Biopreservation and Biobanking

Long-term cryopreservation of human umbilical vein endothelial cells (HUVECs) is important and beneficial for a variety of biomedical research and applications. In this study, we investigated HUVEC's cryobiological characteristics and parameters that are indispensable for predicting and determining an optimal cooling rate to prevent lethal intracellular ice formation (IIF) and severe cell dehydration during the cryopreservation processes. The parameters include cell membrane hydraulic conductivity (i.e., cell membrane water permeability), L_p, cell membrane water permeability activation energy, E_lp, and osmotically inactive volume of a cell V_b. Cryomicroscopy was used to study the IIF phenomena and cell volume excursion at various cooling rates, 1, 10, and 20°C/min, respectively, based on which the cryobiological parameters were determined using biophysical and mathematical models. Results from this research work laid an important cryobiological foundation for the optimization of HUVEC's cryopreservation conditions.

Rapid and uniform rewarming has been proved to be beneficial, and sometimes indispensable for the survival of cryopreserved biomaterials, inhibiting ice-recrystallization-devitrification and thermal stress-induced fracture (especially in large samples). To date, the convective water bath remains the gold standard rewarming method for small samples in the clinical settings, but it failed in the large samples (e.g., cryopreserved tissues and organs) due to damage caused by the slow and nonuniform heating. A single-mode electromagnetic resonance (SMER) system was developed to achieve ultrafast and uniform rewarming for large samples. In this study, we investigated the heating effects of the SMER system and compared the heating performance with water bath and air warming. A numerical model was established to further analyze the temperature change and distribution at different time points during the rewarming process. Overall, the SMER system achieved rapid heating at 331.63 ± 8.59°C min⁻¹ while limiting the maximum thermal gradient to <9°C min⁻¹, significantly better than the other two warming methods. The experimental results were highly consistent, indicating SMER is a promising rewarming technology for the successful cryopreservation of large biosamples.

Animal cloning is an important technique used to produce clones from valuable farm animals, to rescue animals in risk of extinction, and for producing transgenic animals. The objective of this work was to evaluate the effects of refrigeration on bovine ear skin as a strategy to transport biological material for long periods of time to isolate viable fibroblasts. Ears from eight cows were collected after death and stored for 30 days at 5°C. On days 0, 2, 4, 7, 14, 21, and 30, skin biopsies were cultured in vitro for fibroblast isolation. The time for first fibroblast outgrowth, time to reach 100% confluence. and cell concentration before freezing were observed for each period. In addition, plasma membrane integrity, cell apoptosis, and necrosis in cells were evaluated through fluorescent colorant combination in a flow cytometer from all periods after thawing. Fibroblasts obtained after 30 days of storage, considered a critical period, were tested for embryo production using nuclear transfer (NT) with micromanipulators. All time points allowed for cell culture. The time of cell growth onset was longer in samples refrigerated for 14, 21, and 30 days. The time to reach confluence also increased with longer refrigeration periods. Cells from day 0 reached confluence in 24 ± 2 days, while day 30 cells took 31 ± 0 days. Cell concentration and viability dropped with increased storage time and freezing/thawing, respectively. It was found that a long period of sample storage results in cell damage, making cultivation more difficult and decreasing cell viability post-thawing and cell concentration. However, when cells from day 30 were used as nuclei donors in NT, a 26.05% blastocyst rate after 7 days in culture was obtained. In conclusion, refrigeration at 5°C was shown to be efficient in maintaining viable tissue for up to 30 days, and fibroblasts isolated can be used for cloned embryo production.

