Extracellular Cryoprotectants Research Articles

Morphological evaluation of adult domestic cat testicular biopsy after vitrification.

Testicular biopsies (9 mm3) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher's post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO's negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.

Comparison of the effect of Sumbawa honey and sucrose as extracellular cryoprotectants on viability, morphology, and phenotype stability of CD34+ hematopoietic stem cells from umbilical cord blood

Cryopreservation is a common practice for the long-term storage and maintenance of the quality of Hematopoietic Stem Cells (HSC) from Umbilical Cord Blood (UCB). Cryomedium containing 10% DMSO is the standard intracellular cryoprotectant agent (CPA) in HSC cryopreservation. However, DMSO is toxic to both cells and patients during transplantation. Therefore, the concentration of DMSO needs to be reduced by adding extracellular CPAs, such as sucrose or Sumbawa honey. The objective of this study was to compare the ability of Sumbawa honey and sucrose as extracellular CPAs to protect HSC CD34+ during cryopreservation. This in vitro study was designed using cryomedium consisting of 10% DMSO as a control, 5% DMSO + 5% Sumbawa honey, and 5% DMSO + 5% sucrose. The results showed that the cryoprotectant containing 5% DMSO + 5% Sumbawa honey had a positive effect and a significant difference (p&lt;0.05) compared with 5% DMSO + 5% sucrose on the viability and morphology of HSC. However, the mean reduction in phenotype stability as indicated by the decrease in percentage CD34+ in the 10% DMSO (6.90 ± 8.60), 5% DMSO + 5% sucrose (10.60 ± 9.20), and 5% DMSO + 5% Sumbawa honey (8.60 ± 11.50) showed no significant difference (p&gt;0.05). In conclusion, the combination of DMSO and Sumbawa honey was able to maintain the viability, morphology, and phenotype stability of HSC. Therefore, honey can be used as an alternative cryoprotectant for the cryopreservation of hematopoietic stem cells.

Effect of modified CEP-3 diluents with aqueous soybean extract on liquid semen quality in Ongole crossbred bull.

Egg yolk (EY) is commonly used as an extracellular cryoprotectant in semen diluents but has some negative effects. Therefore, this study aimed to assess the potential of lecithin derived from plants, such as soybeans, as an alternative extracellular cryoprotectant and to characterize liquid semen quality of Ongole crossbred bulls using a modified caudal epididymis plasma-3 [CEP-3 (m)] as a base diluent and aqueous soybean extract (ASE). A bull with progressive motility (PM) of fresh semen >70% was used. Two soybean extracts were also used, namely, ASE 1 and ASE 2, obtained by extraction procedures 1 and 2, respectively. The study was conducted using an experimental design with 11 treatments and ten replications, with diluents comprising different levels of ASE 1 and ASE 2, as well as a positive control with 10% EY. The parameters measured were motility (M) and its kinetic parameters, including PM, M, velocity curve linear, velocity straight linear, velocity average pathway, linearity, straightness, wobble, amplitude lateral head beat cross frequency, and hyperactivity using computer-assisted sperm analysis, viability, and spermatozoa abnormalities. The CEP-3(m) diluent formula and ASE 1 at a 30% level maintained the PM of spermatozoa up to day 5 (40.7% ± 16.1%) of cold storage. Meanwhile, the CEP-3(m) diluent formula and ASE 2 could only maintain PM >40% until day 3 (42.1% ± 13.5%) of cold storage at a 30% level. The CEP-3(m) diluent and ASE 1 at a level of 25%-30% supported spermatozoa life (viability) up to day 5 with a value >80% (81.8 ± 3.5; 86.4 ± 2.6). The abnormality value of spermatozoa in various diluents during cold storage on days 0-5 was below 20%. Soybean extracts 1 and 2 can substitute EYs as extracellular cryoprotectants in modified CEP-3 basic diluents. Soybean extract 1 can support the life of spermatozoa up to day 5 but may cause the viscosity and movement of spermatozoa to be hyperactive. Soybean extract 2 can support the life of spermatozoa up to the 3rd day of cold storage and produces progressive (non-rotating) movement patterns. Further, research is recommended with higher levels of ASE 2.

