Post-thaw Culture Research Articles

Influence of different post-thaw culture time on the clinical outcomes of different quality embryos.

In thawed embryo transfer cycles, the most common method is to transfer the embryos after 2 h of culture. Clinical outcomes of frozen-thawed cleavage embryo transfer cycles regarding the embryos status and the culture time of frozen-thawed cleavage embryos were limited and did not elucidate all unclear issues. The objective of this study was to examine the clinical outcomes of frozen-thawed cleavage embryo transfer cycles according to the embryos status and the culture time (2 h or overnight). In this retrospective study (5-year period), 1,654 frozen-thawed embryos were analyzed. Firstly, frozen-thawed cleavage embryos were divided into 2 groups according to their status as follows: with at least 1 optimal embryo and without optimal embryos. Secondly, both of them were divided into 2 groups according to the culture time (2 h or overnight). Age of the female, infertility factors, clinical pregnancy, implantation rate, and live birth rate were compared. There were no statistically significant differences in the pregnancy rate, the implantation rate, live birth rate, the miscarriage rate, and the ectopic pregnancy rate in each group. However, the implantation rate increased after 2 h of incubation (41.1%) compared to overnight incubation (36.0%) in the group with at least 1 optimal day-3 embryo (p < 0.05). The cancellation rate in the suboptimal day-3 embryos group (9.1%) was higher than in the group containing at least 1 optimal embryo (0.2%) for the long (overnight) culture (p < 0.05). The implantation rate can be improved in the optimal day-3 embryos transferred after 2 h of culture, but not for suboptimal day-3 embryos. Some unnecessary transfers can be avoided after overnight culture because of no further cleavage of the embryos.

Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation.

Cryopreservation is an essential tool to meet the increasing demand for stem cells in medical applications. To ensure maintenance of cell function upon thawing, the preservation of the actin cytoskeleton is crucial, but so far there is little quantitative data on the influence of cryopreservation on cytoskeletal structures. For this reason, our study aims to quantitatively describe cryopreservation induced alterations to F-actin in adherent human mesenchymal stem cells, as a basic model for biomedical applications. Here we have characterised the actin cytoskeleton on single-cell level by calculating the circular standard deviation of filament orientation, F-actin content, and average filament length. Cryo-induced alterations of these parameters in identical cells pre and post cryopreservation provide the basis of our investigation. Differences between the impact of slow-freezing and vitrification are qualitatively analyzed and highlighted. Our analysis is supported by live cryo imaging of the actin cytoskeleton via two photon microscopy. We found similar actin alterations in slow-frozen and vitrified cells including buckling of actin filaments, reduction of F-actin content and filament shortening. These alterations indicate limited functionality of the respective cells. However, there are substantial differences in the frequency and time dependence of F-actin disruptions among the applied cryopreservation strategies; immediately after thawing, cytoskeletal structures show least disruption after slow freezing at a rate of 1°C/min. As post-thaw recovery progresses, the ratio of cells with actin disruptions increases, particularly in slow frozen cells. After 120 min of recovery the proportion of cells with an intact actin cytoskeleton is higher in vitrified than in slow frozen cells. Freezing at 10°C/min is associated with a high ratio of impaired cells throughout the post-thawing culture.

Prolonged post-thaw culture of embryos does not improve outcomes of frozen human embryo transfer cycles: A prospective randomized study

Objective: To evaluate the impact of prolonged post-thaw embryos culture on pregnancy outcome during frozen embryo transfer cycles. Methods: This prospective cohort study evaluated 324 thaw transfer cycles with 819 embryos from 269 patients at the Center for Reproductive Endocrinology and Infertility of Hue University Hospital in Vietnam. These frozen embryo transfer cycles were divided into two groups at the time of thawing: the short culture group (2-hour post-thaw culture) and the overnight culture group (overnight culture for 18 h) before the embryo was transferred into the uterus. The rates of embryo intact, grade A embryo at frozen and transfer time and continuing cleavage were recorded. The clinical outcomes including serum beta-human chorionic gonadotropin, clinical pregnancy and implantation rate were evaluated after 14 days, 4 weeks, 6 weeks, respectively, after embryo transfer. Results: Human chorionic gonadotropin positive occurred in 39.5% of patients in the short culture group compared to 25.9% in the overnight culture group with risk difference (RD)=13.6%, relative risk (RR)=1.343, 95% confidence interval (CI) 1.085-1.663, P 35 years) and women who received 3 embryos, pregnancy outcomes were found to be significantly (P Conclusions: The prolonged post-thaw culture period does not increase pregnancy outcome in comparison with the short culture.

