Non-penetrating Cryoprotectants Research Articles

P-182 Validation of single-step warming for human blastocysts shows successful results are independent of the sucrose concentration used

Abstract Study question Can warming of blastocysts in a single step be successfully performed using different sucrose concentrations? Summary answer Blastocyst warming in a single step using sucrose concentrations between 0.25 M and 1.0 M shows similar rates of survival, re-expansion and development in vitro. What is known already Since the introduction of human blastocyst vitrification, excellent survival rates have been reported. Warming procedures usually involve three to five steps with exposure to solutions with decreasing concentrations of non-penetrating cryoprotectants. Successful blastocyst warming procedures are described using high as well as low starting levels of sucrose. Recent data show that a single step warming procedure can be equally effective in terms of survival and pregnancy rates. Moving towards simpler procedures can support further optimization of laboratory procedures. Study design, size, duration Blastocysts donated and consented by patients were used to evaluate a shorter warming procedure. Warming was performed in a single step using different sucrose concentrations. After warming, embryos were assessed for morphological survival and cultured in a time-lapse incubator to monitor re-expansion and development in vitro for 24 h. Three series of tests were performed. A control group using standard warming procedure was included in the first test Participants/materials, setting, methods For warming, carriers containing a single blastocyst were plunged into a warming solution at 37 °C, containing 0.25, 0.5 or 1.0 M sucrose and stayed in the solution for 1 or 2 minutes. Three series of experiments were performed, testing different sucrose concentrations or volume of warming solution. Warmed blastocysts were cultured following several rinsing steps and assessed for morphological survival. Re-expansion after 2 h, embryo characteristics and development were monitored until 24 h post warming. Main results and the role of chance Results of the different tests are summarized in the table, including information on sucrose levels, exposure times, volume of warming medium, numbers of blastocysts warmed, recovered, meeting criteria for embryo transfer, full re-expansion after 2 hours, viable after 24 h culture. No differences were observed between the different groups. Overall, 98 % of warmed blastocysts would be considered transferable after single step warming and 96 % were viable after 24 h culture. Results confirm blastocysts can be warmed at 37 °C in a single step using 0.25 M, 0.5 M or 1 M sucrose with no effect on survival or development in vitro. Limitations, reasons for caution This is a preclinical validation on use of a reduced warming time for vitrified blastocysts. Further evaluation and clinical validation of the findings is required to confirm safety of a simplified warming procedure. Working temperature rather than warming medium volume may be critical when aiming for shorter warming procedures. Wider implications of the findings Reducing the warming time and number of handling steps minimizes exposure of blastocysts to a suboptimal environment and operator or handling related stresses. It also allows further optimization of laboratory protocols and workflow. When confirmed clinically, these findings encourage investigating similar changes for other reproductive cells. Trial registration number not applicable

Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants.

Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs. Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.

Selection of an optimal cryoprotectant for long-term storage of human tendon allografts

Introduction. Tendon grafts are widely demanded in reconstructive plastic surgery. Allogeneic tendons potentially have a number of advantages. However, the method of long-term storage of allogeneic tendons has not been optimized to date.Aim. Selection of an optimal cryoprotectant for the storage of human tendon allografts at ultra-low temperatures, which allows preserving the native tissue structure.Material and methods. We studied M. tibialis anterior tendon grafts taken from tissue donors. Endocellular/penetrating cryoprotectants (dimethyl sulfoxide, polyethylene glycol-400, glycerol) and exocellular/non-penetrating cryoprotectants (glucose solution, albumin solution) were used in the cryopreservation process. Tendon mechanical properties were evaluated using rupture and stretching-shear test, the general morphology of tendons, topography, dense and integrity of collagen fibers, preservation of cellular elements were microscopically evaluated.Results. Histological analysis showed that the safety of collagen and elastin fibers differed depending on the cryoprotectant used. At the same time, micro-fractures of collagen fibers were microscopically detected in all the experiments. In the presence of dimethyl sulfoxide, polyethylene glycol-400 and their combination the structure of collagen fibers and cells did not undergo visible changes compared to the control, whereas in all the experiments with non-penetrating cryoprotectants the topography and orientation of the fibers were clearly disturbed, deformation of many cells in the tendons was also observed.Conclusions. Cryoprotectants based on dimethyl sulfoxide, polyethylene glycol and their combinations allowed us to preserve the structural integrity of allogeneic tendons. Non-penetrating cryoprotectants did not effectively preserve the integrity of collagen fibers and cells in the tendons and cannot be recommended for cryopreservation.