Interactions Among Sucrose and Concentrations of Serum Fetal Bovine on the Cryopreservation of Somatic Cells Derived from Red-Rumped Agoutis

BACKGROUND: The synergistic action among the different extracellular cryoprotectants could improve somatic cell quality after thawing and provide bases for the formation of biobanks for redrumped agoutis. OBJECTIVE: This study evaluated the interactions among sucrose (SUC) and concentrations of serum fetal bovine (FBS) on the cryopreservation of somatic cells derived from redrumped agoutis. MATERIALS AND METHODS: Cells were cryopreserved with 10% dimethyl sulfoxide and different concentrations of FBS (10%, 40%, and 90%) with or without 0.2 M SUC, totaling six comparison groups. Non-cryopreserved cells were used as a control. Cells were evaluated for viability, metabolic activity, proliferative activity, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptosis levels. RESULTS: No difference was observed among cryopreserved with DMSO containing (10FBS, 10FBS-SUC, 40FBS, 40FBS-SUC, 90FBS, 90FBSSUC) and non-cryopreserved groups for viability, metabolic activity, proliferative activity, and ROS levels. Interestingly, only cells cryopreserved with 90% FBS and SUC maintained the ΔΨm like the control. This indicates that at high concentrations of FBS, SUC contributes to the maintenance of this parameter in cryopreserved cells. Moreover, at concentrations of 10% and 40% of FBS, SUC contributed to the maintenance of viability evaluated by the levels of apoptosis evaluated after thawing. In summary, we verified that 90% FBS and 0.2 M SUC promote greater ability of cells after thawing. Additionally, SUC positively acts in cryopreservation solutions containing 10% and 40% FBS. CONCLUSION: This information is essential to an understanding of the mechanisms involved in the interactions of extracellular cryoprotectants in somatic cell cryopreservation solutions of red-rumped agoutis.

Bax/Bcl-2 Expression Ratio Analysis of Rat Ovary Vitrified with Date Juice Concentrate as a Natural Extracellular Cryoprotectant.

The use of extremely low temperatures in vitrification is known to cause cryoinjury so that it can trigger the activation of the intrinsic apoptotic pathway, which can damage the structural integrity of the pre-antral follicle. Based on that, it is necessary to use an appropriate cryoprotectant to protect the preserved cell. This study aimed to identify the potential use of date juice concentrate (DJC) as a natural extracellular cryoprotectant to suppress the rate of apoptosis after vitrification. This experimental research uses 24 samples of ovarian rats. Rats were fed and drank an ad libitum. Ovaries were isolated in the proestrus phase, then processed into slides for immunohistochemistry (IHC) staining using anti-Bax and anti-Bcl-2 antibodies. IHC results were evaluated for the brown colour using ImageJ IHC Profiler. The results were analysed as an optical density and displayed in the Bax/Bcl-2 ratio. All data were statistically analysed with either parametric (analysis of various) or non-parametric (Kruskal-Wallis) tests. The combination of EG 7.5% + DJC 15% (KP2) showed the lowest Bax/Bcl-2 ratio in primordial and primary follicles. Meanwhile, the lowest Bax/Bcl-2 ratio in secondary follicles is found in KP4 (EG 15% + DJC 15%). The DJC is known to contain a dominant amount of glucose. The DJC shows antioxidant activity and contains antioxidant compounds, phenols and flavonoids. The sugar content and antioxidant compounds of DJC can protect against follicle membrane damage, so the rate of intrinsic apoptosis pathway is also suppressed initially with Bax protein suppression in the mitochondrial membrane.

P-442 A new option to thaw slow-frozen human ovarian tissue in cancer patients: efficacy and safety of the combination of different cryopreservation kits

