Cryoprotective Medium Research Articles

From 3D to 3D: isolation of mesenchymal stem/stromal cells into a three-dimensional human platelet lysate matrix

BackgroundMesenchymal stem/stromal cells (MSCs) are considered an important candidate in cell therapy and tissue engineering approaches. The culture of stem cells in a 3D environment is known to better resemble the in vivo situation and to promote therapeutically relevant effects in isolated cells. Therefore, the aim of this study was to develop an approach for the direct isolation of MSCs from adipose tissue into a 3D environment, avoiding contact to a 2D plastic surface. Furthermore, the use of a cryoprotective medium for the cryopreservation of whole adipose tissue was evaluated.Materials and methodsCryopreservation of fresh adipose tissue with and without a cryoprotective medium was compared with regard to the viability and metabolic activity of cells. After thawing, the tissue was embedded in a novel human platelet lysate-based hydrogel for the isolation of MSCs. The migration, yield, viability, and metabolic activity of cells from the 3D matrix were compared to cells from 2D explant culture. Also, the surface marker profile and differentiation capacity of MSCs from the 3D matrix were evaluated and compared to MSCs from isolation by enzymatic treatment or 2D explant culture.ResultsThe cryopreservation of whole adipose tissue was found to be feasible, and therefore, adipose tissue can be stored and is available for MSC isolation on demand. Also, we demonstrate the isolation of MSCs from adipose tissue into the 3D matrix. The cells derived from this isolation procedure display a similar phenotype and differentiation capacity like MSCs derived by traditional procedures.ConclusionsThe presented approach allows to cryopreserve adipose tissue. Furthermore, for the first time, MSCs were directly isolated from the tissue into a soft 3D hydrogel environment, avoiding any contact to a 2D plastic culture surface.

Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage

Study questionDoes cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing?Summary answerVitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing.What is known alreadyA major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa.Study design, size, durationThis was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol.Participants/materials, setting, methodsAll human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25–45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen.Main results and the role of chanceAmong the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000).Limitations, reasons for cautionExtensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing.Wider implications of the findingsThe presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost.

Optimizing cryopreservation of hematopoietic stem cells collected for autologous stem cell transplantation in patients with multiple myeloma

Background & Aim Cryopreservation is a necessary step in the preparation of hematopoietic stem cells (HSC) used to reconstitute hematopoiesis for autologous stem cell transplantation. Cells are collected by leukapheresis, cryopreserved and stored while patients undergo preparative regimen. It is critical to optimize cryopreservation techniques to ensure optimal recovery of viable progenitor cells, especially when target cells are limited. Current methods are adequate, but limitations include inconsistency in components used to formulate media between institutions, increased risk for technical error and contamination. This study sought to optimize DMSO concentration and improve efficiency and compliance by comparing three different cryoprotectant media. Methods, Results & Conclusion Methods Ten HSC products, collected after standard mobilization of multiple myeloma patients, were cryopreserved with PRIME-XV FreezIS (Irvine Scientific) and compared to products previously cryopreserved with two standard use cryoprotectants: Std10 and Std5 (Std10 and Std5 are cryoprotectant media formulated in-house to achieve a final DMSO concentration of 10% and 5% respectively). Cells were processed and cryopreserved using institutional standard procedures, with storage for up to 6 months. At time of infusion, HSC were analyzed for CD34+ recovery and viability. Adverse events (AE) and time to engraftment were also monitored. Results Average CD34+ recovery for HSC cryopreserved with Std10, Std5 and PRIME-XV FreezIS were 39%, 74.7%, and 73.6% respectively (p-value: ≤ 0.001 Std10 vs. FreezIS). Median time to neutrophil engraftment was comparable (11 days) for all 3 media, while platelet engraftment occurred at a median of 20, 19 and 17 days respectively (p-value: >0.05 Std10 vs. FreezIS). Infusion-related AEs decreased from 80% (Std10) to 10% (FreezIS) (p-value: ≤ 0.0001). Discussion PRIME-XV FreezIS is a GMP-grade, pre-constituted cryoprotectant that minimizes cell processor variability and handling. In addition, compared to cryopreservation media prepared in house containing both 5% and 10% DMSO, PRIME-XV FreezIS results in ample CD34+ recovery and similar HSC engraftment. Finally, as anticipated, infusion toxicity caused by DMSO and frequency of AEs decreased significantly in patients infused with PRIME-XV FreezIS and Std5 when compared to products cryopreserved with Std10.

