Vitrification Protocol Research Articles

Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: A study using murine embryonic stem cells

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz micro-capillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1–2 M). The cryoprotectants used included 2 M 1,2-propanediol (PROH, cell membrane permeable) and 0.5 M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post-vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2 M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

Pollination of Dendrobium Hybrids Using Cryopreserved Pollen

Pollination effectiveness was evaluated for pollen (pollinia) from two Dendrobium hybrids, ‘Sena Red’ and ‘Mini WRL’, submitted to cryopreservation using a vitrification protocol. Parameters evaluated included pollinia exposure to a previtrification solution (PVS2) under ice (0 °C) or room (27 ± 2 °C) temperatures from 1 to 4 hours before cryopreservation (LN). On removal from cryopreservation, pollinia were used to pollinate flowers of the same hybrids to verify viability and germination. All pollinia showed high percentages of germination (greater than 80%) after crosses were performed, except for pollinia from Dendrobium ‘Sena Red’ submitted to 3 hours of precooling (0 °C) in PVS2 followed by LN (60%) and for pollinia submitted to PVS2 for 3 hours at room temperature with no precooling (70%). Capsules were formed for both hybrids and seeds were successfully produced. The seed viability test revealed high viability (90% to 95%) for all treatments for both hybrids. Seeds observed under a microscope contained well-formed embryos and no abnormalities were identified. Seeds from all treatments germinated. Germinating seeds developed into healthy seedlings with well-formed leaves and roots. Cryopreservation of pollinia was successfully accomplished either by direct storage in liquid nitrogen without cryoprotection treatments or by using a PVS2 vitrification protocol.

Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO

Vitrification has become common for cryopreservation of embryos. However, the most optimal protocol for vitrification is still to be found. Two vitrification protocols with similar osmolarities were compared: Protocol A, containing dimethyl sulphoxide (DMSO), propane-2-diol, and ethylene glycol, and Protocol B, containing propane-2-diol and ethylene glycol. Viability and the importance of specific incubation times for early embryo recovery, survival, and cleavage were studied. For assessment of cryodamage, embryos were labelled with Alexa Fluor 488-conjugated annexin V and propidium iodide. Vitrification studies on early mouse embryos were followed up with studies on human embryos. The two vitrification protocols did not differ in embryo survival rates and were equally efficient in both mouse and human embryo models. Morphological assessment of embryos directly after vitrification was not a useful tool for assessing survival in this study. Extended exposure of embryos with both vitrification protocols showed that the DMSO-containing vitrification solutions did not lead to cell membrane damage and death as quickly as the DMSO-free vitrification solutions. To assess embryo viability, the authors recommend that vitrification of early embryos should be combined with extended culture and assessment of normal blastocyst development before transferring to patients.

Cryopreservation of shoot-tips by droplet vitrification applicable to all taro (Colocasia esculenta var. esculenta) accessions

The application of the droplet vitrification cryopreservation technique to taro accessions from a range of Asia Pacific countries is presented. The optimum protocol involves excision of about 0.8 mm shoot-tips from in vitro plants, 20–40 min PVS2 exposure at 0°C followed by rapid plunge into liquid nitrogen. Thawing was done at room temperature (25°C) and shoot-tips inoculated on MS medium with 0.1 M sucrose regenerated into plantlets 4–6 weeks later. This new droplet vitrification protocol improved the mean post-thaw regeneration rates to 73–100% from 21–30% obtained with the previous cryo-vial vitrification protocol.

Application of different oocyte vitrification protocols, laser assisted zona slitting, and temperature on post-warming survival of bovine oocyte

Application of different oocyte vitrification protocols, laser assisted zona slitting, and temperature on post-warming survival of bovine oocyte

Comparison of spermatogenesis and steroidogenesis of cryopreserved neonatal mouse testicular tissue – vitrification vs. controlled slow-rate freezing

Ectopic allo- or xeno-grafting of frozen testicular tissue is a promising new approach that can be used to preserve testicular function in mutant animals and cancer patients for future fertility. Rescue of genetic resources and valuable mutant strain associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The aim of present study was to improve new mouse testicular tissue cryopreservation protocols by comparing spermatogenesis and testosterone levels of grafted neonatal testicular tissue after exposed to different vitrification protocols and slow-rate freezing. Experimental basic animal study. Neonatal mouse testes were frozen using 0.7 M DMSO + 5% FBS with a computer controlled slow rate freezer. Three modified vitrification protocols were used: (1) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M Propylene glycol (PG)), (2) 40% Ethylene glycol (EG) + 18% Ficoll + 0.35 M Sucrose, and (3) 15% EG + 15% PG + 0.5 M Sucrose solutions. Frozen-thawed testicular tissues were grafted onto castrated immunodeficient Ncr-nude mice. Fresh neonatal testes were grafted to serve as the positive control. Animals were sacrificed, and histological analyses of grafted tissue and serum hormonal assays were performed 12 weeks post-transplantation. Survival of testicular grafts is superior in the control and computer controlled slow rate freezing groups comparing to the other 3 vitrification groups (P<0.05). Spermatogenesis from the slow rate freezing groups is also comparable to the controls. Testosterone production from the slow rate freezing group is significantly higher than the vitrification protocols tested (P<0.01). There were no significant differences between controlled slow-rate freezing technique and freshly grafted control. Our data shows a clear advantage of the computer controlled slow-rate freezing protocol over the standard vitrification techniques for neonatal mouse testis cryopreservation. Further modifications and improvement in the vitrification process is required before it can be applied to testicular tissue.

Effect of procedure slow-freezing and vitrification protocol on ultra microstructure, ER and MHC-II antigen expressions of rabbit ovarian tissues and the proliferative activity of the follicles

To investigate the effect of PROH procedure slow-freezing protocol and DMSO + EG vitrification protocol on ultra microstructure, ER and MHC-II antigen expressions of rabbit ovarian tissues and the proliferative activity of the follicles.

