Cryoprotectant Dimethyl Sulfoxide Research Articles

Unravelling spermatogenesis in spotted wolffish: Insights from the ultrastructure of juvenile male testes to the cryopreservation of broodstock sperm

The aim of this study was to deepen our understanding of the reproductive biology of male spotted wolffish (Anarhichas minor) using two different experimental approaches involving juvenile and mature broodstock fish. The first approach consisted of a detailed histological examination of the testes to identify the onset of gonadal maturation and characterise the spermatogenic stages in two- and three-year-old juvenile specimens. Light microscopy analysis revealed clear differences between the age groups. Two-year-old fish displayed well-defined interstitial tissue, Sertoli cells and cysts housing spermatogonia stem cells in which meiosis had not yet begun. In contrast, three-year-old fish exhibited cysts containing spermatocytes, spermatids and abundant spermatozoa, indicating the initiation of the spermatogenic cycle, albeit with asynchronous puberty. Histochemical staining revealed a significant presence of smooth myoid cells in the interstitial tissue of sexually mature fish, while electron microscopy further revealed synaptonemal complexes indicating the onset of meiosis and centriolar structures that gave rise to flagella. The second approach focused on optimising semen freezing and cryopreservation procedures in mature broodstock individuals over the age of 10 years. Seven freezing extenders (KT, TS-2, OP, MT, MH, HBSS, or SR), with seawater (SW) as a control, were assessed along with two cryoprotectants dimethylsulfoxide (DMSO) or methanol to evaluate their impact on pre- and post-thaw semen quality. Results showed that the MT and HBSS extenders were superior in total sperm kinetics at 1:3 dilution, and that DMSO showed optimal results in sperm motility and velocity variants. Moreover, the MT and HBSS groups demonstrated consistent sperm viability after cryopreservation, with values similar to fresh samples. Based on the viability results of the SYBR-green-14/PI assay comparing fresh and cryopreserved sperm using MT and HBSS, the MT extender emerged as the most effective freezing medium for cryopreservation of spotted wolffish broodstock sperm. In conclusion, this study provides a comprehensive understanding of the reproductive dynamics of male spotted wolffish, offering valuable insights for both scientific research and aquaculture management.

Systematic cryopreservation study of cardiac myoblasts in suspension.

H9c2 myoblasts are a cell line derived from embryonic rat heart tissue and demonstrate the ability to differentiate to cardiac myotubes upon reduction of the serum concentration (from 10% to 1%) and addition of all-trans retinoic acid in the growth medium. H9c2 cells are increasingly being used as an easy-to-culture proxy for some functions of cardiomyocytes. The cryobiology of cardiac cells including H9c2 myoblasts has not been studied as extensively as that of some cell types. Consequently, it is important to characterize the cryobiological response and systematically develop well-optimized cryopreservation protocols for H9c2 cells to have optimal and consistent viability and functionality after thaw for high quality studies with this cell type. In this work, an interrupted slow cooling protocol (graded freezing) was applied to characterize H9c2 response throughout the cooling profile. Important factors that affect the cell response were examined, and final protocols that provided the highest post-thaw viability are reported. One protocol uses the common cryoprotectant dimethyl sulfoxide combined with hydroxyethyl starch, which will be suitable for applications in which the presence of dimethyl sulfoxide is not an issue; and the other protocol uses glycerol as a substitute when there is a desire to avoid dimethyl sulfoxide. Both protocols achieved comparable post-thaw viabilities (higher than 80%) based on SYTO 13/GelRed flow cytometry results. H9c2 cells cryopreserved by either protocol showed ability to differentiate to cardiac myotubes comparable to fresh (unfrozen) H9c2 cells, and their differentiation to cardiac myotubes was confirmed with i) change in cell morphology, ii) expression of cardiac marker troponin I, and iii) increase in mitochondrial mass.