Intramyocardial Injection of Mesenchymal Precursor Cells and Successful Temporary Weaning From Left Ventricular Assist Device Support in Patients With Advanced Heart Failure

Left ventricular assist device (LVAD) therapy improves myocardial function, but few patients recover sufficiently for explant, which has focused attention on stem cells to augment cardiac recovery. To assess efficacy and adverse effects of intramyocardial injections of mesenchymal precursor cells (MPCs) during LVAD implant. A randomized phase 2 clinical trial involving patients with advanced heart failure, undergoing LVAD implant, at 19 North American centers (July 2015-August 2017). The 1-year follow-up ended August 2018. Intramyocardial injections of 150 million allogeneic MPCs or cryoprotective medium as a sham treatment in a 2:1 ratio (n = 106 vs n = 53). The primary efficacy end point was the proportion of successful temporary weans (of 3 planned assessments) from LVAD support within 6 months of randomization. This end point was assessed using a Bayesian analysis with a predefined threshold of a posterior probability of 80% to indicate success. The 1-year primary safety end point was the incidence of intervention-related adverse events (myocarditis, myocardial rupture, neoplasm, hypersensitivity reactions, and immune sensitization). Secondary end points included readmissions and adverse events at 6 months and 1-year survival. Of 159 patients (mean age, 56 years; 11.3% women), 155 (97.5%) completed 1-year of follow-up. The posterior probability that MPCs increased the likelihood of successful weaning was 69%; below the predefined threshold for success. The mean proportion of successful temporary weaning from LVAD support over 6 months was 61% in the MPC group and 58% in the control group (rate ratio [RR], 1.08; 95% CI, 0.83-1.41; P = .55). No patient experienced a primary safety end point. Of 10 prespecified secondary end points reported, 9 did not reach statistical significance. One-year mortality was not significantly different between the MPC group and the control group (14.2% vs 15.1%; hazard ratio [HR], 0.89; 95%, CI, 0.38-2.11; P = .80). The rate of serious adverse events was not significantly different between groups (70.9 vs 78.7 per 100 patient-months; difference, -7.89; 95% CI, -39.95 to 24.17; P = .63) nor was the rate of readmissions (0.68 vs 0.75 per 100 patient-months; difference, -0.07; 95% CI, -0.41 to 0.27; P = .68). Among patients with advanced heart failure, intramyocardial injections of mesenchymal precursor cells, compared with injections of a cryoprotective medium as sham treatment, did not improve successful temporary weaning from left ventricular assist device support at 6 months. The findings do not support the use of intramyocardial mesenchymal stem cells to promote cardiac recovery as measured by temporary weaning from device support. clinicaltrials.gov Identifier: NCT02362646.

Investigation of the processes of freezing and freeze-drying of bacterial concentrates for the dairy industry

This project is dedicated to the development of finishing equipment for the production of dry bacterial concentrates. One of the main objects is a quick-freezing apparatus designed for freezing bacterial concentrates, as well as a continuous vacuum freeze dryer. In this market, domestic production takes about 9 ... 12%. Creation of high-tech equipment of a new generation for the production of bacterial concentrates for dairy enterprises will allow to solve the problem of import substitution of this type of product. The main directions for the use of bacterial concentrates in the dairy industry. The analysis of used starter cultures, the range of products manufactured using bacterial concentrates. The classification of bacterial concentrates according to the method of storage and use is given. The methods of using bacterial concentrates at dairy enterprises are considered. The ways to improve the production of bacterial concentrates are proposed. To study the effect of technological parameters of vacuum freeze-drying on the quality of the final bacterial concentrate, a series of experiments was performed. Drying was carried out to a final moisture content of 3.0–3.2%, while the residual pressure in the chamber varied in the range of 10–50 Pa, and the heat flux density ranged from 1.2 to 1.8 kW / m2. The expediency of creating domestic production of baconconcentrates on an industrial scale is substantiated. The results of the study of the kinetics of the freezing of bacterial concentrates are given. The rational parameters of the freezing process and the optimal composition of the cryoprotective medium are proposed. Investigations of the process of vacuum-sublimation dehydration of bacterial concentrate in a layer and granulated form have been conducted. The quality indicators of dry bacterial concentrates are given. The results will allow to carry out engineering calculations and design of equipment for freezing and vacuum sublimation plants with various methods of energy supply.