Abstract Study question Is follicular viability of slow-frozen human ovarian tissue preserved if rapid thawing is performed using a solution containing extracellular cryoprotectant only? Summary answer Follicular viability is preserved even using thawing solutions containing extracellular cryoprotectant only, combining kits with different composition. What is known already Ovarian tissue cryopreservation is an alternative option to oocytes or embryos for fertility preservation in cancer patients facing gonadotoxic treatments. To date, each brand producing kits approved for the slow-freezing of human ovarian tissue recommends the use of its own thawing kit. However, a potential single protocol based on the use of any extracellular cryoprotectant has already been proposed for human oocytes and embryos. The current study aims at finding alternative options to thaw cryopreserved human ovarian tissue when the original kit was withdrawn from the market and only one CE-marked kit was available, even with different composition in cryoprotectants. Study design, size, duration Ovarian tissue cryopreservation of ten cancer patients (18.3 ± 7.6 years) undergoing fertility preservation between 2001 and 2012 was performed following a slow-freezing protocol with 1.5M 1,2 PROH and 0.5M Sucrose. Once deceased, for each patient, cortical fragments were prospectively thawed and equally allocated into two groups: i) fragments thawed using 0.5-1M 1,2 PROH and 0.3M Sucrose (PROH+S Group, n = 73); ii) fragments thawed following an adjusted protocol with 0.5M Sucrose only (S Group, n = 73). Participants/materials, setting, methods Post thawing follicular density/mm2, integrity (%) and the presence of interstitial oedema were assessed by histological and ultrastructural analysis performed after formalin fixation and haematoxylin/eosin staining. Follicular viability was evaluated by the expression of markers of proliferation (Ki67) and of vascularization (CD31) by immunohistochemistry after a 24h culture in Iscove’s modified Dulbecco’s medium at 37 °C and 6% CO2. A paired comparison was performed referring to the fresh tissue of the same patient as control. Main results and the role of chance The histological evaluation performed after thawing revealed that follicles were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. A significant reduction of follicular density per mm2 was observed in both study groups (14.2 ± 12.0 vs. 15.1 ± 14.0 for PROH+S and S Group, respectively; p = 0.4) compared to the fresh tissue (27.2 ± 31.6; p = 0.04) as well as a remarkable decreased of the proportion of intact follicles (39.3 ± 17.1 vs. 25.5 ± 9.8; p = 0.2) compared to the fresh tissue (98.1 ± 1.4; p = 0.002). Thawed samples equally showed interstitial oedema and increased stromal cell vacuolization and chromatin clumping. Ki67 positive staining of active proliferating cells revealed a comparable proportion of viable follicles between thawed samples (46.3 ± 20.8 vs. 28.3 ± 27.9 for PROH+S and S Group, respectively; p = 0.2). Finally, the expression of the endothelial marker CD31 in the thawed samples suggested an equivalent number of blood vessels per mm2 (43.8 ± 34.3 vs. 41.7 ± 44.8; p = 0.6). Limitations, reasons for caution Single centre study with a limited sample size. Only 24h of in vitro culture was assessed. The use of the freezing medium corresponding to the Sucrose only solution was not tested. Clinical outcomes after ovarian tissue transplantation should be evaluated before drawing final conclusion. Wider implications of the findings First evidence of the feasible application of a “Universal Warming” protocol, irrespective of brand and cryoprotectants, for the rapid thawing of slow-frozen human ovarian tissue. IVF centres would be provided with alternative options to thaw ovarian tissue for restoring reproductive potential in cancer patient undergoing ovarian transplantation. Trial registration number Not applicable

Addition of bovine serum albumin (BSA) in cauda epididymal plasma-2 (CEP-2) extender to Ongole grade bull sperm motility and membrane integrity during the freezing process

The semen freezing process causes a decrease in quality, especially sperm motility and membrane damage. Decreasing the semen quality during the freezing process can be prevented by adding semen diluents that fulfill the nutritional needs of sperm, such as CEP-2 and extracellular cryoprotectants, such as BSA. Cauda Epididymal Plasma-2 diluents have a composition like bull plasma cauda epididymis and have been shown to be able to maintain the quality of bull liquid semen at 5°C for eight days. The addition of BSA with different levels in the CEP-2 extender was expected to support the function of egg yolk in preventing damage to the sperm membrane due to cold shock during the freezing process. The purpose of this study was to determine the best BSA level to maintain sperm motility and membrane integrity during the freezing process. The research material was a three-year-old Ongole grade bull ejaculate which was collected once a week with an artificial vagina and individual motility of at least 70%. The research method was an experimental laboratory with BSA level 0; 0.2; 0.4; 0.6; 0.8 and 1%. The results showed that the highest sperm motility was 0.6% BSA level (42+2.58%) and the highest membrane integrity at the BSA level was 0.4% (84.30±2.56%). The study concluded that the addition of BSA in CEP-2 diluents increased the motility and membrane integrity during the freezing process, met the Indonesian National Standards. The research suggested the use of a BSA level of 0.6% in CEP-2 diluents for commercial frozen semen production.

Current and Future Perspectives for the Cryopreservation of Cord Blood Stem Cells.

