Cryobiology最新文献

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.

Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in preserving male fertility, especially for patients who need proactive preservation, such as cancer patients before undergoing radiation or chemotherapy. Although cryopreservation does not directly address infertility, effective preservation can support future fertility. However, the process may compromise sperm DNA integrity. Despite their impairment, damaged sperm often retain vitality and may still have the potential to fertilize an egg. Nonetheless, if damaged sperm fertilize an egg, excessive DNA damage could impede embryo implantation and development, despite the egg's repair capabilities. Consequently, precise detection of sperm DNA damage is crucial and urgent. To better address the issue of sperm DNA damage detection, we have introduced a novel fluorescence biosensor technology known as the TDT/SD Probe. This technology utilizes terminal deoxynucleotidyl transferase (TdT) and strand displacement probes to accurately detect the number of sperm DNA breakage points during the cryopreservation process. Experimental results reveal that the number of sperm DNA breakpoints significantly increases after both sperm vitrification (8.17 × 10⁵) and conventional slow freezing (10.80 × 10⁵), compared to the DNA breakpoints of fresh semen samples (5.19 × 10⁵). However, sperm vitrification has the least impact on sperm breakage points. This research provides innovative means for further optimizing sperm preservation techniques by offering a novel DNA damage detection method, enabling more precise assessment of sperm DNA damage during the freezing process.

Isochoric (constant-volume or volumetrically confined) vitrification has shown potential as an alternative cryopreservation-by-vitrification technique, but the complex processes at play within the chamber are yet poorly characterized, and recent investigations have prompted significant debate around whether a truly isochoric vitrification process (in which the liquid remains completely confined by solid boundaries) is indeed feasible. Based on a recent thermomechanical simulation of a high-concentration Me₂SO solution, Solanki and Rabin (Cryobiology, 2023, 111, 9–15.) argue that isochoric vitrification is not feasible, because differential thermal contraction of the solution and container will necessarily drive generation of a cavity, corrupting the rigid confinement of the liquid. Here, we provide direct experimental evidence to the contrary, demonstrating cavity-free isochoric vitrification of a ∼3.5 M vitrification solution by combined isochoric pressure measurement (IPM) and photon-counting x-ray computed tomography (PC-CT). We hypothesize that the absence of a cavity is due to the minimal thermal contraction of the solution, which we support with additional volumetric analysis of the PC-CT reconstructions. In total, this study provides experimental evidence both demonstrating the feasibility of isochoric vitrification and highlighting the potential of designing vitrification solutions that exhibit minimal thermal contraction.

Non-permeable disaccharides are widely used as cryoprotectant agents due to their low cytotoxicity, but their protective effect is insufficient when the disaccharides are present only extracellularly. On the other hand, cryoprotectant agent (CPA)-free cryopreservation has been recently achieved by instantaneously inkjet-freezing cells as tiny droplets. However, CPA-free cryopreservation requires skilled handling operations due to instability of the vitreous water without the CPA. In this study, the effectiveness of separately adding two types of disaccharides in inkjet freezing of 3T3 cells was evaluated and the following results were obtained. First, trehalose showed the highest effect at 0.57 M, twice the plasma osmolarity, with a maximum cell viability of over 90 % when freezing 70 pL droplets. However, higher concentrations of trehalose decreased cell viability due to damage caused by dehydration. Similarly, sucrose gave cell viability close to 90 % at 0.57 M with 70 pL droplets, and higher concentrations decreased cell viability. Next, the relationship between minimum trehalose concentrations to prevent intracellular and extracellular ice crystal formation and droplet size was analyzed. The results indicated that trehalose of less than 0.57 M was able to inhibit intracellular ice crystal formation even in the largest droplet used in this study, 450 pL, while trehalose of nearly 0.57 M was required to inhibit extracellular ice crystal formation in the smallest droplet, 70 pL. In other words, the suppression of extracellular ice crystals by the addition of CPA was shown to be crucial in improving the viability of inkjet superflash freezing.

This study investigated the impact of protein enrichment on the physicochemical, cooking, textural, and color properties of frozen cooked noodles (FCN) stored for 0–3 weeks at −18 °C. Incorporating casein, egg white protein, and soy protein into the noodles significantly increased moisture content, with casein-enriched noodles showing the highest initial moisture levels. The addition of proteins also led to increased ash content, indicating improved nutritional quality. Protein enrichment resulted in reduced cooking loss and enhanced water retention during cooking and frozen storage. Casein-enriched noodles exhibited the highest water absorption capacity and the most substantial enhancement in textural properties, maintaining cohesiveness, gumminess, and elasticity better than egg white protein and soy protein during storage. The results indicated that egg white protein promotes intermolecular interactions, leading to enhanced color stability over time. These findings suggest that enriching with the protein could be a viable approach to elevate the overall quality of FCN.