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

Preparation of cryopreserved chimeric antigen receptor T cells for the locoreogional delivery to the neural axis

Background aimsIntracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents an attractive delivery method to central nervous system tumors. Although IC delivery is actively being employed in early-phase clinical studies, no thaw/wash methods have been published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T-cell products before IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure. MethodsWe developed a thaw/wash procedure that consist of product thaw at 37°C, equilibration for 5 min in 1 volume of preservative-free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS and storage in a syringe at 20–25°C. Final formulated products (FPs) were assessed for quality and safety attributes and stability over 3 h from the completion of the thaw. Stability parameters included CAR T-cell viability, transgene surface expression and cytolytic activity. ResultsThe developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (versus pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%–88%; total nucleated cell recovery mean: 76.5%, range: 65.4%–82.5%). Other prespecified quality assurance/quality control parameters including appearance/ integrity, sterility and endotoxin level (≤1.0 EU/mL), were also met by all FPs (n = 3). Three hours’ post thaw/wash stability was confirmed. All products maintained cell viability greater than 70% (mean, 80.0%; range, 79%–81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared with thawed not diluted/washed control CAR T cells. ConclusionsWe have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.

Viability of mononuclear cells in leukocyte concentrates at the stages of their preparation, freezing, and thawing

Aim. To evaluate the viability of mononuclear cells (MNCs) in leukocyte concentrates (LCs) at the stages of their preparation, freezing, and thawing.Materials and methods. The study material included 44 LCs from donors of allogeneic hematopoietic stem cells (HSCs) and 189 autologous LCs from patients with oncohematological disorders. LCs were obtained from donors and patients by leukocytapheresis after mobilization of HSCs. LCs from patients were frozen with dimethyl sulfoxide (DMSO) used as a cryoprotectant at a final concentration of 5% and stored in liquid nitrogen. LCs were thawed before transplantation. A total of 161 LCs were immediately transfused to the recipient after thawing, and 28 LCs were washed from DMSO before transfusion. Flow cytofluorometry was used to determine the percentage of MNC populations that excluded 7-aminoactinomycin D (7-AAD).Results. The viability of peripheral blood MNCs in donors and patients was close to 100%. It was found that leukocytapheresis and cryopreservation with DMSO did not affect the viability of MNCs. The freezing of LCs with DMSO, storage in liquid nitrogen, and thawing resulted in a significant decrease in the content of viable MNCs (p = 0.0025), while no effect of LC storage duration on the viability of MNCs was revealed. Following DMSO removal from LCs, significantly more HSCs remained in a viable state than without washing (94.4 [94.5; 95.2] % vs. 86.7 [67.6; 92.9] %, (p = 0.0051); for other MNC populations, except monocytes, the differences in the viability index were also statistically significant.Conclusion. The viability of MNCs in LCs is recommended to be used as an independent characteristic of the transplant quality. In obtaining LCs and mixing them with the cryoprotectant DMSO, the viability of MNCs does not decrease, while in thawed LCs, it decreases significantly. Thawing of LCs with removal of DMSO allows to achieve the best viability of HSCs and most MNC populations.

A novel method to pack cryopreserved amniotic membrane for wound dressing—the pathway through validation of a new biological product

Amniotic membrane (AM) has been widely used as a biological dressing for many pathologies and illnesses worldwide, and products derived from this tissue have been commercially available in several countries. In Brazil, regulatory agencies have recently authorized its clinical use as a non-experimental therapy for burns, diabetic and venous stasis ulcers, and intrauterine adhesions. In this study, we present our pathway through validating the first available service in the country of AM cryopreservation, with a protocol for long-term storage in high-efficiency nitrogen cryogenic freezers and a specific way of packing the tissue for optimal clinical handling and efficient storage space utilization while preserving live cells and the tissue’s biological properties. Using gauze as support, cryoprotectant dimethyl sulfoxide and product presentation as a multilayer roll exhibited the best cell viability results and maintained the tissue integrity and presence of stem/progenitor cells. Essential proteins involved in tissue regeneration and immune and antimicrobial control were detected from the secretome of cryopreserved tissue similar to fresh tissue. Furthermore, immunogenic markers, such as human leukocyte antigens, were detected at very low levels in the tissue, confirming their low immunogenicity. Finally, we demonstrate that the tissue can be kept under refrigerated conditions for up to 7 d for further use, maintaining sterility and considerable cell viability. Our cryopreservation and storage protocol kept the AM viable for at least 20 months. In conclusion, this study enabled us to determine a novel efficient protocol for long-term AM preservation for future clinical applications.