Prolonged culture of blastocysts after thawing as a tool for improving prediction of success.

A few years ago, we started to use a new freeze-thaw protocol for the frozen embryo transfer cycles. Instead of thawing the embryos 2-4h prior to the transfer, we started thawing the embryos 20-22h prior to the transfer. The aim of this study was to compare the pregnancy rate in cases of embryos that continued to develop in the post-thawing culture to that of embryos that did not. A retrospective cohort study of blastocyst freeze/thaw cycles vitrified on day 5, thawed and transferred after 20-22h in the culture, between January 2012 and December 2016. A total of 375 patients were included in the analysis. Two hundred twenty-eight embryos graded as good, 87 graded as fair, and 60 graded as poor embryos were transferred. The clinical pregnancy rate (50% vs. 19.5% vs 3.3% p < 0.01) and the ongoing pregnancy rate (38.5% vs. 13.6% vs 1.7% p < 0.01) were higher in cases of good embryo quality compared with fair and poor-quality embryos, respectively. For good embryos, progressing to a better grade during the culture did not change the clinical pregnancy rate (51.3% vs. 46.2% p = NS) or the ongoing pregnancy rate (38.5% vs. 37.5% p = NS). For fair embryos, progressing to a better grade during the culture resulted in a higher clinical pregnancy rate (25.4% vs 9% p = 0.05). The development of the fair embryos in the culture has a highly positive impact on the pregnancy rate and this factor should be taken into consideration before deciding how many embryos to transfer.

RNA-Based dCas9–VP64 System Improves the Viability of Cryopreserved Mammalian Cells

Regenerative therapies require availability of an abundant healthy cell source which can be achieved by efficient cryopreservation techniques. Here, we established a novel approach for improved cell cryopreservation using an mRNA-based dCas9-VP64 gene activation system for transient, yet highly efficient expression of epigenetic related genes in mammalian cells for repression of metabolic activity. Before freezing, mammalian cells were treated by dCas9-VP64-modified mRNA and guide RNAs for upregulation of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and transcriptional co-repressor Sin3A genes. Cell viability, karyotype, pluripotency, and other cell specific functions were analyzed during post-thaw culture. Using conventional cryopreservation protocols, we found improvement of viability in dCas9-VP64 pretreated cells ([Formula: see text]) compared to untreated cells. Combined with dCas9-VP64 system, a reduced amount of cryoprotectant (5% DMSO) did not negatively affect the post-thaw viability. Co-delivering chemically modified dCas9-VP64 mRNA with gRNAs is an efficient gene delivery method compared to DNA-based strategies, without the associated safety concerns. This approach is a simple, yet effective way to accelerate a wide array of cellular research and translational medical applications.

In vitro regeneration of adventitious buds from leaf explants and their subsequent cryopreservation in highbush blueberry