Optimization of practical ways to obtain mature follicles from cryopreserved ovarian tissues, especially in patients suffering from ovarian dysfunction, is very important. In vitro ovarian tissue culture allows faster screening of follicle development and reduces follicle isolation damage. During ovarian tissue culture, controlling oxidative stress is critical to support better follicular development and less damage. Immature Naval Medical Research Institute (NMRI) mouse ovaries (8-days-old) were randomly distributed into four cultured groups; non-vitrified, vitrified, non-vitrified N-acetyl-L-cysteine (NAC)⁺, and vitrified NAC⁺. Ovaries of vitrified groups along with non-vitrified ovaries were cultured on agar gel in the presence or absence of NAC for 5 days. Afterward, morphological evaluations, mRNA expressions of Gdf9, Bmp6, Lif, Amh, Bax, and Bcl2 genes, malondialdehyde, and total antioxidant capacities were compared between four groups at the first and last day of culture. Good preservation of tissue integrity and an increase of follicular development were observed in all groups. In addition, the expression of Gdf9, Lif, Bax, and Bcl2 genes were increased and Amh was decreased in groups cultured in the presence of NAC compared to groups cultured without NAC. Although total antioxidant capacity was not significantly different between the experimental groups, the lipid peroxidation and apoptotic index were significantly reduced in the presence of NAC. Thus, it appears that NAC antioxidant acts as a contributory factor for the ex vivo culture of ovarian tissue and reduces oxidative stress, apoptotic index, and improves follicular development, especially in non-vitrified groups.

The aim of this study was to evaluate the effect of hydrated carbon 60 fullerene (C₆₀HyFn) on ram semen quality during cryopreservation. Three ejaculates from each of seven Akkaraman rams were collected using an artificial vagina during the nonbreeding season and pooled. Pooled semen samples were divided into 10 equal parts and diluted with tris + egg yolk extender not containing (control) and containing 100, 200, 400, and 800 nM and 1, 5, 10, 20, and 40 μM C₆₀HyFn at 37°C. After addition of 5% glycerol and an equilibration process for 3 hours, the samples were frozen in 0.25-mL straws in an automatic freezing device at −140°C and stored in a liquid nitrogen container. Straws were thawed 24 hours after freezing and analyzed immediately with no incubation period. Motility, kinematic parameters, abnormality, vitality, hypo-osmotic swelling test (HOST), and oxidative stress levels were analyzed in thawed semen. Compared with the control, 200, 400, and 800 nM and 1 and 5 μM C₆₀HyFn doses increased motility and HOST values and decreased the dead sperm rate. When compared with the control, addition of C₆₀HyFn significantly decreased malondialdehyde levels (between 200 nM and 40 μM doses) and significantly increased glutathione peroxidase (between 800 nM and 40 μM doses) and catalase (between 1 and 40 μM doses) activities. In conclusion, results of this study show that the C₆₀HyFn nanoparticles are nontoxic to ram semen and their supplementation in the extender is beneficial to sperm motility and membrane integrity after freeze–thawing.

In this study, the effects of trehalose and 1, 3-cyclohexanediol (1, 3-CHD) on the ultrastructure of frozen-thawed ram sperm were assessed and compared. In the control group, sperm were frozen without trehalose and 1, 3-CHD. In the trehalose group, 100 mM trehalose was used for sperm cryopreservation. In the cyclohexanediol group, the freezing extender contained 100 mM 1, 3-CHD. The transmission electron microscope (TEM) was used to observe the ultrastructural alterations of sperm. For verification of the TEM results, the plasma membrane and acrosome integrity of ram frozen sperm was assessed. Three fertility-proven rams were used in this study. Semen collection was repeated 6 times. The collected semen was pooled to preclude the individual difference each time. The sperm collected from a representative ram were used for ultrastructural observation. The TEM results indicated extensive and severe cryoinjuries on the main organelles of ram frozen sperm. Some alterations in plasma membrane, including detachment, rupture, dilation, or loss, appeared in post-thaw sperm. The bending shape and leakage of genetic materials were also observed in the nucleus. In addition, the outer acrosome membrane in some frozen sperm was broken or partly lost. Further, leakage of the inner contents of acrosomes also occurred. Sperm mitochondria was negatively influenced by cryopreservation. With 1, 3-CHD or trehalose, the percentage of sperm with normal acrosomes was 62% or 64%, and it was significantly higher than that of the control (41.51%, p < 0.05). However, different from trehalose, 1, 3-CHD cannot efficiently protect the post-thaw integrity of the plasma membrane (48.09% vs. 26.92%, p < 0.05). The TEM results were consistent with the quality assessment of frozen-thawed sperm. Collectively, trehalose and 1, 3-CHD can mitigate cryoinjuries on sperm ultrastructure. The cryoprotective effects of trehalose on sperm plasma membrane are superior to 1, 3-CHD. Sperm plasma membrane is the most sensitive to cryoinjuries, followed by acrosomes, mitochondria, and nuclei.