Cryopreservation of sperm in the spotted scat, Scatophagus argus (Linnaeus, 1766) - Conservation and aquaculture perspectives

This study emphasizes the need for sperm quality assessment and cryopreservation strategy in the spotted scat Scatophagus argus as the occurrence of male population of this species is limited. Analyses of sperm quality were made including ionic content and osmolality of seminal plasma and blood serum. Sodium chloride (0.9%) was optimized as the extender with a dilution ratio of 1:3 (milt:cryomedium) for all experiments. Cryoprotectants including dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), glycerol (GLY), propylene glycol (PG), ethylene glycol (EG) and methanol (MeOH) were screened at various concentrations and equilibration times. Cryopreservation was performed using the cryoprotectants DMSO, GLY, EG, and MeOH individually as well as in combinations. Experiment 1 was done with DMSO, GLY, EG and MeOH (5, 10 and 15%); experiment 2 was done with DMSO (5%) + GLY (5%), DMSO (5%) + EG (5%), DMSO (5%) + MeOH (5%). Simultaneously, experiment 3 was done with both cryoprotectants and co-cryoprotectants viz. DMSO (5%) + egg yolk (5%), DMSO (5%) + coconut water (5%), DMSO (5%) + tomato water (5%) and DMSO (5%) + honey (5%). The cooling sample was optimized and performed with a one-step freezing protocol of −5 °C/min from 25 °C to −150 °C. The results indicated that samples subjected to experiment 2 using DMSO (5%) + EG (5%) for 60 days of storage registered maximum motility (82.33 ± 2.51%), duration time of sperm motility (10.45± 1.01 min) and viability (84.14 ± 5.29%) of post-thaw cryopreserved sperm. Morphology and ultrastructure of post-thaw sperm showed smooth surface topography and normal architecture of cellular organelles. Biochemical analysis indicated a reduction in the protein content of post-thaw cryopreserved sperm. DNA integrity (Comet assay) showed 6.30 ± 1.00% of tail DNA in post-thaw sperm, whereas fresh sperm registered 2.41 ± 1.85%. Functional integrity using the HOST of post-thaw sperm showed a maximum of 82.66 ± 1.15% with DMSO (5%) + EG (5%). Finally, fertility of post-thaw sperm using mature eggs of wild-caught females indicated the entry of sperm by breaking the vitelline membrane. Overall it was demonstrated that the DMSO (5%) + EG (5%) was amenable to the long-term preservation of sperm. Therefore, this protocol can be used as an alternative for male broodstock development under captive breeding program.

Influence of conditioned media from glial cell cultures on contractility of uterine in rats of different ages

Background. The physiological regulation of the uterine contractile activity changes with age, which leads to an increased number of prolonged labor and emergency caesarean sections in women giving birth at the age of 35+. One of the modern approaches to correct the function of the reproductive system is the use of from cell cultures. CM from glial cell culture contains neurotrophic factors that play an important role in maintaining the contractile function of the uterus. Current cell culture technologies include cryopreservation.Objective: to research experimentally the effect of CM obtained from intact and cryopreserved cultures of glial cells on the contractile activity of the uterus in rats of different reproductive ages.Materials and methods. The monolayer cell culture was obtained from the dorsal root ganglia of neonatal piglets and cryopreserved in the presence of cryoprotectant dimethyl sulfoxide. CM from native and cryopreserved cultures were collected for 28 days, after which fractions with a molecular weight of &lt; 30 kDa were obtained from them by ultrafiltration. Rats at the age of 6 and 14 months, which corresponds to reproductive age and late reproductive age (LRA), were intraperitoneally injected with 0.2 ml of media from intact (ICM) or cryopreserved (CCM) cultures for 9 days. On the 30th – 32nd day after the end of the administration of CM animals were slaughtered and the uterine contractile activity was determined by the organ bath method, the relative area of myometrium and density of myocytes by histological method, the average area of labeling with specific antibodies to smooth muscle actin by immunohistochemical method. The statistical significance of differences was assessed by the Mann–Whitney test.Results. It was found that spontaneous, OT-, and KCl-induced tension of isometric contraction of the uterus in intact LRA rats decreased by 19, 20, and 14%, respectively, compared with intact reproductive aged animals. After the introduction of ICM and CCM in LRA animals, normalization of isometric contraction parameters was observed. This effect was realized against the background of an increase in the area of the myometrium, the density of myocytes, and actin expression.Conclusions. Intra-abdominal administration of CM from glial cell culture increases the uterine contractile activity in LRA rats. This effect is realized by increasing the relative area of the myometrium, the density of myocytes, and the area of expression of smooth muscle actin. The effect of media from intact and cryopreserved cultures on the contractile activity of the uterus was similar, which makes it possible to use low-temperature culture storage technologies to obtain CM without losing its biological effect.

