Cryo letters最新文献

Background: The impacts of suboptimal shipping conditions during transport on cell viability, recovery, and function of cryopreserved samples, have not been well studied.

Objective: The impacts of suboptimal shipping on viability and recovery after the freezing and thawing were investigated using nine cancer cell lines, with particular reference to the approximate level of exposure temperature and exposure time at which adverse effects occur, and whether there are differences in sensitivity between cell types.

Materials and methods: The adverse effects of any set of suboptimal shipping conditions (-80 degree C for 7 d, -65 degree C or -50 degree C for 1, 3, and 7 d) on nine cancer cell lines (CHO-K1, COS-1, HeLa, HepG2, HL-60, Jurkat, MCF7, MDCK, 293T) were compared with data obtained during storage in liquid nitrogen.

Results: No statistically significant decrease in viability was observed in seven of the nine cell lines after freezing and thawing. On the other hand, a statistically significant decrease in the cell recovery was observed after 2 d post freezing and thawing in the nine cell lines, except CHO-K1 at higher exposure temperatures and longer exposure times. Visualization of the adverse effects on the cell lines using a heat map showed that the impacts tended to be more pronounced under the condition of exposure at -50 degree C for three or more days.

Conclusion: These results will contribute to the development of standardized protocols and best practices for the optimal shipping of frozen animal cells. https://doi.org/10.54680/fr25210110212.

Background: Airflow drying of mammalian cells has not been successful yet, with one obstacle being the improper drying time of cells-laden trehalose droplets.

Objective: To evaluate the major factors affecting the drying kinetics of aqueous trehalose droplets.

Materials and methods: A numerical analysis was applied to the evaporation behavior of aqueous trehalose droplets for the preservation of biomaterials. Factors such as convection caused by droplet contraction, vapor diffusion, and Stefan flow around droplets were taken into account in the analysis.

Results: Reducing the size and initial concentration of the droplets helps to achieve rapid drying of droplets. Forced convection can effectively enhance the initial drying rate of droplets, which may mitigate cell damage caused by hypertonic solutions. Upon drying, however, the rapid formation of an outer glassy shell inhibits the further drying of trehalose droplets.

Conclusion: Simple enhancement of convection does not facilitate complete droplet vitrification due to the formation of an outer glassy shell that prevents water movement. https://doi.org/10.54680/fr25210110712.

Three-dimensional (3D) culture systems, which include spheroids (SPs), provide a unique platform for studying complex biological processes in vivo and for enhancing the capabilities of in vitro test systems. Their uniqueness lies in the 3D organization of cells and in the reproduction of complex intercellular interactions, similar to those in native tissues and organs. These mini-organs can be used for fundamental research, tissue-engineering constructs, development of preclinical models for testing pharmacological drugs, etc. Important and current issues regarding SPs involve improving methods for their production and cryopreservation. Solving these issues will expand the range and effectiveness of their use in tissue engineering. Here, we describe the authors' research and experience on factors influencing the formation of SPs, which can enhance the understanding of their correct application and standardization. A crucial aspect of this review is the information on applying theoretical approaches based on physico-mathematical calculations to improve the quality of existing cryopreservation protocols for SPs. https://doi.org/10.54680/fr25110110112.

Background: The skin, the largest organ in the human body, is composed of complex layers that include subcutaneous adipose tissue. Understanding the characteristics of this skin structure is essential to optimize therapeutic interventions, such as cryolipolysis, aiming for more effective and personalized results.

Objective: To evaluate the immunohistochemical effects of skin tissue in adult women undergoing cryolipolysis.

Materials and methods: We carried out an experimental and blind study with immunohistochemical analysis in women with localized abdominal fat, categorized based on the constitution of the skin as flaccid or firm according to the Investigator Assessment Skin Laxity Scoring System scale. Participants were randomized before undergoing the cryolipolysis procedure. Forty-five days after the procedure, they underwent abdominoplasty, with collection of biological material. We evaluated the inflammatory markers EBF-1, TNF-alpha, and CD68, as well as Caspase 3, cleaved Caspase 3, apoptotic BCL2, Ki-67 for fibroblast proliferation, and FIS1 for mitochondrial proliferation.

Results: Six women were included, divided into two groups; three women with loose and three with firm skin. We observed that after cryolipolysis, the group with flaccid skin showed higher expression of the Casp3, TNF-alpha, BCL2, and FIS1 markers compared to those with firm skin.

Conclusion: Cryolipolysis may act differently according to tissue morphology, suggesting that its apoptotic response is more pronounced in the group with flaccid skin. https://doi.org/10.54680/fr25110110212.

Background: Carnitine reduces reactive oxygen species-induced apoptosis and DNA fragmentation through its antioxidant effect.