Trehalose in Extenders for Cryopreservation of Tambaqui (Colossoma Macropomum) Sperm

BACKGROUND: Sugars may act as either energy substrates or non-penetrating cryoprotectants. OBJECTIVE: Inclusion of non-penetrating trehalose was tested in extenders for the cryopreservation of Tambaqui ( Colossoma macropomum) sperm. MATERIALS AND METHODS: Sperm was extended 1/9 (v/v) in Beltsville Thawing Solution (BTS) with 10% DMSO (control) or 50, 100, 150 and 200 mM trehalose without 10% DMSO. Post-thawed sperm quality was evaluated, including fertilization and hatching rates, sperm motility, motility period and viability, integrity of sperm membrane and DNA, and mitochondrial functionality. RESULTS: Extenders with 100 ∼ 150 mM trehalose achieved fertilization and hatching rates similar to those of the 10% DMSO-treated sperm samples. Trehalose at 100 and 150 mM provides better protection than 10% DMSO treatment for sperm motility, viability, DNA integrity and mitochondrial functionality. Fertilization and hatching rates were highly correlated (r = 0.95, P < 0.001). CONCLUSION: The addition of 100 ∼ 150 mM trehalose in extender can replace 10% DMSO for the cryopreservation of C. macropomum sperm.

Development of assisted reproductive technologies for the conservation of Atelopus sp. (spumarius complex)

Amphibians are in peril, given the ongoing sixth mass extinction of wildlife. Thus, Conservation Breeding Programs (CBPs) are attempting to breed some species under laboratory conditions. The incorporation of assisted reproduction technologies (ARTs), such as hormonal stimulation, sperm collection and cryopreservation, and in vitro fertilization is contributing to successful CBPs. The objective of this study was to apply ARTs in sexually mature individuals of an undescribed species of Atelopus (spumarius complex) (harlequin frog). Our procedure involves hormonal induction of gametogenesis in this species. We were able to induce gamete release through administration of human chorionic gonadotropin (hCG) in males, and in females this has been achieved through the sequential administration of hCG (priming doses), and combinations of hCG with gonadotropin releasing hormone analogue, GnRHa (ovulary dose). We standardized sperm cryopreservation by performing toxicity tests of cryoprotectants, fast/slow freezing and thawing, as well as supplementation of non-penetrating cryoprotectants (sugars). Next, we performed in vitro fertilization, evaluated the fertilization capacity of the cryopreserved sperm, and describe external features of fresh and cryopreserved sperm. We found that 10 IU/g hCG induced the release of the highest sperm concentrations between 3 and 5 h post-injection, while 2.5 IU/g hCG induced the release of eggs in most treated females. Under cryopreservation conditions, the highest recovery of forward progressive motility or FPM was 26.3 ± 3.5%, which was obtained in cryosuspensions prepared with the 5% DMF and 2.5% sucrose. Cryopreserved sperm showed narrower mitochondrial vesicles after thawing, while in frozen samples without cryodiluent showed 31% of spermatozoa lost their tails. In most cases, our attempts of in vitro fertilization were successful. However, only ∼10% of embryos were viable. Overall, our study demonstrates that the development of ARTs in individuals of Atelopus sp. (spumarius complex) bred in laboratory can be successful, which result in viable offspring through in vitro fertilization. Our study provides a baseline for assisted breeding protocols applicable to other harlequin frogs of the genus Atelopus.

Potentialities of using cryoprotectants in gluten-free frozen dough and microwave baking as an emerging technology

Disorders related to human health involving proteins that form the gluten network are increasingly in society, justifying the need to offer bakery products for this market niche. This study aimed to gather important information about the challenges involving technological production in the gluten-free bakery market. The approach encompasses the development of frozen dough for gluten-free baking, the use of cryoprotectants to maintain cell viability during the freeze-thawing step, the application of emulsifiers to improve the quality of the bread and the baking by an unconventional method using the microwaves. The introduction of frozen dough in the gluten-free bakery sector represents an interesting tool. However, these bread formulations require a higher amount of water compared to a traditional bread formulation (70 to 120 %), which makes the freezing and thawing steps extremely challenging, especially when it comes to the biopreservation of yeast cell viability. Cryopreservation with isolated and/or combined application of penetrating (glycerol) and non-penetrating (sucrose, fructo-oligosaccharide, trehalose, proteins and hydrocolloids) cryoprotectants promotes changes in the viscosity of the medium and is an excellent alternative for the maintenance of the microorganism responsible for the fermentation process. The appropriate choices of cryoprotectants, as well as the control of the freezing and thawing and baking processes, are important steps in the production line, to guarantee products with adequate technological and sensory qualities.

High-speed centrifugation of extender of freeze-thaw boar semen.