We report a new cryopreservation method for highbush blueberry (Vaccinium corymbosum) using small leaf squares-bearing adventitious buds. Leaf explants were cultured on adventitious bud regeneration medium composed of Woody Plant Medium (WPM) supplemented with 20 µM zeatin. After 21 days of adventitious bud regeneration, small leaf squares (SLSs, 2 × 3 mm), each bearing multiple adventitious buds, were cut from the leaf explant, precultured on WPM containing 0.3 M sucrose for 24 h and were treated for 30 min with a loading solution composed of WPM containing 1.0 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 at 0 °C for 40 min. Each of dehydrated SLS was then transferred onto an aluminum foil with small holes and PVS2 was dropped until it covered the SLS, prior to a direct immersion in liquid nitrogen. Cryopreserved SLSs were re-warmed in WPM containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, more than 23 adventitious buds were produced in each leaf explant, and 100% of SLSs were able to survive and resume shoot regrowth, with more than six shoots per SLS obtained following cryopreservation in three highbush blueberry cultivars. In ‘Misty’, the morphology of plantlets regenerated from cryopreserved SLSs was identical to that of the in vitro-derived ones. No polymorphic bands were detected using inter-simple sequence repeat markers and random amplified polymorphic DNA in plantlets of ‘Misty’ recovered from cryopreservation. The use of SLSs-bearing adventitious buds for cryopreservation reported in the present study eliminates shoot tip excision. This cryopreservation method can be considered an efficient cryopreservation of plant shoot tips, and has potential applications to other plant species.

Epigenetic status of buffalo fibroblasts treated with sodium butyrate a chromatin remodeling agent.

The somatic cells having higher levels of DNA methylation and reducing it in donor cells before nuclear transfer (NT) by treating them with chemicals, may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Therefore, the present study was planned to understand mechanism of epigenetic changes in donor cells (buffalo fibroblasts) treated with different concentration of sodium butyrate (NaBu)-a chromatin remodeling agent. The cultured fibroblasts purity and lineage were confirmed by fibroblast specific protein and gene markers (Vimentin, Tubulin and Cytokeratin) at different passages using immuno-staining and qPCR respectively. The buffalo fibroblast cells were treated with 1, 3 and 5 mM of NaBu and observations were taken on their morphological changes, population doubling time and cell proliferation after 48 h of treatment. The epigenetic changes were observed using acetylation (H3K9ac) and methylation (H3K27me3) markers expression. The fibroblast cells derived from new born ear tissue started emerging and anchoring to cell culture flasks within 24 h and showed spindle-shaped morphology. The confluent culture was cryopreserved at different time interval. The post thaw culture behavior of the cryopreserved cells was also observed at different time interval and passages. The morphology of NaBu treated cells were changed with increase of dosages of NaBu treatment. The increase population doubling times and decreases the proliferation rate in the dose dependent manner. The NaBu treatment showed that the significantly increased the acetylation (H3K9ac) and methylation (H3K27me3) level over the control. Based on the observations of fibroblast characterization as well as epigenetic modifications of these cells after treatment with NaBu, results suggest that the cells may provide a useful approach for better epigenetic reprogramming in SCNT embryos.

Cryopreservation of small leaf squares-bearing adventitious buds of Lilium Oriental hybrid ‘Siberia’ by vitrification

We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L−1 α-naphthalene acetic acid and 0.5 mg L−1 thidiazuron. Small leaf squares (SLSs, 3 × 4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species.

Cryopreservation and re-culture of a 2.3 litre biomass for use in a bioartificial liver device.

For large and complex tissue engineered constructs to be available on demand, long term storage using methods, such as cryopreservation, are essential. This study optimised parameters such as excess media concentration and warming rates and used the findings to enable the successful cryopreservation of 2.3 litres of alginate encapsulated liver cell spheroids. This volume of biomass is typical of those required for successful treatment of Acute Liver Failure using our Bioartificial Liver Device. Adding a buffer of medium above the biomass, as well as slow (0.6°C/min) warming rates was found to give the best results, so long as the warming through the equilibrium melting temperature was rapid. After 72 h post thaw-culture, viable cell number, glucose consumption, lactate production, and alpha-fetoprotein production had recovered to pre-freeze values in the 2.3 litre biomass (1.00 ± 0.05, 1.19 ± 0.10, 1.23 ± 0.18, 2.03 ± 0.04 per ml biomass of the pre-cryopreservation values respectively). It was also shown that further improvements in warming rates of the biomass could reduce recovery time to < 48 h. This is the first example of a biomass of this volume being successfully cryopreserved in a single cassette and re-cultured. It demonstrates that a bioartificial liver device can be cryopreserved, and has wider applications to scale-up large volume cryopreservation.