The use of non-cryopreserved hematopoietic stem cells for autologous transplantation in multiple myeloma patients

Background . An autologous stem cell transplant (ASCT) is the standard of treatment young and fit patients with multiple myeloma (MM). The using this method of treatment is limited due to the high consumption of economic and intellectual resources, the low availability of Cryobank departments in regions of Russia. Objective : to evaluate the efficacy and safety of using non-cryopreserved peripheral blood stem cells (non-CRYO PBSCs) in patients with multiple myeloma who underwent autologous transplantation. Materials and methods . We conducted a prospective study of the efficacy and safety of using non-CRYO PBSCs in patients with MM who underwent ASCT. The number of PBSCs apheresis procedures in 82.9 % (n = 29 pts) was 1 day, and in 17.1 % (n = 6 pts) was 2 days. After apheresis, PBSCs were stored at a temperature of +4…+6 °C in a blood bank refrigerator for up to 72 hours. During 4 years, ASCT using non-CRYO PBSCs was performed in 35 patients with MM. The average number CD34+ cell dose was 2.63 × 106 /kg. The median time to neutrophil engraftment was 11 days (range from 9 to 14). The median time to platelet engraftment was 12 days (range from 8 to 19). The control group included 43 patients with MM, who underwent PBSCs ASCT according to the traditional method, including preparation and cryopreservation of the apheresis product of PBSCs with cryoprotectant dimethyl sulfoxide, as well as subsequent defrosting on the day of transplantation (day 0). Results . There were no significant differences comparing the safety of ASCT in non-CRYO and CRYO PBSCs groups among such parameters as the viability of PBSCs, the frequency of complications, the time of hematopoietic engraftment, the need for platelet and red blood cells transfusion therapy, and time of hospitalization. Conclusion . Hence, the method of short-term storaging non-CRYO PBSCs is not inferior to the traditional method of controlled freezing, is more economical and can be used in medical organizations that do not have a Cryobank in their structure.

Inertial Microfluidic Purification of CAR-T-Cell Products.

Chimeric antigen receptor T (CAR-T) cell therapy is rapidly becoming a frontline cancer therapy. However, the manufacturing process is time-, labor- and cost-intensive, and it suffers from significant bottlenecks. Many CAR-T products fail to reach the viability release criteria set by regulators for commercial cell therapy products. This results in non-recoupable costs for the manufacturer and is detrimental to patients who may not receive their scheduled treatment or receive out-of-specification suboptimal formulation. It is demonstrated here that inertial microfluidics can, within minutes, efficiently deplete nonviable cells from low-viability CAR-T cell products. The percentage of viable cells increases from 40% (SD ± 0.12) to 71% (SD ± 0.09) for untransduced T cells and from 51% (SD ± 0.12) to 71% (SD ± 0.09) for CAR-T cells, which meets the clinical trials' release parameters. In addition, the processing of CAR-T cells formulated in CryStor yields a 91% reduction in the amount of the cryoprotectant dimethyl sulfoxide. Inertial microfluidic processing has no detrimental effects on the proliferation and cytotoxicity of CAR-T cells. Interestingly, ≈50% of T-regulatory and T-suppressor cells are depleted, suggesting the potential for inertial microfluidic processing to tune the phenotypical composition of T-cell products.