In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Much has been developed in the search for alternatives for this biological product. This work aimed to evaluate the processed egg yolk through ultracentrifugation and/or sonication in the cryopreservation of swine semen. Twenty-seven semen doses were purchased from a commercial boar stud and processed for cryopreservation using egg yolk lactose 11% (control) extender, processed using two different methods: high-speed centrifugation and sonication. Then, they were submitted to freeze-thawing protocol and were assessed for kinematic and cell structural parameters. Samples in which extenders underwent centrifugation had better results in velocity parameters, meanwhile those that only sonication was performed had poorest results in this parameter. The preservation of the membrane and mitochondria structure had better results when the diluent was only centrifuged in comparison with the other treatments. Therefore, centrifugation of extender containing egg yolk is important for better cryopreservation of swine semen.

Molecular mechanisms of cell cryopreservation with polyampholytes studied by solid-state NMR

Polyampholytes are emerging macromolecular membrane non-penetrating cryoprotectants; however, the mechanism behind their cryopreservation remains unclear. Here, we investigated the mechanism using solid-state NMR spectroscopy. The polymer-chain dynamics and the water and ion mobilities in the presence of various membrane penetrating and non-penetrating cryoprotectants were monitored at low temperatures to mimic cryopreservation conditions. NMR experiments revealed that the water, Sodium-ion, and polymer-chain signals in a carboxylated poly-ʟ-lysine (COOH-PLL) solution broadened upon cooling, indicating increasingly restricted mobility and increased solution viscosity. Moreover, strong intermolecular interactions facilitated the COOH-PLL glass transition, trapping water and salt in the gaps of the reversible matrix, preventing intracellular ice formation and osmotic shock during freezing; this reduced cell stress is responsible for cryoprotection. This simple NMR technique enabled the correlation of the cryoprotective properties of polymers that operate through mechanisms different from those of current cryoprotectants, and will facilitate the future molecular design of cryoprotectants.

Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. II: Effect of the addition of saccharides to freezing medium on sperm function

Saccharides have bioprotective properties, with a high capacity to preserve biological proteins and membranes during sperm cryopreservation. The aim of this study was to evaluate how replacing the lactose of cryopreservation media by sucrose (SUC) or trehalose (TRE) at concentrations of 0.2 M (SUC-1 and TRE-1) and 0.25 M (SUC-2 and TRE-2) affects frozen/thawed pig spermatozoa. The media used were composed of medium A (saccharide/egg yolk) and B (saccharide/egg yolk/glycerol), their osmolality being determined prior to freezing. Cell viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), lipid peroxidation, thiol group oxidation, total reactive oxygen species (ROS), peroxynitrite and superoxide anion (O2●-) were determined through flow cytometry; total motility (TM), progressive motility (PM) and kinetic parameters motility were determined immediately after thawing (T0) and again 30 (T30) and 60 (T60) minutes later. The SUC-2 and TRE-2 groups maintained viability significantly and presented fewer lipid membrane disorders, respectively, both with a significant increase in MMP. The production of O2●- and peroxynitrite was lower in the TRE-2 groups compared to the control (P < 0.05). Total motility at T0 was greater in the TRE-2 group (P < 0.05). Sperm kinetics was not affected by the treatment. The use of saccharides SUC and TRE at a concentration of 0.25 M improves sperm quality, so that both non-penetrating cryoprotectants can be utilized in pig sperm freezing media.

Криоконсервирование сперматозоидов человека в ПВП и сахарозе

В данном исследовании изучали уровень жизнеспособности спермиев человека после криоконсервирования с непроникающими криопротекторами, которые не требуют удаления из суспензии клеток перед применением сперматозоидов во вспомогательных репродуктивных технологиях (ВРТ). В результате проведенных исследований установили, что использование сахарозы и поливинилпирролидона (ПВП) в различных комбинациях в качестве криозащитных агентов при замораживании сперматозоидов позволяет получить их выживаемость на уровне 60–80 %. Таким образом, криоконсервирование спермиев человека с использованием сахарозы и ПВП является перспективным для ВРТ, поскольку спермии можно использовать сразу после отогрева для оплодотворения ооцитов in vitro

Raman Cryomicroscopic Imaging and Sample Holder for Spectroscopic Subzero Temperature Measurements.