凍結保存したイネ培養細胞の再培養初期の生存活性について(平成16年度 第50回低温生物工学会研究報告)

凍結保存したイネ培養細胞の再培養初期の生存活性について(平成16年度 第50回低温生物工学会研究報告)

9. 凍結保存した植物培養細胞の生存率と培地の成分の関係(平成12年度第46回低温生物工学会研究報告)

9. 凍結保存した植物培養細胞の生存率と培地の成分の関係(平成12年度第46回低温生物工学会研究報告)

An efficient droplet-vitrification cryopreservation for valuable blueberry germplasm

We report a droplet-vitrification for efficient cryopreservation of valuable germplasm of blueberry. In this protocol, in vitro stock shoots were cold-hardened in the dark at 4°C for 3 weeks. Shoot tips (1.5–2.0mm in length) including 4–5 leaf primordia were excised from the cold-hardened stock shoots and precultured with 0.3M sucrose for 1day. Precultured shoot tips were treated with a loading solution containing 1.0M sucrose and 2M glycerol for 30min, followed by exposure to PVS2 for 40min at 0°C. Dehydrated shoot tips were then transferred onto 3.5μL PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1h. Frozen foils with shoot tips were re-warmed in 1.2M sucrose for 20min at room temperature, followed by post-thaw culture for a recovery medium containing 0.3mgL−1 zeatin for shoot regrowth. With optimized parameters, shoot regrowth levels were 46.7% for ‘O’ Neal’ and 91.7% for ‘Misty’, with a mean shoot regrowth level of 66.5% obtained across the five valuable blueberry genotypes tested. Histological observations showed that many cells in the upper part of apical dome and some in leaf primordia 1–3 survived, while those in lower part of apical dome and leaf primordia 4–5 were seriously damaged, following cryopreservation. No polymorphic bands were detected using inter-simple sequence repeat markers (ISSR) and random amplified polymorphic DNA (RAPD) in regenerants of ‘O’ Neal’ and ‘Misty’ recovered from cryopreservation. To the best our knowledge, this is the first report on blueberry cryopreservation by droplet-vitrification, which would provide a technical platform for establishment of cryo-banks of valuable blueberry germplasm.

Cryopreservation and evaluations of vegetative growth, microtuber production and genetic stability in regenerants of purple-fleshed potato

Buds of in vitro-grown shoots of two purple-fleshed potato genotypes were successfully cryopreserved by encapsulation-vitrification (Encap-vitri) and droplet-vitrification (Drop-vitri). Optimal time durations of exposure to PVS2 for shoot regrowth of cryopreserved buds were 5–7 h and 6 h for ‘E03-2677’ and for ‘Blue Congo’, respectively, in Encap-vitri, and 30–50 min and 40 min for the former and the latter, respectively, in Drop-vitri. Higher rates of shoot regrowth were obtained in 1.5–2.0 mm-buds than in 1.0–1.4 mm-ones in Encap-vitri for ‘E03-2677’ and ‘Blue Congo’, while opposite results were found in Drop-vitri. In ‘Blue Congo’, only apical shoot tips survived and developed into shoots, with one shoot produced in one cryopreserved bud in Encap-vitri and Drop-vitri. In ‘E03-2677’, survival and shoot regrowth patterns were similar to those of ‘Blue Congo’ in Encap-vitri. However, both apical and axillary shoot tips survived and developed into two shoots in one bud in Drop-vitri. In ‘E03-2677’, histological observations revealed only apical shoot tips survived following Encap-vitri, while both apical and axillary shoot tips survived following Drop-vitri. Vegetative growth in shoots regenerated from Encap-vitri and Drop-vitri after 3 weeks of post-thaw culture was significantly lower than that from control, but markedly increased after 6 months of post-thaw culture. In both ‘E03-2677’ and ‘Blue Congo’, number of microtubers per shoot, per vessel and ≥3 mm in diameter were significantly greater in shoots regenerated from Encap-vitri than in those from the control. Assessments by ISSR and RAPD of genetic stability did not find any polymorphic bands in regenerants recovered from Encap-vitri and Drop-vitri. To the best of our knowledge, this is the first report on cryopreservation of purple-fleshed potato by vitrification-based procedures. Results reported here would provide valuable basic and technical information on cryopreservation of purple-fleshed potato.