Hematopoietic stem cell (HSC) transplantation is a well-established procedure for the treatment of many blood related malignancies and disorders. Before transplantation, HSC are collected and cryopreserved until use. The method of cryopreservation should preserve both the number and function of HSC and downstream progenitors responsible for long- and short-term engraftment, respectively. This is especially critical for cord blood grafts, since the cell number associated with this stem cell source is often limiting. Loss of function in cryopreserved cells occurs following cryoinjuries due to osmotic shock, dehydration, solution effects and mechanical damage from ice recrystallization during freezing and thawing. However, cryoinjuries can be reduced by 2 mitigation strategies; the use of cryoprotectants (CPAs) and use of control rate cooling. Currently, slow cooling is the most common method used for the cryopreservation of HSC graft. Moreover, dimethyl-sulfoxide (DMSO) and dextran are popular intracellular and extracellular CPAs used for HSC grafts, respectively. Yet, DMSO is toxic to cells and can cause significant side effects in stem cells' recipients. However, new CPAs and strategies are emerging that may soon replace DMSO. The aim of this review is to summarise key concepts in cryobiology and recent advances in the field of HSC cryobiology. Other important issues that need to be considered are also discussed such as transient warming events and thawing of HSC grafts.

Cryopreservation of coho salmon sperm (Oncorhynchus kisutch): Effect on sperm function, oxidative stress and fertilizing capacity

Sperm cryopreservation is an important tool for reproductive management; however, it has not been possible to perform this procedure for coho salmon. This study was designed to assess the effect of cryopreservation on sperm functional parameters, oxidative stress markers and fertilizing capacity of coho salmon. Eight extenders were tested, consisting of two intracellular cryoprotectant agents (CPA-I): dimethylsulfoxide (DMSO) at 8 and 10% and methanol (MeOH) at 9 and 12%, two extracellular cryoprotectants (CPA-E): 0.14 M trehalose and 0.18 M glucose, supplemented in all cases with 10% egg yolk and Storfish®. Milt samples from 18 males were evaluated in three trials. In the first trial, the toxic effect of cryoprotectants on motility was evaluated after 0, 15, 30, and 60 min of equilibration time. The second trial was conducted to select the extender which produced the best post-thaw sperm motility. In both trials, sperm motility was analysed by a subjective method. In the third trial, milt samples were frozen in the three best extenders; post-thawed samples were assessed for motility by computer-assisted sperm analysis CASA, viability, superoxide anion production (O⁠2⁠•−), mitochondrial membrane potential (ΔΨm) by flow cytometry, lipid peroxidation (malondialdehyde, MDA) and fertilizing capacity were also analysed. In Trial 1, no negative effect of the extenders tested was observed on sperm motility during the different equilibration times. In Trial 2, the post-thaw sperm motility was significantly affected by CPA-I, CPA-E and their interaction, in other words, the protection capacity was dependent on the type of CPA-I and at the same time on the CPA-E. Thus, the best results were obtained with DMSO combined with glucose, with mean motility of 16.44 ± 12.55% (8% DMSO + glucose) and 11.57 ± 10.23% (10% DMSO + glucose). In Trial 3, all the sperm parameters were significantly affected after freezing. Mean post-thaw sperm motility varied from 20.50 ± 15.83% to 35.80 ± 17.64%, cell viability ranged from 56.23 ± 11.05% to 59.42 ± 12.00% and the ΔΨm varied from 12.50 ± 8.9% to 14.63 ± 10.09%, without significant differences between the three extenders assessed in this trial. On other hand, the percentage of living spermatozoa producing O⁠2⁠•− and the MDA levels increased significantly after freezing, which suggests that sperm cryodamage was caused in part by oxidative stress. The combination of either 8% or 10% DMSO with glucose yielded the best fertility rates (30.00 ± 11.73% and 31.00 ± 10.25% respectively). In conclusion, DMSO in combination with glucose and hen egg yolk is the most suitable extender for preserving the sperm quality and fertilizing capacity of coho salmon.

"Universal Warming" protocol for vitrified oocytes to streamline cell exchange for transnational donation programs: a multi-center study.

To investigate the clinical efficacy of a "Universal Warming" protocol, based on subsequent steps with 1M and 0.5M concentration of extracellular cryoprotectant (ECCP), on shipped oocytes. Oocytes are vitrified using different brands of ready-to-use kits which recommend that the use of their own warming kit and combining different vitrification/warming kits may have legal consequences for assisted reproductive (AR) centers, until this practice has been validated with clinical studies. Retrospective multi-center transnational observational study. Number of oocytes warmed 1.898. Vitrification performed with vitrification kit (Kitazato, Japan); warming carried out randomly with two different kits: Kitazato warming kit and Vit Kit®-Thaw (FujiFilm Irvine, USA). Warmed oocytes were assigned to 2 groups: KK (Kitazato/Kitazato) 939, and KI (Kitazato/Irvine) 959. Primary endpoint: survival rate. Secondary endpoints: fertilization rate; blastulation rate; implantation rate; live birth rate. Survival was comparable between the groups: 84.6% (795/939) in group KK vs 82.1% (787/959) in group KI. Fertilization rate was lower (P= 0.027) in group KK (75.7%-602/795) than in group KI (80.4%-633/787). Blastulation and implantation and live birth rates were all statistically comparable between the study groups: blastulation rate was 58.5% (352/602) vs 57.8% (366/633); implantation rate was 41.5% (80/193) vs 45.9% (84/183); live birth rate was 52.5% (62/118) in KK and 45.0% (54/120) in KI. The use of this "Universal Warming" protocol simplifies vitrified oocyte exchange between AR centers in different countries, and overcomes potential regulatory/commercial/availability differences affecting clinical practice.