The effect of follicle size and cryoprotectants on nuclear maturation and early embryonic development of vitrified - thawed Awassi sheep oocytes (Ovis aries)

In this study, two experiments were conducted to study the effect of both the follicle size and the cryoprotectants dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the main phases of nuclear maturation (Experiment I), cleavage stages and embryo quality (Experiment II) of Awassi sheep oocytes. Follicles were classified into two groups: small follicles (SF) (1-2 mm) and large follicles (LF) (&gt; 2 mm). Oocytes were vitrified in three solutions: A (30% DMSO), B (30% EG) and C (15% DMSO and 15% EG). In Experiment I, the resulting vitrified-thawed oocytes in solution C achieved the best rates after the control group (fresh), respectively as the rates of maturation, germinal vesicle (GV), metaphase II(M-II), arrest, and lyses were 85.71% (P = 0.04), 8.33% (P = 0.02), 72.92% (P = 0.04); LF group, 15.25% (P = 0.04), and 5.08% (P = 0.04); SF group, respectively. In Experiment II, the same group of oocytes achieved the best rates after the control group, as the rates of fertilization, cleavage, 2-16 cell, Type3, blastocyst, and Type1 embryos were 63.28% (P = 0.001), 57.46% (P = 0.001), 40.38% (P = 0.04), 38.46% (P = 0.04); LF group, 30.00% (P = 0.01), and SF group 36.67% (P = 0.001), respectively, while the vitrified-thawed oocytes in A solution (SF group) reached the highest rate of Type 2 embryo quality (58.06%; P = 0.01). No significant differences were noticed in the germinal vesicle breakdown (GVBD), metaphase I (M-I) and morula stage. Vitrification of oocytes obtained from follicles with a diameter of more than 2 mm in a cocktail solution of DMSO (15%) and EG (15%) led to a significant increase in the yield and quality of the resulting sheep embryos.

Generation of reproductively mature offspring from the endangered green and golden bell frog Litoria aurea using cryopreserved spermatozoa.

Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen-thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians.

Cryopreservation of Mammalian Oocytes: Slow Cooling and Vitrification as Successful Methods for Cryogenic Storage.

Two basic methods for the laboratory-focused cryopreservation of mammalian oocytes are described, based on work with murine oocytes. One method uses a relatively low concentration of the cryoprotectant propanediol plus sucrose and requires controlled rate cooling equipment to achieve a slow cooling rate. This method has also produced live births from cryopreserved human oocytes. The second method, which is described here, employs a high concentration of the cryoprotectant dimethyl sulfoxide plus a low concentration of polyethylene glycol. This is a vitrification method, which involves ultra-rapid cooling by plunging standard straws into liquid nitrogen vapor, hence avoiding the need for specialized equipment, but requires technical ability to manipulate the oocytes quickly in the highly concentrated cryoprotectant solutions. Murine oocytes that have been vitrified using this technique have resulted in live births. Vitrification using other cryoprotectant mixtures is now a popular clinically accepted method for cryobanking of human oocytes.

Exploring novel cell cryoprotectants based on neutral amino acids

Cryoprotectants play a key role in cell cryopreservation because they can reduce cryoinjuries to cells associated with ice formation. To meet the clinical requirements of cryopreserved cells, cryoprotectants should be biocompatible, highly efficient and easily removable from cryopreserved cells. However, integration of these properties into one cryoprotectant still remains challenging. Herein, three biocompatible neutral amino acids, including β-alanine, γ-aminobutyric acid and ε-aminocaproic acid, are first reported to have the potential as such ideal cryoprotectants. The results demonstrate that they can inhibit ice formation and reduce osmotic stress to provide extracellular and intracellular protection, thereby ensuring high cryopreservation efficiency for both anuclear and nucleated cells. More importantly, due to the remarkable osmotic regulation ability, the neutral amino acids can be rapidly removed from cryopreserved cells via a one-step method without causing observable damage to cells, superior to the current state-of-the-art cryoprotectants—dimethyl sulfoxide and glycerol. This work provides a new perspective to develop novel cryoprotectants, which may have dramatic impacts on solvent-free cryopreservation technology to support the cell-based applications, such as cell therapy and tissue engineering, etc.