Objective: To investigate the effect of carnitine on capacitation, mitochondrial activity, acrosomal integrity, reactive oxygen species (ROS), membrane fluidity, and DNA fragmentation during the cryopreservation of Hariana bull spermatozoa.

Materials and methods: Thirty-two semen ejaculates were obtained using artificial vagina (AV) from four seemingly healthy Hariana bulls. Following dilution, the diluted semen samples were split into four aliquots: Group I, the control, included no carnitine; Groups II, III, and IV were the aliquots that contained carnitine supplements of 2.5, 5, and 10 mM, respectively. These four diluted semen samples were then processed immediately for freezing and equilibration.

Results: Regarding post-thaw sperm parameters, such as viability, motility, velocity parameters, capacitation status, mitochondrial activity, acrosomal integrity, reactive oxygen species (ROS), membrane fluidity, and DNA fragmentation, Groups II and III, containing 2.5 mM and 5 mM carnitine respectively, had significantly (P < 0.05) improved parameters compared to the Group I (control). At 5 mM, there was a substantial (P < 0.05) decrease in early apoptotic-like alterations in sperm cells, accompanied by a greater population of sperm cells with high mitochondrial membrane potential.

Conclusion: Carnitine has been shown to have cryoprotective properties in semen extenders. For improved post-thaw sperm quality, carnitine may be added to a Hariana bull semen extender at a dose of 5 mM. https://doi.org/10.54680/fr25110110712.

Background: There is a crucial need to develop appropriate cryopreservation solutions so that somatic resource biobanks of wildlife can be established.

Objective: Here, we propose a cryopreservation protocol to optimize the preservation of skin-derived fibroblasts from six-banded armadillos (Euphractus sexcinctus Linnaeus, 1758) by comparing different concentrations of fetal bovine serum (FBS) in the absence or presence of sucrose as non-permeable cryoprotectants.

Materials and methods: Cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium (DMEM) with 10% dimethyl sulfoxide (DMSO), varying concentrations of FBS (10, 20 and 40%) without or with 0.2 M sucrose, totaling six comparison groups. Cells not subjected to cryopreservation were used as a control. Cells were evaluated for morphological characteristics, viability, metabolism, apoptosis levels, proliferative activity and mitochondrial membrane potential.

Results: Cells maintained similar fusiform morphology and demonstrated high viability (> 90%) before and after cryopreservation in all groups. Cryopreserved cells with 10 and 40% of FBS without sucrose showed lower metabolism, but, when sucrose was added, this parameter was maintained as in the control group. This effect was not observed in the 20% FBS groups in the absence or presence of sucrose, with viability similar to that of the non-cryopreserved group. The addition of sucrose maintained apoptosis levels, while the 20 and 40% FBS without sucrose groups showed alterations in viable, early apoptosis and necrosis stages. Nevertheless, all cryopreserved groups showed lower proliferative activity with a higher population doubling time (16.2-19.9 h) than the non-cryopreserved group (15.2 h). Finally, the 20% FBS groups, in the absence or presence of sucrose, maintained the mitochondrial membrane potential.

Conclusion: We demonstrated that 20% FBS with sucrose was the most suitable cryopreservation solution for six-banded armadillo skin-derived fibroblast lines, promoting high cell survival after thawing. https://doi.org/10.54680/fr25110110412.

Background: Resveratrol (Res) (3,5,4'-trihydroxystilbene) is a natural polyphenol that exhibits important biological activities.

Objective: To assess the effects of resveratrol (Res) on freeze-thawed survival of semen from Honamli and Hair Bucks.

Materials and methods: Six bucks, aged 2-3 years (three from each breed), were included in the study. Semen was collected from each breed and mixed separately after removing seminal plasma. The mixed semen was diluted with different Res concentrations (0 uM as control, 25 µM, 50 uM, 100 µM, 500 uM, and 1 mM) in Tris diluent and subjected to cryopreservation in liquid nitrogen vapor and frozen. After thawing, the samples were evaluated for motility and some spermatologic quality parameters by flow cytometry.

Results: Data were analyzed separately for Honamli and Hair breeds. The results showed that the Res 1 mM group had the lowest motility in all assessments (P < 0.05). However, no significant differences were observed between the other Res and control groups (P > 0.05). In terms of apoptosis, Hair bucks exhibited a statistically significant difference in late apoptotic parameters, with the control showing the highest values (P. < 0.05). The Res 25 uM group (similar to the control group) showed lower mitochondrial oxidative stress than the Res 1 mM group (P. < 0.05).

Conclusion: Res at a dose of 1 mM did not protect most sperm functional and biochemical parameters except for apoptosis and performed worse than the control group. When all parameters are evaluated collectively, concentrations lower than 1 mM should be used for freezing Honamli and Hair Buck semen with resveratrol. https://doi.org/10.54680/fr25110110312.