The fundamental interactions between plant cells and cryoprotectants during vitrification are understudied in the field of plant cryopreservation. Within this area of research, real time cryoprotectant permeation into plant cells is even less documented. In this study, we monitor the real time permeation of individual cryoprotectants into rice callus cells when in mixtures with other cryoprotectants. Specifically, we use coherent anti-Stokes Raman scattering (CARS) microscopy to observe the permeation of individually deuterated DMSO, ethylene glycol, and glycerol in plant vitrification solution 2 (PVS2) by probing vibrational frequencies that correspond to C-D stretching modes of the cryoprotectant molecules. Additionally, we measure cell plasma membrane responses to PVS2 exposure using brightfield microscopy. We conclude that the permeation of PVS2 components into plant cells occurs faster than the first cell plasma membrane responses observed and therefore permeation and cell plasma membrane response do not appear to be directly correlated. In addition, we observe that cryoprotectant permeation into plant cells occurs more quickly and more uniformly when cryoprotectants are in PVS2 solution than when they are in single component aqueous solutions.

Glycans are carbohydrates present in every organism that bind to specific molecules such as lectins, a diverse group of proteins. Glycans are vital to cell proliferation and protein trafficking. In addition, embryogenesis is a critical phase in the development of marine organisms. This study investigated the effects of chilling and cryoprotective agents (CPAs) on glycans in the embryos of Stenopus hispidus. The glycan profiles of embryos of S. hispidus at the heartbeat stage were analyzed using lectin arrays. The results of analyses revealed that mannose was the most abundant glycan in the S. hispidus embryos; mannose is crucial to cell proliferation, providing the energy required for embryonic growth. Additionally, the results reveled that chilling altered the content of several glycans, including fucose and Gla-GlcNAc. Chilling may promote monosaccharide accumulation, facilitating osmotic regulation of cells and signal molecules to aid S. hispidus embryos in adapting to cold conditions. Changes were also observed in the lectins NPA, orysata, PALa, ASA, discoidin II, discoidin I, UDA, PA-IIL, and PHA-P after the samples were treated with different CPAs. DMSO may minimize cell damage during exposure to chilling by preserving cell structures, membrane properties, and functions. The present study is the first to investigate the profiles and functions of glycans in shrimp embryos subjected to low-temperature injuries. This study enhances the understanding of cell reproduction during embryogenesis and provides valuable information for the study of glycans in embryos.

Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to −4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.

Victims of severe accidental hypothermia are frequently treated with catecholamines to counteract the hemodynamic instability associated with hypothermia-induced cardiac contractile dysfunction. However, we previously reported that the inotropic effects of epinephrine are diminished after hypothermia and rewarming (H/R) in an intact animal model. Thus, the goal of this study was to investigate the effects of Epi treatment on excitation-contraction coupling in isolated rat cardiomyocytes after H/R. In adult male rats, cardiomyocytes isolated from the left ventricle were electrically stimulated at 0.5 Hz and evoked cytosolic [Ca²⁺] and contractile responses (sarcomere length shortening) were measured. In initial experiments, the effects of varying concentrations of epinephrine on evoked cytosolic [Ca²⁺] and contractile responses at 37 °C were measured. In a second series of experiments, cardiomyocytes were cooled from 37 °C to 15 °C, maintained at 15 °C for 2 h, then rewarmed to 37 °C (H/R protocol). Immediately after rewarming, the effects of epinephrine treatment on evoked cytosolic [Ca²⁺] and contractile responses of cardiomyocytes were determined. At 37 °C, epinephrine treatment increased both cytosolic [Ca²⁺] and contractile responses of cardiomyocytes in a concentration-dependent manner peaking at 25–50 nM. The evoked contractile response of cardiomyocytes after H/R was reduced while the cytosolic [Ca²⁺] response was slightly elevated. The diminished contractile response of cardiomyocytes after H/R was not mitigated by epinephrine (25 nM) and epinephrine treatment reduced the exponential time decay constant (Tau), but did not increase the cytosolic [Ca²⁺] response. We conclude that epinephrine treatment does not mitigate H/R-induced contractile dysfunction in cardiomyocytes.

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3′UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.