In vitro evaluation of Labrador dog spermatozoa cryopreserved in Tris-citric acid-fructose buffer supplemented with different combinations of extracellular and intracellular cryoprotectants

Aim of this study was to compare different combinations of penetrating intracellular CPAs, i.e., glycerol (G), ethylene glycol (EG), propylene glycol (PG), dimethyl formamide (DM), and methyl acetamide (MA) and extracellular [egg yolk (EY), egg yolk plasma (EYP), low-density lipoproteins (LDL), and coconut water (CW)] in Tris-citric acid-fructose buffer (T) for Labrador dog semen cryopreservation. The study was conducted in two parts, first trial was conducted to assess optimum glycerol concentration (5–7%) in TEY and equilibration time (ET, 2–4 hrs) for Labrador dog semen cryopreservation. Secondly, compatibility of 15% TEY, 15% TEYP, 13% TLDL, and 25% TCW with G, DMF, MA, D + M, EG, and PG was evaluated for in vitro sperm function tests. Decline in sperm attributes, i.e., motility, viability, plasma membrane integrity (PMI), and acrosome integrity (AI)) was significantly (p < 0.05) less in 7% TEY-G and 4 h compared to other concentrations and ET at post-thaw. There was significantly (p < 0.05) less decline in sperm attributes in TEY-G, TEYP-G, TLDL-G, TLDL-D, TLDL-EG, and TCW-D extenders compared to other combinations at post-thaw. However, these parameters were significantly (p < 0.05) high in TEY-G and TEYP-G compared to TEYP-D, TLDL-G, TLDL-D, TLDL-EG, and TCW-D extenders at post-thaw. However, decline in motility, viability, PMI, and AI was identical in these seven extenders. This study concluded that glycerol at a concentration of 7% in TEY and 4 h ET were optimum for successful cryopreservation and besides TEY-G, other combinations of protectants may be an alternative for canine semen cryopreservation.

A monocentric analysis of the efficacy of extracellular cryoprotectants in unfrozen solutions for cleavage stage embryos

BackgroundIn the absence of international guidelines indicating the usage of vitrification rather than slow-freezing, the study aim was to analyze a large cohort of slow-frozen/thawed embryos to produce a rationale supporting the standardization of IVF cryopreservation policy.MethodsThis retrospective analysis included 4779 cleavage stage embryos cryopreserved by slow-freezing/thawing from September 2009 to April 2017 at a single Center. Biological and clinical outcomes of three different commercial kits adopted sequentially, i.e. Vitrolife Cleave Kit® from Vitrolife (kit 1) vs. K-SICS-5000 Kit® and K-SITS-5000 Kit® from Cook Medical (kit 2) and Freeze/Thaw 1™ Kit® from Vitrolife (kit 3) were collected and compared in the light of cryoprotectants composition.ResultsKit 3 compared to kit 1 and kit 2 showed significantly (P < 0.001) higher embryo survival (79.9% vs. 75.6 and 68.1%, respectively) and frozen embryo replacement (91.5% vs. 86.5 and 83.3%, respectively) rates, and significantly (P < 0.001) lower blastomere degeneration rate (41.5% vs. 43.6 and 52.4%, respectively). No significant difference for clinical outcomes was observed among kits. Only a slight positive trend was observed for kit 3 vs. kit 1 and kit 2 on delivery rate per thawing cycle (7.12% vs. 4.19 and 4.51%, respectively; P < 0.058) and live birth rate (3.07% vs. 2.59 and 1.93%, respectively, P < 0.069). Thawing solutions of kit 3 were similar to those of any warming protocol.ConclusionsA defined concentration of extracellular cryoprotectants in thawing/warming solutions had a beneficial effect on the embryo cryosurvival rate. Results could provide the rationale for the adoption of a single standardized warming protocol.

Methods of Cryoprotectant Preservation: Allogeneic Cellular Bone Grafts and Potential Effects.