Raman spectroscopy has been gaining in popularity for noninvasive analysis of single cells. Raman spectra and images deliver meaningful information regarding the biochemical, biophysical, and structural properties of cells in various states. Low-temperature Raman spectroscopy has been applied to verify the presence of ice inside a frozen cell and to illustrate the distribution of both penetrating and non-penetrating cryoprotectants. This chapterdelineates Raman cryomicroscopic imaging of single cells as well as sample handling for spectroscopic measurements at subzero temperature. The experimental setup is depicted with a special emphasis on a custom-built temperature-controlled cooling stage. The use of Raman cryomicroscopic imaging is demonstrated using Jurkat cells cryopreserved in a sucrose solution. Moreover, strategies for determining intracellular ice formation (IIF) and analysis of sucrose partitioning across the cell membrane are presented.

Protocol for Cryopreservation of Endothelial Monolayers.

One of the major challenges in the preservation of complex tissues is the cryosensitivity of the endothelium, the single layer of cells lining blood vessels, corneas, and other tissues. The increasing importance of endothelial monolayers in tissue-engineered constructs for transplantation and research warrants the need to develop protocols for the successful cryopreservation of cells in monolayers. In this chapter, we describe a recently published cryopreservation protocol that we developed based on examination of various factors that influence the post-thaw recovery of endothelial monolayers. To efficiently investigate cryopreservation protocol parameters, we employed an interrupted slow-cooling procedure (graded freezing) that allows dissecting loss of cell viability into contributions from slow-cooling injury and rapid-cooling injury. Our optimized protocol involves culturing cells on Rinzl plastic coverslips, using a combination of a penetrating cryoprotectant (5% dimethyl sulfoxide) and a non-penetrating cryoprotectant (6% hydroxyethyl starch), addition of 2% chondroitin sulfate, controlled cooling at 0.2°C/min or 1°C/min, and removal of cryoprotectant immediately after thaw. The protocol has been validated for human umbilical vein and porcine corneal endothelial cell monolayers.

Soy lecithin as a potential alternative to powdered egg yolk for buck sperm cryopreservation does not protect them from mitochondrial damage

The aim of this study was to address whether soy lecithin (SL) was an effective non-penetrating cryoprotectant for buck sperm cryopreservation in the presence of seminal plasma. There was also an attempt to determine the optimal concentration of BHT as an antioxidant in powdered egg yolk (PEY) or in SL based media. Two ejaculates were collected from six bucks and mixed ejaculates were aliquoted into washed, using centrifugation procedures, and unwashed samples. In Experiment 1, washed sperm were re-suspended in PEY (15%) or SL (1%) media, while unwashed semen was only diluted in SL medium. In Experiment 2, washed and unwashed sperm were diluted in PEY and SL media, respectively, with there being different BHT concentrations (0.6, 2.0 and 5.0 mM). In both experiments, after 4 h of refrigeration, there were no differences neither in sperm viability nor plasma membrane functional integrity (HOST) between groups when there were evaluations using eosin-nigrosine staining. After thawing, however, there was a negative effect on motility of washed sperm preserved in SL media. Furthermore, results from cytometry evaluations indicated there was a larger population of thawed sperm with intact plasma (SYBR-14+/PI-) and acrosome (PE-PNA-) membranes, but inactive mitochondria (Mitotracker deep red-) when SL media were used. When there was BHT supplementation, there was only a slight enhancement of motility of spermatozoa preserved in PEY media with 5 mM BHT. In conclusion, when effectiveness and efficiencies are considered, PEY is the non-penetrating cryoprotectant that should be utilized for buck sperm cryopreservation.

Cryopreservation of human spermatozoa with penetrating and non-penetrating cryoprotectants

Сryopreserved spermatozoa are widely used in infertility treatment by assisted reproductive technologies. However, the spermatozoa survival rate remains low in patients with oligoastenoteratozoospermia. Therefore the development of effective cryopreservation methods for spermatozoa from pathospermia is relevant. The effectiveness of cryopreservation spermatozoa from oligoastenoteratozoospermia man using penetrating and non-penetrating cryoprotectants was compared. Sperm motility, viability and morphological characteristics were evaluated after cryopreservation with glycerol and polyvinylpyrrolidone. The average number of spermatozoa count in fresh ejaculate was (11.0±0.2) mln/ml. After isolation of active motile fraction the number of cells was (3.8±0.3) mln/ml and (84.3±8.4) % from them were motile (group 3). (78.8±6.6) % of spermatozoa cryopreserved with glycerol (group 1) and (41.4±8.1) % cryopreserved with polyvinylpyrrolidone (group 2) remained active motile. The spermatozoa viability after cryopreservation was (82.1±8.6) % and (89.6±8.6) % in group 1 and 2, respectively. Despite the high rate of spermatozoa survival in group 1 the number of motile cells decreased to (27.3±4.8) % after cryoprotectant removing stage. Morphological analysis revealed that the incidence of spermatozoa head abnormalities was (25.97±2.67), (19.21±2.67) and (20.57±1.19) % in group 1–3, respectively. The differences of spermatozoa midpiece and tail abnormalities in the study groups were statistically insignificant. The use of polyvinylpyrrolidone as a cryoprotectant allows preserving 90 % of survived spermatozoa from oligoastenoteratozoospermia men after freeze/thawing. The set of spermatozoa head, neck and midpiece abnormalities is significantly lower after cryopreservation with polyvinylpyrrolidone compared with routine method with glycerol. Two-stage spermatozoa cryopreservation method with polyvinylpyrrolidone is promising for assisted reproductive technologies since spermatozoa can be used immediately after warming for oocyte fertilization without cryoprotectant removing step.