Comparison of conventional and automated freezing methods on PB2 rooster semen cryopreserved with glycerol and dimethylsulfoxide tris coconut-water extender

BackgroundPoultry semen cryopreservation remains an easy and promising way of preserving and transferring poultry germplasm. Standardizing and optimizing freezing procedure with natural extender may be a reliable step towards achieving better post-thawed sperm quality. This study was conducted to investigate the effects of four different freezing protocols (FP) on frozen rooster semen extended with tris coconut water extender (TCWE). A total of 20 roosters were used and trained for semen collection. TCWE was prepared by adding coconut water to tris buffer and kept at 37 °C. Semen was collected and pooled from the roosters and was evaluated for motility before dilution procedure. Three different concentrations (8, 10, and 15%) of two intracellular cryoprotectants glycerol and dimethylsulfoxide (DMSO) were supplemented in TCWE. Pooled semen was divided into six equal fractions, and TCWE containing cryoprotectants in different concentrations were diluted with the semen in ratio 1:2 (semen:extender). Diluted semen was manually filled in 0.25 ml straws and sealed. Semen straws were equilibrated for 4 h at 4 °C. Each straw fraction was further divided into four parts, and subjected to four FP (slow freezing 1, 2, 3 and fast freezing 4). Each FP was done on samples containing 8, 10, and 15% glycerol and 8, 10, and 15% DMSO. After each protocol, semen straws were finally deep into liquid nitrogen – 196 °C. After 48 h, the straws were thawed individually to evaluate post/thawed motility, viability, and membrane integrity. The experiment consists of three trials.ResultsAt 8% glycerol concentration, FP1 has significantly (P > 0.05) the highest percentage motility (73.33%) compared to FPs 2, 3, and 4 (56.68, 50.00, and 23.33% respectively). At 10% glycerol, FP2 had the highest motility (48.33%) and HOST (64.00%). At 15% glycerol, FP4 (fast freezing) had the highest percentage motility (71.67%), viability (76.33%), and HOST (71.67%). At 8%, DMSO concentrations FP4 had the highest significant (P > 0.05) motility, viability, and HOST. Ten percent and 15% DMSO revealed no significant (P > 0.05) difference in most parameters among all the FPS. DMSO performed better than glycerol irrespective of concentrations in all FPS on most parameters evaluated.ConclusionsFast freezing performed better with 15% concentration of glycerol, while slow and fast freezing performed better with 10% and 15% DMSO concentrations, which implies that DMSO may serve as better cryoprotectant for poultry semen freezing irrespective of freezing protocols adopted.

Антиоксидант глутатіон підвищує стійкість ядровмісних клітин кордової крові під час кріоконсервування з диметилсульфоксидом

Abstract: The cryopreservation effi ciency of nucleated cells (NCs), in particular hematopoietic progenitor (HPCs) and cord blood (CB) cells was comprehensively assessed in protective mixtures, containing intracellular cryoprotectant dimethyl sulfoxide (DMSO) and antioxidant glutathione in different concentrations assessed immediately after thawing and further transfer to the in vitro conditions, simulating physiological ones. The use of 1 and 3 mM glutathione in cryoprotectant medium with 7.5 and 10% DMSO enabled preserving 85.9 and 91.2% of viable NCs and HPCs, respectively, immediately after thawing and 75–80% of viable NCs/HPCs after 1-hour incubation in the antioxidant-free Hank’s solution, which exceeded the control values. At the same time, the recovery and viability of NCs and HPCs after cryopreservation in 5% DMSO with 1mM or 3 mM glutathione were the same or even higher than those in 7.5 and 10% DMSO without glutathione. The NCs cryopreservation in the glutathione-containing solutions was esta-blished to reduce the number of DCF+ cells. The supplementation of 7.5% DMSO with 1 or 3 mM glutathione reduced the cell number with excess reactive oxygen species from 19% (in glutathione-free samples) to 11% (in glutathione-contained ones), even after transfer to the in vitro conditions, simulating physiological. Probl Cryobiol Cryomed 2020; 30(1): 058–067

Seminal characteristics and sensitivity of Astyanax lacustris (Characiformes: Characidae) sperm to cryoprotective solutions based on dimethylsufoxide and methylglicol

