Cryo letters最新文献

Despite the routine use of cryopreservation for the storage of biological materials, its outcomes are often sub-optimal (including reduced post-thaw viability, recovery, and functionality) due to the damage caused by uncontrolled ice growth. Traditional cryoprotective agents (CPAs), including dimethyl sulfoxide (DMSO), fail to prevent damage caused by ice growth and concerns over CPA cytotoxicity have fostered an increased interest in developing improved CPAs and cryoprotection strategies. The inhibition of ice recrystallization by natural antifreeze (glyco)proteins [AF(G)Ps] to improve cryopreservation outcomes has been examined; however, the ice binding properties of these substances and their challenging large-scale production make them poor CPA candidates. Therefore, the development and deployment of biocompatible, small-molecule ice recrystallization inhibitors (IRIs) for use as CPAs is a worthwhile objective. Extensive structure-activity relationship studies on AF(G)Ps revealed that simple carbohydrate derivatives could inhibit ice recrystallization. It was later discovered that this activity could be fine-tuned by delicately balancing the molecule's hydrophobicity and hydrophilicity. Current generation small-molecule IRIs have been meticulously designed to avoid binding to the surface of ice and subsequent biological testing (for both cytotoxicity and cryopreservation efficacy) has demonstrated significant improvements to the cryopreservation outcomes of several cell types. However, an individualized cell-specific approach for the simultaneous assessment of multiple cryopreservation outcomes is necessary to realize the full potential of IRIs as CPAs. This article provides a detailed overview of the development of small-molecule carbohydrate-based IRIs and highlights the crucial cell-specific biological considerations that must be taken into account when assessing cryopreservation outcomes. https://doi.org/10.54680/fr24210110112.

Background: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants.

Objective: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts.

Materials and methods: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance.

Results: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect.

Conclusion: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

Background: Nanotechnology can benefit livestock industries, especially through postharvest semen manipulation. Zinc oxide nanoparticles (Np-ZnO) are potentially an example.

Objective: To investigate how the addition of zinc oxide nanoparticles (Np-ZnO) affected the characteristics of post-thawed goat semen.

Materials and methods: Seminal pools from four Saanen bucks were used. Semen was diluted in Tris-egg yolk extender, supplemented with Np-ZnO (0, 50, 100 or 200 ug/mL), frozen and stored in liquid nitrogen (-196 degree C), and thawed in a water bath (37 degree C / 30 s). Semen samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), and assessed for other functional properties by epifluorescence microscopy, such as plasma membrane integrity (PMi), acrosomal membrane integrity (ACi) and mitochondrial membrane potential (MMP).

Results: For total motility (TM), the group treated with 200 ug/mL Np-ZnO was superior to the control. In straight-line velocity (VSL), the control was better than the group containing 200 ug/mL of Np-ZnO. For average path velocity (VAP), the control was higher than with 100 ug/mL Np-ZnO. For linearity (LIN), the control was higher than with 200 μg/mL Np-ZnO. In straightness (STR), the control and 100 μg/mL Np-ZnO were higher than with 200 ug/mL Np-ZnO. In wobble (WOB), the control was better than the 50 μg/mL Np-ZnO treatment. In PMi, ACi and MMP no significant differences were found.

Conclusion: The addition of Np-ZnO (200 ug/mL) to the goat semen freezing extender improved the total motility of cells, whilst negatively affecting sperm kinetics. https://doi.org/10.54680/fr24210110512.

Background: Stem cell-laden hydrogel microcapsules construction is important for a wide application in tissue engineering and cell-based medicine, such as building an ideal immune barrier. Challenges are emerging for effectively storing such microcapsules by cryopreservation, and a large proportion of research has been on the cryopreservation of single cells encapsulated into microcapsules without a core-shell structure.

Objective: To achieve the effective cryopreservation of stem cell-laden hydrogel microcapsules with a core-shell structure.

Materials and methods: A novel core-shell alginate hydrogel encapsulation method was used to produce mesenchymal stem cell-laden microcapsules by microfluidic technique.