Debridement of the bone surface during a surgical fusion procedure initiates an injury response promoting a healing cascade of molecular mediators released over time. Autologous grafts offer natural scaffolding to fill the bone void and to provide local bone cells. Commercial bone grafting products such as allografts, synthetic bone mineral products, etc., are used to supplement or to replace autologous grafts by supporting osteoinductivity, osteoconductivity, and osteogenesis at the surgical site. To assure osteogenic potential, preservation of allogeneic cells with cryoprotectants has been developed to allow for long-term storage and thus delivery of viable bone cells to the surgical site. Dimethyl sulfoxide (DMSO) is an intracellular cryoprotectant commonly used because it provides good viability of the cells post-thaw. However, there is known cytotoxicity reported for DMSO when cells are stored above cryogenic temperatures. For most cellular bone graft products, the cryoprotectant is incorporated with the cells into the other mineralized bone and demineralized bone components. During thawing, the DMSO may not be sufficiently removed from allograft products compared to its use in a cell suspension where removal by washing and centrifugation is available. Therefore, both the allogeneic cell types in the bone grafting product and the local cell types at the bone grafting site could be affected as cytotoxicity varies by cell type and by DMSO content according to reported studies. Overcoming cytotoxicity may be an additional challenge in the formation of bone at a wound or surgical site. Other extracellular cryoprotectants have been explored as alternatives to DMSO which preserve without entering the cell membrane, thereby providing good cellular viability post-thaw and might abrogate the cytotoxicity concerns.

Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods &amp; Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture.

PF779 VIABILITY OF HEMATOPOETIC STEM CELLS FOLLOWING STORAGE AT −80OC WITH 5% DIMETHYLSULFOXIDE AND HEMATOLOGIC RECOVERY IN TRANSPLANTED MYELOMA PATIENTS

Background:Dimethylsulfoxide (DMSO) is a major intracellular cryoprotectant, used for cryopreservation of stem cells. It is toxic to both cells and patients at temperatures above 0oC. Reduction of this effect is achieved by either washing of cells after thawing or by reduction of DMSO during freezing and storage. The latter requires addition of extracellular cryoprotectants to the freezing media.Aims:We assessed the effect of low DMSO concentration and different hematocrits of the frozen cells on cell viability and hematologic recovery in patients, transplanted for multiple myeloma.Methods:Cells were non‐programmed frozen and stored at −80oC in a cryoprotectant solution achieving final concentrations of 5% DMSO, 3.6% of hydroxyethyl starch (HES, Weight average molecular weight 450 000 Da) and 3% of human serum albumin. The cell concentration in the frozen product for the first 77 patients (84 transplantations) varied between 100x106 and 250x106 cells/ml. In an attempt to reduce the amount of DMSO infused, for the rest of the patients (n = 172; 192 transplantations) we further decreased the volume of the freezing suspension by removal of the entire plasma.The average age of the transplanted patients was 55 (35 ‐ 71). The cells were bedside thawed at 37oC water bath. The average cell dose was 2,95x106/kg (1,3 – 9,2x106/kg).Results:Viability of the stem cells following thawing assessed by trypan blue exclusion was 95,34% (70–99). The hematocrit of the frozen cells had no effect on cell viability (94,80%(low) vs 95,52%(high)). The major complaints, if any, during stem cell infusion were coughing and an increase in nausea and vomiting induced by the prior conditioning.The average time for hematological recovery was 11,66 days (between 9 and 19) for the neutrophils, and 12,17 (between 9 and 20) days for the platelets. There was no significant difference in viability and hematologic recovery (11,86 and 12,60 vs 11,59 and 12,02) between patients receiving cells frozen with low or high hematocrit.Summary/Conclusion:Dimethylsulfoxide, despite its cryoprotective properties, is toxic for stem cells at temperatures above zero C and induces many side effects (cardiac, neurologic, respiratory, etc.) in the patients. To reduce those side effects we use lower DMSO concentration, high hematocrit resulting in lower volume of the frozen cell suspension, thus reducing the final quantity of DMSO to be infused to the patients. This does not affect the cell viability or the hematologic recovery of patients after transplantation. Our easily performed method for unprogrammed freezing of stem cells with final DMSO concentration 5% at −80oC is safe, well tolerated, and provides cryopreservation, which allows high viability and stable cell engraftment, while reducing the undesired side effects of DMSO.

