively reduce the ice crystal size during the recrystallization process at low concentration by adsorption and lattice matching. The nanocomposite with a 10:1 mass ratio of PVA and GO exhibited the best ice control effect, which could reduce the ice crystal size to only 17 % of that of pure water. Surprisingly, the modification of PVA significantly enhanced the absorption value of GO in Visible and Near-Infrared light (380 nm–980 nm), giving it excellent photothermal conversion efficiency (39.7 %), accelerating ice thawing rate, and thus synergistically reducing the mechanical damage caused by ice recrystallization. Finally, using the prepared PVA17-g-GO as cryoprotectants (CPA), the cryopreservation experiments on Hela cells and A549 cells demonstrated that this material at low concentration of 20 μg/ml could achieve high cell survival efficiency (>85 %). The preparation and development of this efficient ice-recrystallization-inhibiting nanomaterial with bidirectional synergy can provide new ideas and methods for the safety and progress of current cryopreservation technology.

Development of heart organoid cryopreservation method through Fe3 O4 nanoparticles based nanowarming system.

Beyond single cell two-dimensional (2D) culture, research on organoids that can mimic human organs is rapidly developing. However, there are still problems in commercialization and joint research using organoids due to the lack of technology to safely store organoids. Since organoids are 3D complex structures with a certain size (0.1-5mm) beyond the size of cells, the conventional cell-level cryopreservation method using cryoprotectant (CPA) cannot overcome the damage caused by volume change due to osmotic pressure difference and ice nucleation. Herein, we attempted to solve such limitations by applying a nanowarming system using CPA with high cell permeability and Fe3 O4 nanoparticles. By performing beat rate measurement, histological analysis, contractility analysis, and multi-electrode array, it was verified that the developed method could significantly improve functional recovery and survival of heart organoids after freezing and thawing. In this study, we demonstrated a successful organoid cryopreservation method based on a Fe3 O4 nanowarming system. The developed technology will provide clues to the field of tissue cryopreservation and spur the application of organoids.

Cryoprotectants for red blood cells: Evaluate safety and effectiveness by in vitro measures

AbstractThe transfusion of red blood cells (RBCs) is crucial in current medicinal research. The shelf‐life of donated RBCs preserved by the normal method is very short, limiting their clinical application. Cryopreservation is a reliable and effective technology for the long‐term storage of RBCs. During the process, ice formation will cause irreversible injuries to RBCs. Glycerol is used as a cryoprotectant (CPA) for RBCs to mitigate cryoinjuries. But it severely induces RBC hemolysis and deformation. This review introduces some biocompatible and effective CPAs used for RBC cryopreservation, outlining recent advances. Currently, there is no uniform standard to determine whether a CPA is suitable for RBCs. According to previous studies, we summarize for the first time comprehensive methods to evaluate the performance of CPAs by ensuring their safety and effectiveness. The safety of CPAs is defined as the degree of damage to RBCs, while the effectiveness is demonstrated by the properties of thawed RBCs, including membrane properties, protein activities, rheological properties, and metabolites levels. A novel CPA that is confirmed suitable for RBCs by these methods may be applied to other cells. We believe comprehensive methods can thoroughly evaluate the performance of CPAs and promote the development of transfusion therapy in clinic.

Osmotic response during kidney perfusion with cryoprotectant in isotonic or hypotonic vehicle solution.

Organ cryopreservation would revolutionize transplantation by overcoming the shelf-life limitations of conventional organ storage. To prepare an organ for cryopreservation, it is first perfused with cryoprotectants (CPAs). These chemicals can enable vitrification during cooling, preventing ice damage. However, CPAs can also cause toxicity and osmotic damage. It is a major challenge to find the optimal balance between protecting the cells from ice and avoiding CPA-induced damage. In this study, we examined the organ perfusion process to shed light on phenomena relevant to cryopreservation protocol design, including changes in organ size and vascular resistance. In particular, we compared perfusion of kidneys (porcine and human) with CPA in either hypotonic or isotonic vehicle solution. Our results demonstrate that CPA perfusion causes kidney mass changes consistent with the shrink-swell response observed in cells. This response was observed when the kidneys were relatively fresh, but disappeared after prolonged warm and/or cold ischemia. Perfusion with CPA in a hypotonic vehicle solution led to a significant increase in vascular resistance, suggesting reduced capillary diameter due to cell swelling. This could be reversed by switching to perfusion with CPA in isotonic vehicle solution. Hypotonic vehicle solution did not cause notable osmotic damage, as evidenced by low levels of lactate dehydrogenase (LDH) in the effluent, and it did not have a statistically significant effect on the delivery of CPA into the kidney, as assessed by computed tomography (CT). Overall, our results show that CPA vehicle solution tonicity affects organ size and vascular resistance, which may have important implications for cryopreservation protocol design.

Dynamics of Organic Acids during the Droplet-Vitrification Cryopreservation Procedure Can Be a Signature of Oxidative Stress in Pogostemon yatabeanus.

Cryopreservation in liquid nitrogen (LN, -196 °C) is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. In previous studies, a systematic approach was used to develop a droplet-vitrification (DV) cryopreservation protocol for Postemon yatabeanus shoot tips that includes preculture with 10% sucrose, osmoprotection with C4-35%, cryoprotection with A3-80% vitrification solution, and a three-step regrowth starting with the ammonium-free medium. The tricarboxylic acid (TCA) cycle is a crucial component of plant cell metabolism as it is involved in redox state regulation and energy provision. We hypothesized that organic acids (OAs) associated with the TCA and its side reactions indirectly indicate metabolism intensity and oxidative stress development in shoot tips under the cryopreservation procedure. In this study, the contents of 14 OAs were analyzed using gas chromatography-tandem mass spectrometry (GC-MS/MS) in P. yatabeanus shoot tips in a series of treatments including individual steps of the DV procedure, additional stress imposed by non-optimum protocol conditions (no preculture, no osmoprotection, various vitrification solution composition, using vials instead of aluminum foils, etc.) and regrowth on different media with or without ammonium or growth regulators. The possible relation of OA content with the total cryoprotectant (CPA) concentration and shoot tips regeneration percentage was also explored. Regeneration of cryopreserved shoot tips reduced in descending order as follows: standard protocol condition (91%) > non-optimum vitrification solution (ca. 68%) > non-optimum preculture (60-62%) > regrowth medium (40-64%) > no osmoprotection, cryopreservation in vials (28-30%). Five OAs (glycolic, malic, citric, malonic, and lactic) were the most abundant in the fresh (control) shoot tips. The dynamic pattern of OAs during the DV procedure highly correlated (r = 0.951) with the total CPA concentration employed: it gradually increased through the preculture, osmoprotection, and cryoprotection, peaked at cooling/rewarming (6.38-fold above control level), and returned to the fresh control level after 5 days of regrowth (0.89-fold). The contents of four OAs (2-hydroxybutyric, 3-hydroxypropionic, lactic, and glycolic) showed the most significant (10-209-fold) increase at the cooling/rewarming step. Lactic and glycolic acids were the major OAs at cooling/rewarming, accounting for 81% of the total OAs content. The OAs were categorized into three groups based on their dynamics during the cryopreservation protocol, and these groups were differently affected by protocol step modifications. However, there was no straightforward relationship between the dynamics of OAs and shoot tip regeneration. The results suggest that active modulation of OAs metabolism may help shoot tips to cope with osmotic stress and the chemical cytotoxicity\ of CPAs. Further intensive studies are needed to investigate the effect of cryopreservation on cell primarily metabolism and identify oxidative stress-related biomarkers in plant materials.

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.