Results: This microcapsule could inhibit ice formation to achieve vitreous cryopreservation with a low concentration (2 M) of penetrating cryoprotectants.

Conclusion: Cell laden hydrogel microcapsules may have the potential to be the basis of a new strategy of cell cryopreservation and applications. https://doi.org/10.54680/fr24210110212.

Background: Examining semen cryopreservation in Calomys laucha offers valuable insights for reproductive research and species conservation.

Objective: To determine the most effective sugar for the cryopreservation of C. laucha semen.

Materials and methods: Using 36 epididymides from C. laucha, semen samples were diluted in a 3% skimmed milk medium supplemented with one of four sugars (glucose, fructose, lactose, or sucrose) at a concentration of 0.3 M. These mixtures underwent a conditioning phase at 37 degree C for 10 min, cooled to -80 degree C for another 10 min, and were subsequently stored in liquid nitrogen.

Results: Upon thawing, samples treated with lactose and glucose solutions show superior sperm motility, achieving 8.2% and 10.0% respectively, in contrast to the fructose (2.0%) and sucrose (4.1%) mixtures. Furthermore, samples preserved in glucose registered the highest sperm penetration rates, reaching 44.9%.

Conclusion: Our findings suggest that a cryopreservation medium containing 0.3 M glucose can contribute to the safeguarding C. laucha rodent semen. https://doi.org/10.54680/fr24210110612.

Background: 'Dingjiaba' is an important Prunus persica cultivar (cv) mainly grown in the Hexi corridor in northwest China, which has an inherited strong cold tolerance.

Objective: To compare the transcriptome and physiology data of leaves of cvs 'Dingjiaba' (D) and 'Kanoiwa' (K) following cold treatment at different time periods, in order to gain new insights into the mechanisms of cold adaptation in 'Dingjiaba'.

Materials and methods: We analyzed the transcriptomic and physiological data of leaves of D and K cvs exposed to 0 h (D0/K0), 2 h (D2/K2), 6 h (D6/K6) and 12 h (D12/K12) cold stress.

Results: Low temperature stress caused membrane damage and led to increased rate of electrolyte leakage and increased MDA content. Cold stress induced the accumulation of soluble sugars, soluble proteins and proline in leaves of both cvs, with a lower increase in K compared to D. Transcriptome analysis identified 4,631, 5,069, 5,662 and 3,886 differentially expressed genes (DEGs) between D0 and K0, D2 and K2, D6 and K6 and D12 and K12, respectively. The differentially expressed genes significantly enriched in metabolic pathways and biosynthesis of secondary metabolites. We further validated the reliability of sequencing data of the RNA-Seq with Real-Time Quantitative PCR, which suggested that the expression trend of the RNA-Seq were same as RT-PCR.

Conclusions: These results provide novel insights into a series of molecular mechanisms underlying physiological metabolism and defense. https://doi.org/10.54680/fr24210110312.

Background: Human donor skin is processed to make the acellular dermis matrix (ADM) for tissue repair and regeneration. There is no data on the viscoelastic properties of ADM at room and subzero temperatures.

Objective: The work evaluated the temperature dependence of viscoelastic properties of freeze-dried ADM.

Materials and methods: Donor skin was de-epidermized, de-cellularized and freeze-dried with trehalose as the lyo-protectant. Glass transition of freeze-dried ADM was measured by differential scanning calorimeter (DSC), and viscoelastic properties were examined by dynamic mechanical analyzer (DMA).

Results: At the low moisture range (1.4 +/- 0.5%), the glass transition temperature (Tg) of freeze-dried ADM was 90 degree C to 100 degree C. As the moisture content increased, the Tg decreased steadily. At the high moisture range (10.8 +/- 2.9%), the Tg was 40 degree C to 60 degree C. There were large donor-to-donor variations in viscoelastic properties of freeze-dried ADM as demonstrated by the changes in storage modulus (G'), loss modulus (G") and damping factor tan delta (G"/G'). However, the trends of the temperature dependence for G', G" and tan delta were similar among all 8 donors. For each donor, changes in G' and G" were relatively small between -90 degree C and 40 degree C, and G' was at least one order of magnitude greater than G". Two viscoelastic relaxations were observed in freeze-dried ADM, one at -20 degree C and the other at -60 degree C respectively.