Sumbawa black honey potency as a potential cyropreservative additive for hematopoetic stem cells (HSC) cryostorage

Background & Aim Cryopreservation or freezing (banking) HSC opens opportunities for future clinical applications (autologous HSC transplantation) or for parents and siblings (allogeneic HSC transplants). In the cryopreservation process, the cell is transferred into the cryo medium. Cryo medium comprises of basal medium added with a diluent medium (mainly fetal bovine serum, human serum albumin or umbilical cord blood serum) and DMSO cryoprotectant agent. DMSO is toxic to cells at room temperature and during transplant to the patient's body. Reports of side effects that arise include symptoms in the gastrointestinal tract, heart and blood vessels such as nausea, vomiting, ECG abnormalities, bradycardia and hypotension. DMSO is used in concentrations of 5% or 10% as cryoprotectant. DMSO concentration of 2% or less decreases the cell membrane integrity so that the cells are susceptible to lysis thus low cryopreservative potency. Extracellular cryoprotectants such as sucrose or trehalose combined with DMSO can reduce DMSO concentrations to 2.5%. This can reduce the toxicity caused by the DMSO cryoprotectant formula. Sumbawa black honey is a local product by Sumbawa honey farm that is known to contain high sucrose. This study aims to investigate the potency of Sumbawa black honey as cryopreservative additive to lower the concentration of DMSO in the cryo medium thus limit the toxicity potential. Methods, Results & Conclusion This is an experimental in vitro study from November 2018 until February 2019. The protocol has been approved by ethical committee Faculty of Medicine Universitas Indonesia and UCB donors have signed informed consent of the study. Enriched CD34+ hematopoetic stem cells were obtained by MACS CD34+ separation after ficoll density centrifugation. Cell count performed using tryphan blue exclusion methods. Flow cytometry analysis of CD34+ cells were performed after isolation and after thawing. Cryo medium composed of RPMI 1640, 10% DMSO and 10% UCB serum was used as control. Cryo medium composed of RPMI 1640, 10% UCB serum and either 5% DMSO or 2.5% DMSO added with 5% or 7.5% filtered Sumbawa black honey (for a total percentage of cryopreservative agent 10%).

Estudios preliminares de las características espermáticas y de criopreservación del coral formador de arrecifes Orbicella faveolata

Cryopreservation has been recently applied to coral gametes and tissue with successful results that can be applied for different purposes on coral conservation and restoration. In this study, we decided to determine the sperm morphology of the coral Orbicella faveolata and assess the feasibility of sperm cryopreservation using a combination of intracellular (1,2-Propadiol) and extracellular (milk) cryoprotectants, and two frozen treatments for 24 h. Mature spermatozoa had a triangular-like head shape measuring 4.10 ± 0.69 μm(mean ± SD) and long flagellum (43.24 ± 7.99 μm). Fresh sperm remained viable and mobile for more than five hours after being released from the gamete bundles. After cryopreservation, all post-thaw sperm components assessed (morphology, motility and viability) showed no difference in contrast to fresh sperm. This study is the first report of cryopreservation of O. faveolata sperm, however further research is needed to increase the success of the cryopreservation protocol for broad-scale application.

Testing the efficacy and efficiency of a single \u201cuniversal warming protocol\u201d for vitrified human embryos: prospective randomized controlled trial and retrospective longitudinal cohort study