Background: Despite advancements in bovine embryos cryopreservation techniques, challenges remain, warranting further investigation into their impact on embryo morphology and viability so that outcomes can be improved.

Objective: To analyze, through transmission electron microscopy (TEM), in vitro-produced bovine embryos vitrified using the Cryotop method.

Materials and methods: Groups of embryos were transferred to a stabilization solution (SS) containing 7.5% EG, 7.5% DMSO in maintenance medium (TCM-199 supplemented with 20% FBS) for 2 min, and then transferred to a vitrification solution (VS) containing 15% EG, 15% DMSO, and 0.5 M sucrose in maintenance medium. Warming was performed in five stages with decreasing concentrations of sucrose. After warming, the blastocysts were cultured for 24 h for subsequent survival analysis and ultrastructural evaluation. In vitro-produced bovine embryos that did not undergo the vitrification process were used as a fresh control.

Results: Blastocoel reestablishment was observed in 52.3% (66/126) of vitrified embryos 24 h after warming, demonstrating the method's effectiveness in post-cryopreservation survival. Ultrastructural analysis of embryos from the fresh control group showed flattened trophoectodermal cells with prominent nuclei, well-preserved mitochondria, and Golgi complexes were also evident. Microvilli were observed in some regions near the zona pellucida. Embryos vitrified using the Cryotop method exhibited lesions consistent with the cryopreservation process, such as intracellular disorganization, mitochondrial injuries, and dispersion of microvilli.

Conclusions: Ultrastructural evaluation of in vitro-produced bovine embryos vitrified using the Cryotop method is an effective tool for increased understanding of the injuries caused to embryonic cells during the cryopreservation process. https://doi.org/10.54680/fr25110110612.

Background: Single sperm cryopreservation (SSC) is a method that preserves the limited number of spermatozoa in testicular sperm. But testicular spermatozoa are characterized with low movement, which is not ideal for sperm selection before SSC.

Objective: This study was designed to investigate in vitro incubation (IVC) as a sperm selection technique before SSC on biological factors of testicular spermatozoa.

Materials and methods: Testicular tissue was obtained from 15 azoospermia men. One part of the testicular samples was used as a Control group, which was assessed fresh. One portion was cryopreserved by a vitrification (Vit) method and the two other portions were in vitro cultured for 24 h, with (IVC-Vit) or without (IVC) vitrification. Sperm motility, viability, morphology, DNA fragmentation and mitochondrial membrane potential were evaluated.

Results: Sperm motility and viability were better maintained in the IVC-Vit group compared to the Vit group (P=0.04 and P= 0.003, respectively). Sperm morphology, the fresh, Vit, IVC, and IVC-Vit groups all showed similar results (P > 0.05). Mitochondrial activity was significantly lower in the Vit group compared to the Control fresh group (P = 0.0001). The IVC group had a significantly higher DFI as compared to the Control (P < 0.0001). Compared to the IVC group, the IVC-Vit sperm had a significant increase in DFI (P= 0.0009). There was a statistically significant difference between post warm DFI of the Vit group and IVC-Vit group (P < 0.0001).

Conclusion: IVC as a sperm selection method increased motility and viability of testicular spermatozoa before single sperm vitrification. As DNA fragmentation increased by this technique, this method is not ideal for selecting viable sperm. https://doi.org/10.54680/fr25110110512.

Background: Long-term storage of cowpea pollen is important for the fertilization of spatially or temporally isolated female parents, especially during cowpea crop improvement and wide hybridization programs.

Objective: Experiments were conducted to determine pollen longevity at different storage temperatures and to develop a cryopreservation protocol for pollen of different cowpea accessions.

Materials and methods: The investigation was carried out at the Research Farm of ICAR-NBPGR, New Delhi, India, during the kharif (rainy) season of 2022. Pollen viability was studied after storage for 1, 3, 6, 12 and 24 h, 1 and 2 weeks and 1, 3 and 5 months at three different temperatures (4, -20 and -196 degree C).

Results: Fresh pollen viability ranged from 78 to 91 %. The optimal pollen moisture content was 12-14 % and the optimal air desiccation period under the laminar air flow chamber (22 +/- 1 degree C) was 5 min for subsequent preservation at -196 degree C. Pollen viability was lost completely at 4 and -20 degree C after 1 and 2 weeks of storage, respectively. Pollen stored in liquid nitrogen (-196 degree C) retained vaiblity similar to that of fresh pollen for > 5 months storage. Pollination using cryostored pollen resulted in normal fertilization.

Conclusion: This finding opens a gateway for cowpea haploid germplasm conservation and wide hybridization programs. https://doi.org/10.54680/fr24610110312.