Freeze-Concentrated Phase and State Transition Temperatures of Mixtures of Low and High Molecular Weight Cryoprotectants

Although numerous studies have been conducted on the use of cryoprotectants to prevent the deterioration of food during freezing and frozen storage, scarce reports exist on the thermal transition properties of aqueous cryoprotectant solutions at frozen temperatures. The selection of a suitable cryoprotective medium for the long-term preservation of food requires knowledge of the effects of cryoprotectants and their concentration on the freeze-concentrated unfrozen phase and state transition temperatures known asTg′,Tm′, andTm. Calorimetric measurements were conducted to determine theTg′,Tm′, andTmvalues of thirty frozen aqueous solutions containing maltodextrin, polydextrose, and glucose, in which a distance-based experimental design was used for mixtures of four components to establish their corresponding mass fractions in the mixtures. Thermograms, measured during heating/rewarming from –70 to 20°C, were used to identify the glass transition and freezing temperatures. Mathematical expressions forTg′,Tm′, andTmas a function of the mass fractions of cryoprotectants and water and their interactions (p< 0.05) were developed to aid the formulation of cryoprotective media involving more than two cryoprotectants for adequate frozen conservation of high and intermediate moisture foodstuffs.

ВИКОРИСТАННЯ ТРОМБОЦИТАРНОГО ЛІЗАТУ ЯК КОМПОНЕНТА КРІОЗАХИСНИХ СРЕДОВИЩ ДЛЯ КРІОКОНСЕРВУВАННЯ МЕЗЕНХІМАЛЬНИХ СТРОМАЛЬНИХ КЛІТИН

Mesenchymal stromal cells (MSCs) due to their unique properties are widely used in regenerative medicine. Standard cryopreservation methods that are based on the use of penetrating cryoprotectant dimethyl sulfoxide (DMSO) and fetal bovine serum (FS) can ensure high cell survival, but limit the possibility of therapeutic application because of the risk of adverse reactions. The toxicity of high DMSO concentrations and FS immunogenicity require significant optimization of cryopreservation approaches. In the current study, freezing of human MSCs in cryoprotective media (CPM) with different compositions was performed. Twenty-four hours prior to freezing, cells were pretreated by addition of 100 mM sucrose into the culture medium. CPM with 200 mM sucrose were supplemented with 10% FS or 10% platelet lysate (PL) in the presence or absence of 1% DMSO. The cells frozen without any cryoprotectants were used as a negative control. The MSCs cryopreserved in media containing 10% DMSO and 10% FS were chosen as a positive control group. The MSCs were frozen in cryogenic vials with a cooling rate of 1 deg/min to -80°C with the following immersion into liquid nitrogen. The cell survival was determined by trypan blue staining; metabolic activity was assessed using the Alamar Blue test. It was revealed that viability of MSCs after freezing in CPM containing 200 mM sucrose, 10% FS or 10% PL without DMSO addition were 59±3.3% and 58±2.5%, respectively. The metabolic activity of cells in the PL group exceeded the results of the FS group by 12%. When 1% DMSO was added into the CPM containing 200 mM sucrose and 10% PL, the cell survival rate and metabolic activity were by 7% and 13% higher than in the presence of 10% FS. The obtained results indicate that replacement of FS with PL in the CPM without penetrating cryoprotectant DMSO allows to maintain MSCs viability and increase their metabolic activity after freeze-thawing.