This study dealt with the development of a sperm cryopreservation protocol of Tor tor. Sperm was collected from hormone-induced males and the concentration and pH of sperm were estimated as 4.3 ± 0.1 × 10¹⁰ cells/mL and 8.6 ± 0.2, respectively. Activation of sperm motility was evaluated in different osmolalities of NaCl solution where motility of sperm decreased with increasing osmolality of extenders, and was completely inhibited at 319 mOsmol/kg. Similarly, the swimming duration of activated sperm was affected as the osmolality of the extender increased. The duration of initial motility of sperm was recorded as 8.4 ± 0.4 minutes at 48 mOsmol/kg, while the highest motility was recorded as 68.0 ± 7.2 minutes at 128 mOsmol/kg. To evaluate the toxicity of cryoprotectants, sperm was incubated with dimethyl sulfoxide (DMSO) and methanol at 5%, 10%, and 15% concentrations, respectively, for 5–45 minutes. Alsever's solution with 5% and 10% DMSO produced better motility during 5–10 minutes of incubation and 15% DMSO seemed toxic to sperm. For the cryopreservation of sperm, Alsever's solution, egg yolk citrate, and urea egg yolk were used as extenders with DMSO and methanol. Alsever's solution with 10% DMSO provided the highest equilibration (90.0% ± 3.5%) and post-thaw (80.0% ± 3.5%) motility followed by that of 87.0% ± 2.0% and 79.0% ± 1.9% with egg yolk citrate plus DMSO, and 82.0% ± 2.6% and 78.0% ± 2.0% with urea egg yolk plus DMSO, respectively. The sperm cryopreservation protocol developed through this study can be applied for long-term preservation of genetic materials of the critically endangered T. tor, and eventually, it will be an effective tool for protecting them from extinction.

Cryopreservation of spermatozoa is a general procedure to preserve viable sperm for an indefinite period. Despite the efficiency of sperm cryopreservation, excessive reactive oxygen species (ROS) production during cryopreservation can induce structural and functional changes in spermatozoa. Also, cryopreservation has been shown to decrease the spermatozoa's antioxidant activity inducing them to be more sensitive to damage caused by ROS. Experimental evidence suggests that astaxanthin (AXT) has essential activities such as antioxidant, antibacterial, and antithrombotic properties. Therefore, this study aimed to evaluate the effect of AXT on the sperm quality of healthy men during freezing–thawing. In the first phase, 10 semen samples with different concentrations of AXT (0.0, 0.5, 1, and 2 μM) were cryopreserved to achieve an optimal dose of AXT. Then, motility, viability, and phosphatidylserine (PS) externalization were evaluated. In the second phase, 25 samples were collected and divided into 3 groups: fresh group, control group (untreated frozen–thawed samples), and AXT group (treated frozen–thawed with AXT). Then, samples were cryopreserved in freezing media supplemented with or without the optimal concentration of AXT (1 μM). After thawing, the levels of sperm parameters, including motility (using a computer-assisted sperm analyzer), viability (eosin–nigrosin), early apoptotic change (annexin V/propidium iodide), ROS (flow cytometry), and lipid peroxidation (LPO) (using enzyme-linked immunosorbent assay), were evaluated. Our results showed that the addition of 1 μM AXT to sperm freezing media improved all parameters of sperm motility and viability (p ≤ 0.05). Furthermore, it could reduce the levels of ROS parameters (intracellular hydrogen peroxide and superoxide) compared with the control group (p ≤ 0.05). Also, AXT significantly decreased the level of PS externalization (p ≤ 0.05) and LPO (p ≤ 0.05) after the freezing–thawing process. In conclusion, our findings demonstrated that human semen treatment with 1 μM AXT before the freezing–thawing process has protective effects against oxidative stress and could diminish the destructive effects of this process on sperm quality.