Abstract This study aimed to determine the semen characteristics of Astyanax lacustris after hormonal induction and to evaluate the sensitivity of the species sperm to cryoprotective solutions based on the cryoprotectants dimethyl sulfoxide and methyl glycol. Volume, color, sperm concentration, total motility and aspects of sperm movement were analyzed using “Integrated Semen Analysis System”. Three different extenders were tested: A) glucose 5%+egg yolk 10%, B) BTS®5% and C) glucose 5% and two permeable cryoprotectants: dimethyl sulfoxide (Me2SO) and methyl glycol (MTG). Fresh A. lacustris semen presented total motility of 76.6±11.2%, motility duration of 33.0±2.2s, sperm concentration of 7.22±3.2×109sptz/mL and seminal osmolality of 219±0.03mOsm/kg-1. The toxicity test showed the highest total motility values at the MTG15%+A, Me2SO15%+B and Me2SO10%+C dilutions, and the Me2SO10%+C and Me2SO15%+C dilutions presented the highest values for curvilinear velocity, linear velocity and average velocity. The tested protocol was not effective at maintaining the viability of A. lacustris semen after freezing because no motility was observed in any of the dilutions. However, the Comet Assay demonstrated that cryoprotectant solutions were effective in protecting the genetic material of cells, as DNA damage levels were low, with no difference between control and Me2SO10% + A, dilutions MTG10%+C, Me2SO10%+B and Me2SO15%+B.

A Method to Efficiently Cryopreserve Mammalian Cells on Paper Platforms.

This protocol describes a simple method to cryopreserve mammalian cells within filter papers as an alternative to conventional slow-freezing approach. The method involves treating paper fibers with fibronectin, using low concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), and slow freezing cells to -80 °C at a 1 °C min-1 rate. In our method, the biocompatibility, large surface area, 3D porosity and fiber flexibility of the paper, in combination with the fibronectin treatment, yield recovery of cells comparable to conventional approaches, with no additional fine-tuning to freezing and thawing procedures. We expect that the paper-based cryopreservation method will bring several advantages to the field of preserving mammalian cells, including accommodation of a higher number of cells within a unit volume and no cell loss after release. The method requires a minimal storage space, where paper platforms with large areas can be rolled and/or folded and stored in stocks, and allows for efficient transportation/distribution of cells in an on-demand manner. Moreover, an additional feature of this method includes the formation and cryopreservation of cellular spheroids and 3D cell cultures.

Evaluation of Bull Semen Quality and Sperm DNA Integrity during Cryopreservation with Different Cryoprotectants

The fertility rate in dairy cattle with cryopreserved semen significantly depends on the reduction of the toxic effects on the sperms induced during cryopreservation. DNA integrity in sperm is vital for the precise transmission of genetic information and therefore the production of high yielders and maintenance of good health in future generations. The reliable and consistent assessment of sperm motility can be succeeded by Computer Assisted Semen Analyzer (CASA). Ten ejaculates were collected from Frisian bulls kept at Centre of excellence for bovine genetics (CEBG) Renala Khurd District Okara. The deleterious effects of three cryoprotectants glycerol, dimethylsulfoxide (DMSO) and ethylene glycol as a constituent of tris-egg yolk-citrate extender were compared on diluted post thawed cryopreserved Frisian bull semen. The quality of semen in terms of viability/motility/progressive velocity was determined with computerized external real image optical system (CEROS) and sperm DNA fragmentation was quantified with comet assay using single cell gel electrophoresis (SCGE) technique. Glycerol was the least followed by DMSO and ethylene glycol was the most toxic cryoprotectant both for semen quality and sperm DNA fragmentation. The present study suggests that sperm DNA fragmentation is an associated feature of semen cryopreservation resulting into cell death (non-motile sperms) and glycerol as a cryoprotectant constituent of tris-egg yolk citrate extender offers a better protection for storage of cryopreserved Frisian bull semen in liquid nitrogen.

Crioprotetores na criopreservação de sementes de Passiflora mucronata Lam

Brazil is one of the centers of diversity of the genus Passiflora L . and therefore has great potential for the utilization of the genetic resources in agronomy. Passiflora mucronata Lam. is a wild species that presents great characteristics for ornamental use. This work aimed to evaluate the effect of different concentrations of cryoprotectants dimethylsulfoxide (DMSO) and sucrose (0, 7, 14, 21 and 28%) on the cryopreservation of Passiflora mucronata seeds. After the application of the cryoprotectants, the seeds were stored in liquid nitrogen for 5 days, thawed, and later, seeded in vermiculite and maintained at a temperature of 27.7 ± 1.9°C. The cryoprotectant type and the application of increasing doses affect the physiological quality of seeds after storage in liquid nitrogen. Cryopreservation may be used for the long-term conservation of seeds, without the use of cryoprotectants.