Conclusion: Freeze-dried ADM was protected in the glassy carbohydrate matrix. DMA observed two viscoelastic relaxations (i.e., alpha process at -20 degree C and beta process at -60 degree C). Overall changes in G' and G'' of freeze-dried ADM were relatively small within one order of magnitude between -90 degree C and 40 degree C. https://doi.org/10.54680/fr24110110612.

Background: Semen preservation by cooling is less expensive, simpler and results in less sperm damage than freezing does. However, spermatozoa can only be preserved for a short period due to the excessive formation of reactive oxygen species (ROS). Although several antioxidants can protect sperms from ROS damage during storage at low temperatures, the use of natural antioxidants derived from plants would be a better alternative.

Objective: To assess the effects of chamuangone, which can reduce oxidation reactions in cells, on cat semen quality after preservation at 4 degree C for 15 days.

Materials and methods: Epididymal sperm samples were collected before being diluted with tris-citric-fructose-egg yolk (TCFE) extender containing different concentrations of chamuangone (0, 50, 100, 150 and 200 ug/mL) and preserved at 4 degree C. Semen samples were evaluated before chilling and then every 3 days after chilling for up to 15 days. Each sample was assessed for sperm motility, viability, DNA integrity, plasma membrane integrity and percentage of spermatozoa with intact acrosomes.

Results: A significantly higher sperm motility was observed in the group supplemented with 100 ug/mL chamuangone compared to the control after 6 days of storage. However, the chamuangone concentration at 200 ug/mL did not significantly increase the sperm motility when compared to the control for the entire storage period.

Conclusion: 100 µg/mL chamuangone can improve sperm characteristics during 15 days of preservation at 4 degree C, keeping sperm alive (49.3 ± 5.2%) and moving (7.1 ± 2.4%). These results can be used for the development of breeding programs using technologically advanced reproductive procedures in domestic and wild cats. https://doi.org/10.54680/fr24110110212.

Background: The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time.

Objective: To develop a more efficient freezing method using cryovials.

Materials and methods: Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 x 10⁸ sperm/mL: Group 1 spermatozoa were cooled in cryovials and extender 1 (E1) and extender 2 (E1 +1 M glycerol) at 4 degree C for 50 min and then frozen over LN₂ for 20 min; Group 2 sperm was cooled and frozen in cryovials with a mixture of E1 and E2 (1:1) in a deep freezer (-80 degree C) for 30 min; Group 3 sperm in cryovials and E1 were cooled at 4 degree C for 20 min, cooled for an additional 20 min after addition of E2 (E1:E2, 1:1), and then frozen using LN_2/ vapour for 20 min. The control (Group 4) consisted of spermatozoa in straws being frozen using the conventional freezing method using two-step dilution. All groups were plunged and stored in LN₂ after freezing and their functional performance and gene expression determined.

Results: Progressive motility and acrosome integrity were highest (P < 0.05) in Groups 2, 3 and 4 (only acrosome integrity). Viability in Group 3 was significantly better that in the other Groups, and the reactive oxygen species (ROS) level and phosphatidylserine (PS) translocation index were significantly lower in Group 2 than the other Groups. The expression of sperm mitochondria-associated cysteine-rich protein (SMCP) and anti-apoptotic B-cell lymphoma 2 (BCL2) genes was highest (P < 0.05) in Group 2 and the expression of pro-apoptotic Bcl2-associated X protein (BAX) was lowest (P < 0.05) in Group 4.

Conclusion: The sperm frozen using cryovials, one step dilution and the deep freezer (Group 2) proved to be a simple and suitable cryopreservation method for dog sperm. https://doi.org/10.54680/fr24110110312.

Background: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos.

Objective: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance.

Materials and methods: In vivo derived mouse embryos were cultured with 100 uM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified.

Results: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification.

Conclusion: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation. https://doi.org/10.54680/fr24110110512.