Cryopreservation of spermatogonial stem cells (SSCs) is an important method to restore and maintain fertility in preadolescent children suffering from cancer. For protection of SSCs from cryoinjury, various antioxidant agents have been used. The aim of this study was to assess the antiapoptotic and antioxidant effects of melatonin in frozen-thawed SSCs. SSCs were isolated from testes of neonatal mice (3–6 days old) and their purities were measured by flow cytometry with promyelocytic leukemia zinc finger protein. After culturing, the cells were frozen in two groups (1) control and (2) melatonin (100 μM) and stored for 1 month. Finally, the cell viability, colonization rate, expression of Bcl-2 and BAX gene, and intracellular reactive oxygen species (ROS) were evaluated after freezing-thawing. Melatonin increased the viability and colonization of SSCs and Bcl-2 gene expression. It also diminished BAX gene expression and intracellular ROS. The results of this study show that melatonin with antioxidant and antiapoptotic effects can be used as an additive for freezing and long-term storage of cells and infertility treatment in the clinic.

Cryoprotectants are crucial factors in cell cryopreservation. Trehalose (Tre), a nontoxic, nonreducing, and natural disaccharide, has the potential to protect cells as a cryoprotectant. As an inducer of autophagy, Tre can influence the development of many diseases and may also have an effect on cell cryopreservation through this mechanism. In this study, human aortic endothelial cells were preserved in different cryopreservation fluids with or without dimethyl sulfoxide and Tre was added. Subsequently, the expression of the main autophagy-related genes LC3, BECN, and P62, cell death and apoptosis, and the proliferation rate were measured in different groups after cryopreservation. Our data showed that Tre can improve the expression of the autophagy-related genes LC3 and BECN and reduce the expression of P62. Dead/alive staining and flow cytometry showed that cell death and cell apoptosis were reduced during cryopreservation with Tre. In addition, the cell proliferation rate after thawing was increased in the Tre group when compared with others. These results all indicated that there might be a connection between Tre-triggered autophagy and the protective role of Tre in cell cryopreservation. Furthermore, strategies to regulate autophagy to reduce apoptosis in this process should be investigated in future research.

The objective of the study was to evaluate the integrity of cat testicular tissues after vitrification with different devices followed by different warming conditions. The influence of vitro culture for 24 hours after warming also was examined. Testicular tissues from adult domestic cats were dissected in small fragments that were vitrified using Cryotop^® or threaded on fine needles, warmed (directly at 37°C or with a preliminary 10 seconds exposure to 50°C), and/or cultured in vitro for an additional 24 hours. For each treatment group, tissues were assessed based on histology, apoptosis, and sperm DNA integrity. Results showed that fragments of testicular tissues were efficiently cryopreserved (maintaining the quality of all cell types) with vitrification with Cryotop followed by direct warming at 37°C, and additional culture of 24 hours at 38.5°C. These encouraging results are paving the road to optimize preservation protocols and use them for systematic banking of tissues from genetically valuable felids.

High concentrations of cryoprotective agents (CPAs) are required to achieve successful vitrification of articular cartilage; however, CPA cytotoxicity causes chondrocyte death. To reduce CPA toxicity, supplementation with research-grade additives, in particular chondroitin sulfate (CS) and ascorbic acid (AA), have previously been shown to improve chondrocyte recovery and metabolic function after exposure to CPAs at hypothermic conditions. However, it is necessary to evaluate the pharmaceutical equivalent clinical grade of these additives to facilitate the supplementation of additives into future vitrification protocols, which will be designed for vitrifying human articular cartilage in tissue banks. We sought to investigate the effectiveness of clinical-grade CS, AA, and N-acetylcysteine (NAC) in mitigating toxicity to chondrocytes during CPA exposure and removal, and determine whether a combination of two additives would further improve chondrocyte viability. We hypothesized that clinical-grade additives would exert chondroprotective effects comparable to those of research-grade additives, and that this protective effect would be enhanced if two additives were combined when compared with a single additive. The results indicated that both clinical-grade and research-grade additives significantly improved cell viability (p < 0.10) compared with the negative control (CPA with no additives). CS, AA, and NAC+AA increased cell viability significantly (p < 0.10) compared with the negative control. However, NAC, NAC+CS, and CS+AA did not improve cell viability when compared with the negative control (p > 0.10). We demonstrated that supplementation with clinical-grade CS or AA significantly improved chondrocyte viability in porcine cartilage subjected to high CPA concentrations, whereas supplementation with clinical-grade NAC did not benefit chondrocyte viability. Supplementation with clinical-grade additives in CPA solutions can mitigate CPA toxicity, which will be important in translating previously developed effective protocols for the vitrification of articular cartilage to human tissue banks.