PurposeTo study the efficacy and efficiency of a “universal warming protocol” for vitrified human embryos, based on subsequent steps with 1 and 0.5 M concentration of extracellular cryoprotectant (ECCP).MethodTwo studies on patients undergoing fertility treatments via ICSI: a prospective randomized controlled trial (RCT) and a retrospective cohort study (CS). Setting: Private assisted reproductive (AR) center.RCT: duration 01/03/2017–01/10/2017; 315 embryos at blastocyst stage obtained from 169 patients. Each patient’s embryos were first randomized for vitrification with two different kits: Vitrification Kit (Kitazato, Japan) and Sage Vitrification Kit (Origio, Denmark). The embryos were randomly warmed with either Kitazato (K) or Sage (S) warming kits, specifically: group A (KK), group B (KS), group C (SK), and group D (SS). Primary outcome measure: survival rate (number of embryos surviving per number of embryos warmed). Secondary: implantation rate (number of embryos implanted per number of embryos transferred).CS: duration 01/01/2013–31/12/2015 embryos from patients’ own oocytes; 10/04/2015–31/07/2017 embryos from donors’ oocytes. A total of 1055 embryos vitrified at cleavage stage obtained from 631 warming cycles: 847 of these obtained from patients’ own oocytes, 208 egg-donation-derived embryos. Each patient’s embryos were vitrified and warmed in various combinations of three different vitrification/warming kits: Kitazato (K), Sage (S), or made in-house in our laboratory (H). Vitrification/warming kits from different manufacturers are routinely used in our AR center, and the warming procedures are randomly performed with any available kit on a “first-in-first-out” basis, irrespective of the kit used for vitrification. Group names: KK, KS, SK, SS, SH, HK, HS, HH (embryos from patients’ own oocytes); eKK, eKS, eSK, eSS (egg-donation-derived embryos).ResultsCryo-survival rates were comparable in all study groups.RCT. Group A 99.0% (96/97), group B 98.8% (83/84), group C 98.4% (61/62), and group D 98.6% (71/72).CS. Embryos from patients’ own oocytes: KK 96.4% (54/56), KS 100.0% (13/13), SK 98.8% (80/81), SS 97.2% (174/179), SH 97.6% (40/41), HK 95.2% (20/21), HS 99.5% (187/188), and HH 97.4% (261/268). Egg-donation-derived embryos: eKK 100.0% (91/91), eKS 98.4% (60/61), eSK 100.0% (26/26), and eSS 96.7 (29/30).Implantation was generally comparable in all study groups—exceptions were in CS: KS vs. SK (P = 0.049), SS (P = 0.012), HS (P = 0.010), HH (P = 0.025); and SH vs. SS (P = 0.042), HS (P = 0.035).ConclusionWorldwide, millions of embryos have been cryopreserved using different vitrification kits; these studies establish that it is possible to combine different kits for vitrification and warming using a universal warming protocol. This can optimize costs, simplify lab routines, and favor embryo exchange between IVF centers.RCT registration numberISRCTN12342851.

Vitrification of domestic cat (Felis catus) ovarian tissue: Effects of three different sugars

We aimed to evaluate the effect of three extracellular cryoprotectants on the morphology of vitrified feline preantral follicles. Feline ovarian fragments (0.5 × 2 × 2 mm) collected from five domestic adult cats subjected to ovariohysterectomy for routine castration were vitrified with ethylene glycol (EG) 40% combined or not with sucrose (0.1 or 0.5 M), trehalose (0.1 or 0.5 M), or raffinose (0.1 M). After vitrification using the solid-surface method and warming of the tissues, cryoprotectants were washed out of the ovarian tissues, which were fixed for histological analysis. The percentages of normal follicles were similar to the control (fresh) (62.9 ± 4.1%) only for tissues exposed and cryopreserved with EG + trehalose at concentrations of 0.1 (35.8 ± 8.3%) and 0.5 M (33.4 ± 5.4%). All the other sugars decreased the percentages of morphologically normal follicles as compared to the control group and the trehalose groups. Based on the results of the present study, we recommend the use of trehalose as the extracellular cryoprotectant for the vitrification of feline ovarian tissue.

Low Density Lipoproteins at 2% Concentration Can Replace Whole Egg Yolk in TES-Tris-Milk Extender for Freezing Buffalo Sperm Cells

Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryoprotectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its interaction with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm.Materials, Methods &amp; Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tourbillon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected. All other kinetic parameters presented significantly better results in extenders containing 2%, 4% and 8% LDL, compared with the control. There was no difference between treatments regarding the integrity of membranes or fragmentation of the DNA after freezing/thawing procedures.Discussion: The molecules of low density lipoproteins of egg yolk have the known action of sequestering BSP ( binder of sperm proteins) protein, due to the chemical affinity of their main components, phosphatidylcholine and phosphatidylethanolamine. The BSP are responsible for removing cholesterol from the plasma membrane of the sperm, preparing it for the sperm capacitation phase. Low density lipoproteins by inhibiting the action of the BSP increase the stability of the plasma membrane during the freeze-thaw process. The milk caseins micelles have a similar ability to bind the BSP, but with lower affinity. The present study has shown that the use of purified low density lipoproteins has an advantage over the use of whole egg yolk, especially with regard to the kinetic parameters of the spermatozoa after thawing. Furthermore, it was observed that low concentrations of low density lipoproteins, such as 2%, in extenders containing skim milk, could preserve the sperm cell during the freeze-thawing process like that higher LDL concentrations and whole egg yolk. Further studies are needed to determine if the cryoprotectant action found in this study was a result of the synergistic action of skim milk with lipoproteins or even at low concentrations like 2% the lipoproteins can provide protection to the buffalo spermatozoa during freezing, as it has been reproduced in other domestic species. In conclusion, our results indicated that the extender TES-Tris-skim milk containing 2% LDL extracted from egg yolk could be used successfully in the cryopreservation of buffalo sperm cells.