Effect of Cryopreservation on Different Passages of Porcine Dorsal Root Ganglion Cell Culture

The cell culture obtained from dorsal root ganglia (DRG) is a valuable model used in biology or medical research. However, the effect of cryopreservation on the properties of DRG-derived cell culture of different passages remains unclear up to date. The objective of the study is to assess the effect of cryopreservation with various concentrations of cryoprotectant dimethyl sulfoxide (DMSO) on the viability and morphological features of porcine neonatal DRG cell culture of different passages. Cell suspension was obtained from DRG of neonatal piglets and cultured in an α-MEM nutritional medium supplemented with 10% fetal calf serum (0, 1st, and 2nd passages). Cells of different passages were cryopreserved at a cooling rate of 0.5°C/min to – 20°C (step 1) and 1°C/min to – 80°C (step 2) followed by immersion into liquid nitrogen. Cryoprotective solutions based on α-MEM nutrient medium and 25% fetal calf serum (FCS) containing 5%, 7.5%, and 10% DMSO were used. It was established that the primary culture (passage 0) consisted of four cell morphological types: large rounded cell bodies of sensory neurons (SN) and three types of non-neuronal cells, namely, polygonal cells with pronounced elongated processes (type 1), spindle-shaped cells (type 2), and multipolar flattened fibroblastoid cells (type 3). As the number of passages increases, an elimination of SN from the culture, a decrease in the relative number of 1st and 2nd cell types, and expansion of 3rd cell type were observed. DRG cell culture had sufficiently high resistance to cryopreservation as cell viability was in the range from 83% to 90% using different concentrations of DMSO. The cells of passage 1 were more resistant to cryopreservation in comparison with primary culture cells (passage 0). The best result was achieved by freezing the culture of passage 1 in the cryoprotective medium with 7.5% DMSO, where 90.6% of viable cells were observed after thawing.

New cryoprotective media for cryopreservation of mammal erythrocytes

PURPOSE: To compare the effectiveness of a newly designed multi-component cryoprotective medium (15% PEO-1500, 10% DMSO, 5% 1.2-propanediol, 5% sucrose) with three known single- and two-component ones containing DMSO (20 %), PEO-1500 (30 %), and glycerol-mannitol (30 % and 4 %, respectively) in the cryopreservation of human, equine, bovine and rabbit erythrocytes. METHODS: Cryopreservation was achieved plunging the cryotubes with erythrocyte suspensions into liquid nitrogen (-196°C) at an average cooling rate of 200 deg/min. During the three cryopreservation stages; equilibration, freeze-thaw and final removal of cryoprotectants, the survival of erythrocytes was estimated by the level of hemolysis, measured spectrophotometrically. RESULTS: With the erythrocytes of all investigated mammals the designed medium produced a higher level of survival at all cryopreservation stages. CONCLUSION: The tested multi-component cryopreservation medium, combining both penetrating and non-penetrating cryoprotectants, diminished their concentration and accompanying toxic effects and achieved a higher survival of human and mammal erythrocytes at all stages of their cryopreservation.

Cryopreservation of Sperm: Effects on Chromatin and Strategies to Prevent Them.

Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at -196°C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols.Despite its importance for assisted reproduction technology (ART) and its success in terms of babies born, this procedure can cause cell damage and impaired sperm function. Various studies have evaluated the impact of cryopreservation on chromatin structure, albeit with contradictory results. Some, but not all, authors found significant sperm DNA damage after cryopreservation. However, studies attempting to explain the mechanisms involved in the aetiology of cryopreservation-induced DNA damage are still limited. Some reported an increase in sperm with activated caspases after cryopreservation, while others found an increase in the percentage of oxidative DNA damage. There is still little and contradictory information on the mechanism of the generation of DNA fragmentation after cryopreservation. A number of defensive strategies against cryoinjuries have been proposed in the last decade. Most studies focused on supplementing cryoprotectant medium with various antioxidant molecules, all aimed at minimising oxidative damage and thus improving sperm recovery. Despite the promising results, identification of the ideal antioxidant treatment method is still hampered by the heterogeneity of the studies, which describe the use of different antioxidant regimens at different concentrations or in different combinations. For this reason, additional studies are needed to further investigate the use of antioxidants, individually and in combination, in the cryopreservation of human sperm, to determine the most beneficial conditions for optimal sperm recovery and preservation of fertility.

Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats.

Cryopreservation of testis tissue is a promising approach to save fertility in prepubertal boys under going gonadotoxic cancer therapies. The using biopolymers as a basis of cryoprotective medium can be effective for the optimization of cryopreservation protocols of immature testicular tissue. The research purpose was to determine morphological parameters and metabolic activity of seminiferous tubules of immature rat testes under exposure to cryoprotective solution (DMSO) based on collagen or fibrin gels (CG or FG) as one of the first stages of developing the cryopreservation protocol. It was found that 30-min exposure of tissue samples to CG and FG with 0.6M DMSO did not impair the spermatogenic epithelium and metabolic activity of the cells (MTT test and total lactate dehydrogenase activity). The use of FG at the time of exposure of 45min did not lead to significant changes in the metabolic activity in contrast to other groups. The findings could be used to substantiate and develop the effective techniques for cryopreservation of immature seminiferous tubules.

Effect of different extender on the quality of sexing sperm before freezing in limousin cattle

The aim of this study was to determine the quality of sexing sperm before freezing by Percoll density gradient centrifugation method using egg yolks tris aminomethan and andromed® extender. This research method was experimental laboratory studies, the treatment egg yolks tris aminomethan and andromed® extender, parameters observed in the study include motility, percentage of viability and abnormalities fresh sperm before freezing. Sexing results were analyzed using completely randomized design, each treatment consisted of 10 replications. The results showed that both sperm motility and abnormalities using andromed had significant (P <0.05) than using egg yolk tris aminomethan. Glicerol in andromed as a cryoprotectant in the groove mechanism of the reaction cell preservation were to a decrease in freezing cryoprotectant medium, to the protect cell membranes, to curb the influence of increasing concentration, as well as to change the shape and size of ice crystals. It might be concluded that the quality of spermatozoa using andromed better than using egg yolk tris aminomethan.

Development of Enzyme-Immunoassay for &lt;i&gt;Bacillus anthracis&lt;/i&gt; Detection

Objective of the study was to develop enzyme-immunoassay test-kit for the detection of Bacillus anthracis spores. Materials and methods. Microbial cultures from the State Collection of Microorganisms at the premises of Affiliated Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation and BALB/c mice were used in the research. Hybridization of B-lymphocytes with SP2/0-Ag14 myeloma cells was performed according to G. Kohler and C. Milstein procedure in De St. Fazekas and P. Scheidegger modification. Hybridomas were cultured in the peritoneal cavity of BALB/c mice. Ascitic fluids were isolated from mice, precipitated with ammonium sulfate and purified by means of ion-exchange chromatography for preparation of monoclonal antibodies. Specific activity of hybridoma’s supernatants, ascitic fluids, purified monoclonal antibodies was studied by «sandwich» ELISA. Specific components of test-kit were lyophilized in suitable cryoprotective medium. Results and conclusions.We have obtained new hybrid cell lines producing specific monoclonal antibodies against Bacillus anthracisspore antigens and ascitic fluids from which immunoglobulins were isolated. Optimum combinations of monoclonal antibodies as a sensitizer and a component of immunoperoxidase conjugates have been selected. Monoclonal antibodies 272E10G1-272F7A10 provide the highest sensitivity of ELISA for the detection of anthrax microbe spore antigens. Our enzyme-immunoassay test allows for identification of Bacillus anthracis spores in concentrations up to 5,0·105 spores per milliliter. No cross reaction with closely related saprophytes and other heterologous microorganisms in concentrations of 1,0·108 CFU per milliliter is observed.