Comparative analysis of cryopreservation methods in Chlamydomonas reinhardtii

Chlamydomonas is a model organism used for studies of many important biological processes. Traditionally, strains have been propagated on solid agar, which requires routine passaging for long-term maintenance. Cryopreservation of Chlamydomonas is possible, yet long-term viability is highly variable. Thus, improved cryopreservation methods for Chlamydomonas are an important requirement for sustained study of genetically defined strains. Here, we tested a commercial cryopreservation kit and directly compared it's effectiveness to a methanol-based method. We also tested thaw-back procedures comparing the growth of cells in liquid culture or on solid agar media. We demonstrated that methanol was the superior cryopreservation method for Chlamydomonas compared to the commercial kit and that post-thaw culture conditions dramatically affect viability. We also demonstrated that cryopreserved cells could be successfully thawed and plated directly onto solid agar plates. Our findings have important implications for the long-term storage of Chlamydomonas that can likely be extended to other algal species.

Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts

This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells.

Cryopreservation of Human Wharton's Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study.

ObjectivesTo compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol.MethodsThe mesenchymal stem cells isolated from human Wharton’s jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation.ResultsThe cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2±0.58% whereas 10% PVP and cocktail solution have shown 62.87±0.35% and 72.2±0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic BAX and p53 genes were higher whilst p21 was lower in all the cryopreserved groups when compare to the control group of WJMSCs.ConclusionAlthough 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.

An evaluation of continuous human embryo culture using the wow dish

The WOW dish (LinKIDTMculture dish,DNP) 25 microwells that allows group culture under a single drop of medium. Through its design it is possible to manage embryos separately whilst in group culture. There are several reports (SUGIMURA et al, 2013) suggesting that the WOW dish improved bovine embryo culture results. Therefore, we investigated whether the WOW dish is suitable for continuous human embryo culture without exchange of culture medium. Retrospective study. The study consisted of three experimental groups. “15ul drop culture” group (control) was the standard embryo culture method with exchange from single medium to single medium at Day3 and Day5 using normal 35mm dish. The other 2 groups were “15ul drop continuous culture” and “WOW dish continuous culture” without exchange of culture medium. A total of 143 pronuclear embryos from 12 patients who consented to post-thaw culture were studied. Pronuclear stage frozen embryos were thawed and cultured across the 3 groups for up to 7days (n=47, 48, 48). We compared the incidence of good blastocysts (≥ 3BB,Gardner) and blastocyst formation rate at Days 5, 6 and 7 in the 3 groups. In the control group, blastocyst formation rate at Days 5, 6 and 7 was 46.8%, 57.4% and 59.6%, respectively. The incidence of good blastocysts was 25.5%, 44.7%, and 48.9% (n = 47) respectively. In the “15ul drop continuous culture” group, blastocyst formation rate at Days 5, 6 and 7 was 45.8%,54.2% and 54.2%. The incidence of good blastocysts was 14.6%,31.3%,33.3% (n = 48). In the “WOW dish continuous culture” group, blastocyst formation rate at Days 5, 6 and 7 was 52.1%,58.3% and 58.3%. Incidence of good blastocysts was 25.0%,43.8% and 47.9% (n = 48). Between each observation time interval, there was no significant differences in both the incidence of good blastocysts and the blastocyst formation rate. These results suggest that the WOW dish is suitable for continuous culture without exchange of culture medium compared to the 15ul drop control method.