Cryopreservation of placenta tissue allows isolating viable mesenchymal and hematopoietic stem cells

Background & Aim Cryopreserved placenta tissue could be prospective source of mesenchymal and hematopoietic stem cells for cell therapy and regenerative medicine. The aim of our study was to determine methods of placental cryopreservation with high stem cells recovery. Methods, Results & Conclusion Chorions (n=4) were isolated from full-term human placentas of healthy donors. One part of tissue from every specimen was seeded as a native control and the other part was frozen. Nine variants of cryopreservation medium composition were selected by combination of different concentration of dimethyl sulfoxide (5%, 7.5%, 10%) and sucrose (0.1 M, 0.2 M) as penetrating and non-penetrating cryoprotectants. The samples were frozen in cryogenic vials in a controlled rate freezer (IceCube, Austria). Placenta-derived multipotent cells (PDMCs) were subcultured from native and thawed chorion explants. Colony forming units (CFU) assay was carried out on passages 0, 2, 6 for PDMCs isolated from native and thawed tissue. Hematopoietic stem cells (HSCs) of full-term human placental tissue were obtained by enzymatic method with following depletion of dead cells. Immunophenotyping was performed on BD FACSAria (USA). MethoCult (StemCell Technologies, Canada) was used for differentiation assays. XY karyotype of PDMCs indicates their foetal origin. The results of flow cytometry analysis revealed CD90+ CD105+ CD73+ CD34− CD45− PDMCs phenotype. PDMCs were able to differentiate into osteogenic and adipogenic lineages. CFU assay showed that number of native tissue-derived colonies on passage 0 was significantly higher in comparison with thawed tissue for the nine variants of cryoprotectants. However, number of CFU for fresh-isolated cells on passages 2 and 6 was not significantly differ in comparison with PDMCs from thawed tissue. Flow cytometry analysis showed that the HSCs content among CD45+ cells of the total light-density cell fraction from the cryopreserved and native placental tissue did not significantly differ. CFU study demonstrated the presence of myeloid progenitors, quantitative analysis revealed difference in efficiency of HSCs preservation between certain variants of cryoprotectants. The best variant reached up to 50% of CFU recovery compared to native sample. Controlled cryopreservation of placental tissue under GMP requirements allows to obtain proliferative active stem cells with suitable for clinical issues characteristics.

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.

Effect of different penetrating and non-penetrating cryoprotectants and media temperature on the cryosurvival of vitrified invitro produced porcine blastocysts.

The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 invitro produced (IVP) porcine blastocysts. The post-warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide+17% ethylene glycol [EG]+0.4mol/L sucrose) and commercial media did not differ, nor did the post-warm survival rate of blastocysts vitrified in medium containing 1,2-propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p<.05). Furthermore, the post-warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non-penetrating cryoprotectant did not differ. There was also no significant difference in post-warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post-warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p<.05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post-warm survival of IVP porcine blastocysts. The improved post-warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.

Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants.

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization.

Влияние инкубации с криопротекторами на количество эпитопов Gal-α-1,3-Gal в клетках линии PK-15

Transplantation of bioprostheses derived from bovine and porcine tissues is an advanced method of correcting defects in blood vessels and valves, however when using it a hyperacute immune response associated with the presence of Gal-alpha- 1,3-Gal (I±-Gal epitope) on the surface of cell membranes can develop. We have studied the effect of penetrating and nonpenetrating cryoprotectants on the amount of I±-Gal epitopes on membranes of PK-15 cell line of porcine origin. Cell labeling with BSIB4 lectin conjugated with FITC and flow cytometry revealed a significant increase in the number of fluorescent cells after incubation with various concentrations of dimethyl sulfoxide and polyethylene glycol with MW of 1500 (PEG-1500). The revealed effect may be associated with an additional exposure of I±-Gal epitopes on cell membrane, locally deformed due to the action of cryoprotectants.Probl Cryobiol Cryomed 2016; 26(4): 331–339

Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture

This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1–2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65–80%).