Кинетика фазовых превращений в процессе охлажденияотогрева клеток Saccharomyces cerevisiae в альгинатсодержащих криозащитных средах

The effect of sodium alginate on the viability and kinetics of phase transformations of Saccharomyces cerevisiae cells during cryopreservation was investigated. There were determined the optimal conditions for yeast cell cryopreservation such as: the cooling rate of 1 deg/min in alginate-containing cryoprotective media (cell viability of (90.79 ± 2.01)% and (88.11 ± 1.75)% in solution of 1% sodium alginate and in combination of 5% dimethyl sulfoxide (DMSO) and 1% sodium alginate, respect tively). Using cryomicroscopy, the presence of sodium alginate in cryoprotective medium was shown to change the crystallization performance and to shift the phase transition zone towards lower temperatures. The beginning of phase transformations in the specimens, cooled in 5% DMSO solution was recorded at –8.5°C, in those, containing 1% sodium alginate and in a three-component system, comprising the combination of 1% sodium alginate and 5% DMSO, it was found at –16 and –15°С, respectively. Probl Cryobiol Cryomed 2018; 28(3): XXX–XXX

Patient-derived xenograft cryopreservation and reanimation outcomes are dependent on cryoprotectant type

Patient-derived xenografts (PDX) are being increasingly utilized in preclinical oncologic research. Maintaining large colonies of early generation tumor-bearing mice is impractical and cost-prohibitive. Optimal methods for efficient long-term cryopreservation and subsequent reanimation of PDX tumors are critical to any viable PDX program. We sought to compare the performance of “Standard” and “Specialized” cryoprotectant media on various cryopreservation and reanimation outcomes in PDX tumors. Standard (10% DMSO media) and Specialized (Cryostor®) media were compared between overall and matched PDX tumors. Primary outcome was reanimation engraftment efficiency (REE). Secondary outcomes included time to tumor formation (TTF), time to harvest (TTH), and potential loss of unique PDX lines. Overall 57 unique PDX tumors underwent 484 reanimation engraftment attempts after previous cryopreservation. There were 10 unique PDX tumors cryopreserved with Standard (71 attempts), 40 with Specialized (272 attempts), and 7 with both (141 attempts). Median frozen time of reanimated tumors was 29 weeks (max. 177). Tumor pathology, original primary PDX growth rates, frozen storage times, and number of implantations per PDX model were similar between cryoprotectant groups. Specialized media resulted in superior REE (overall: 82 vs. 39%, p < 0.0001; matched: 97 vs. 36%, p < 0.0001; >52 weeks cryostorage: 59 vs. 9%, p < 0.0001), shorter TTF (overall 24 vs. 54 days, p = 0.0051; matched 18 vs. 53 days, p = 0.0013) and shorter TTH (overall: 64 vs. 89 days, p = 0.009; matched: 47 vs. 88 days, p = 0.0005) compared to Standard. Specialized media demonstrated improved REE with extended duration cryostorage (p = 0.048) compared to Standard. Potential loss of unique PDX lines was lower with Specialized media (9 vs. 35%, p = 0.017). In conclusion, cryopreservation with a specialized cryoprotectant appears superior to traditional laboratory-based media and can be performed with reliable reanimation even after extended cryostorage.

Диэлектрические свойства криозащитных сред на основе ДМСО и глюкозы в присутствии диоксида церия

Диэлектрические свойства криозащитных сред на основе ДМСО и глюкозы в присутствии диоксида церия

Changes to Extender, Cryoprotective Medium, and In Vitro Fertilization Improve Zebrafish Sperm Cryopreservation.

Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

Fertility preservation in male patients subjected to chemotherapy; innovative approaches for further progress

About 4% of male patients with cancer are under the age of 35 years. With the current increase in efficacy and safety of therapies, a growing number of young adults can achieve long-term survival. In male patients receiving systemic chemotherapy and or bone marrow transplantation, a permanent loss of fertility is a common adverse effect. The only possibility to preserve the patient’s fertility is to spare the gametes or gamete-forming cells from the chemotherapeutic effect. In adults, this can be achieved by the cryopreservation of spermatozoa with the subsequent application of assisted reproductive technology. Sperm cryopreservation is currently performed using slow-rate cryopreservation as a standard method, in which sperm cells are incubated with a cryoprotective medium and slowly subjected to hypothermia in liquid nitrogen (LN) vapor before they are placed in LN. Another technique called vitrification relies on the direct placement of the cells into LN, after being suspended in a vitrification medium. Many studies compared the clinical outcomes of both techniques and revealed equivalent results. This paper sheds light on some innovative approaches for further progress.