Freeze-all at the blastocyst or bipronuclear stage: a randomized clinical trial

To compare outcomes for patients randomized to have all embryos cryopreserved at the blastocyst stage or at the bipronuclear stage with subsequent post-thaw culture to the blastocyst stage. Randomized controlled trial. Private fertility center. This study included 140 women, age 18-40 years, with at least eight antral follicles, and day 3 FSH <10 IU/L undergoing IVF. After oocyte retrieval, subjects were randomized to have entire embryo cohorts cryopreserved at either the bipronuclear stage (2PN Cryo group) or at the blastocyst stage (Blast Cryo group). Ongoing pregnancy (viable fetal heart motion at 10 weeks' gestation) per oocyte retrieval through the first transfer attempt. No significant differences were observed between the two study groups in age at retrieval, body mass index, antral follicle count, day 3 FSH level, or IVF cycle parameters. No significant differences were observed in ongoing pregnancy rate per retrieval (62.0%; 95% confidence interval [CI], 50.3%-72.4%) in the 2PN Cryo group; and 55.1%; 95% CI, 42.6%-67.1% in the Blast Cryo group), implantation rate (60.0% vs. 62.7%), ongoing pregnancy rate per thaw (62.0% vs. 59.4%), ongoing pregnancy rate per transfer (67.7% vs. 69.1%), and the cumulative ongoing pregnancy rate per retrieval from all thaws to date of embryos derived from the study retrieval cycle (64.8% vs. 60.9%). Freeze-all at the blastocyst stage or at the bipronuclear stage has similar efficacy and IVF outcomes. The choice between them may depend primarily on logistical factors. NCT01247987.

Post-thaw non-cultured and post-thaw cultured equine cord blood mesenchymal stromal cells equally suppress lymphocyte proliferation in vitro.

Multipotent mesenchymal stromal cells (MSC) are receiving increased attention for their non-progenitor immunomodulatory potential. Cryopreservation is commonly used for long-term storage of MSC. Post-thaw MSC proliferation is associated with a lag-phase in vitro. How this lag-phase affect MSC immunomodulatory properties is unknown. We hypothesized that in vitro there is no difference in lymphocyte suppression potential between quick-thawed cryopreserved equine cord blood (CB) MSC immediately included in mixed lymphocyte reaction (MLR) and same MSC allowed post-thaw culture time prior to inclusion in MLR. Cryopreserved CB-MSC from five unrelated foals were compared using two-way MLR. For each of the five unrelated MSC cultures, paired MLR assays of MSC allowed five days of post-thaw culture and MSC included in MLR assay immediately post-thawing were evaluated. We report no difference in the suppression of lymphocyte proliferation by CB-MSC that had undergone post-thaw culture and MSC not cultured post-thaw (p<0.0001). Also, there was no inter-donor variability between the lymphocyte suppressive properties of MSC harvested from the five different donors (p = 0.13). These findings suggest that cryopreserved CB-MSC may have clinical utility immediately upon thawing. One implication hereof is the possibility of using cryopreserved CB-MSC at third party locations without the need for cell culture equipment or competencies.

Effect of the antifreeze protein from the arctic yeast Leucosporidium sp. AY30 on cryopreservation of the marine diatom Phaeodactylum tricornutum.

Antifreeze proteins are a group of proteins that allow organisms to survive in subzero environments. These proteins possess thermal hysteresis and ice recrystallization inhibition activities. In the present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Arctic yeast Leucosporidium sp. AY30, LeIBP, in cryopreservation of the marine diatom Phaeodactylum tricornutum, which is one of the classical model diatoms and has most widely been studied with regard to its ecology, physiology, biochemistry, and molecular biology. P. tricornutum cells were frozen by either a fast or two-step freezing method in freezing medium containing 10 % dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol, respectively, with or without LeIBP supplement. When cells were frozen using the two-step freezing method, cell survival was significantly increased and statistically the same as that of unfrozen native cells in the presence of 0.1 mg/ml LeIBP in 10 % propylene glycol or 10 % ethylene glycol at day 11 of post-thaw culture. In the presence of LeIBP, the concentration of chlorophyll a was dramatically increased to 14-, 48-, 1.6-, and 8.8-fold when cells were frozen in freezing medium containing dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. Scanning electron microscopy observations demonstrated that the cells were also successfully preserved and epitheca or hypotheca were not deformed. These results demonstrate that LeIBP was successfully applied to improve cryopreservation of the marine diatom P. tricornutum.