Vitrification of isolated pre-antral follicles from the mouse ovary: comparison of vitrification protocols

Vitrification has not been extensively studied for ovarian follicle cryopreservation. The simplicity of the technique and the promising results seen with oocyte vitrification suggest that this technique may be very valuable for ovarian follicle banking. The mouse ovary provides an excellent system to assess the effect of vitrification on subsequent pre-antral follicle development and maturation. In the present study we applied two different vitrification protocols to mouse pre-antral follicles and studied subsequent maturation upon thawing.

Effect of pre-vitrification zona slitting, temperature variation and cryoprotectant selection on survival, fertilization potential and blastocyst development of warmed human oocytes

Human oocytes have been successfully cryopreserved using both conventional freezing and vitrification protocols. However, regardless of the method used, post-thaw results have been inconsistent and disappointing, limiting clinical application of oocyte cryopreservation. We examined the effect of pre-vitrification oocyte zona slitting, temperature variation and the effect of two different cryoprotectants (DMSO and EG) on post-warmed oocyte viability, subsequent fertilization potential and blastocyst development.

Comparison of two different cryoprotectant formulations for oocyte vitrification

Studies have suggested that using a combination of cryoprotectants (CPA) at lower concentrations can lower the toxicity of vitrification solutions and yield better overall results. In this investigation we compared oocyte vitrification protocols with either ethylene glycol (EG) alone or else in combination with dimethylsulfoxide (DMSO).

S 3 vitrification: a safe, simple, and successful method for blastocyst vitrification

Vitrification is becoming an established way of storing embryos, but a safe, easy, and effective procedure has eluded researchers due to shortcomings of current techniques. We examined the effectiveness of a different vitrification method for human blastocysts. Current vitrification protocols, although successful, have shortcomings making them difficult to use effectively. Problems include use of dimethylsulfoxide, a relatively toxic cryoprotectant; micro-sized containers that are often open to contamination and difficult to manipulate; and short exposure times that are technically difficult leaving no room for error. We devised a different technique (S3 vitrification) that avoids these problems by using a large, sterile, sealable container, and longer exposure and handling times. Blastocysts from IVF patients that consented for freezing or research were vitrified. Also, bovine blastocysts were vitrified. Blastocysts were incubated in 2 solutions with ethylene glycol at 23°C for 5 min each, transferred to a vitrification solution of glycerol and ethylene glycol for 2 min while loading into a sterile 0.25 cc straw, heat-sealed, and vitrified. Straws were thawed for 5 sec in air then 10 sec in 30°C water. Cells were washed for 5 min each through 5 drops of medium with decreasing sucrose (0.85 M to 0 M). Blastocysts were graded and transferred to recipients 0–2 hours post-thaw. Implantation and pregnancy rate were examined. Bovine and human blastocysts (donated for research) were cultured 24 h after thaw and stained to analyze survival of all cells. Blastocyst survival was 92.7%, 57.1% implantation, and a clinical pregnancy of 54.2% (Table 1). Human and bovine blastocysts had a high rate of survival (83.3% n = 24 and 96.1% n = 102, respectively) and intact cells (86.4% n = 721 and 93.6% n = 6775, respectively). TableS3 Blastocyst Vitrification DataClinicThawedIntactReplacedImplantedFHBOngoing/DeliveredPreg/TransferA88 (100)87 (87.5)7 (87.5)74/4 (100)B3330 (90)2713 (48.1)12 (44.4)1211/17 (67.4)Total4138 (92.7)3520 (57.1)19 (54.2)1915/21 (71.4) Open table in a new tab The results show that blastocysts can be vitrified using a safe and simple method. We obtained high survival, pregnancy, and birth rates without dimethylsulfoxide; using a large, sealed container allowing easy manipulation with ample time for exposure and loading. The data suggest that S3 vitrification may be a promising, user-friendly replacement for current techniques.

Effects of vitrification on functional recovery of ovarian tissues

Effects of vitrification on functional recovery of ovarian tissues

Preliminary studies on the vitrification of red sea bream ( Pagrus major) embryos

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream ( Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO + 10% PG + 10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6 ± 16.7% (mean ± S.D.) and 77.8 ± 15.5%, were achieved by the straw vitrifying method (20.5% DMSO + 15.5% acetamide + 10% PG, thawing at 43 °C and washing in 0.5 M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 °C and washing in 0.5 M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.

Osmotic responses and tolerance limits to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes

Oocyte cryopreservation remains a realistic objective, provided that more systematic approaches are applied, such as thorough analysis of the oocyte oolemma permeability to water and diverse cryoprotectants. We prospectively investigated volume changes over time at different temperatures (30 degrees C, 22 degrees C and 8 degrees C) of human metaphase II (MII) oocytes (obtained in stimulated ICSI cycles and matured in vitro from the germinal vesicle stage) when exposed to changes in external osmolality. We also investigated human in vitro matured (IVM) oocytes membrane permeability characteristics at 22 degrees C to 1,2-propanediol (PG) and dimethylsulphoxide (DMSO) and at 30 degrees C, 22 degrees C and 8 degrees C to ethylene glycol (EG), and calculated corresponding oocyte oolemma permeability coefficients (Lp and Pcpa). Furthermore, we investigated the osmotic tolerance limits of IVM oocytes exposed to changes in external osmolality as assessed by their developmental competence during the course of 72 h after ICSI. The results of our studies describe human oocyte membrane permeability coefficients for EG at 30 degrees C (2.85+/-0.15x10(-3) cm/min), 22 degrees C (1.17+/-0.60x10(-3) cm/min) and 8 degrees C (0.37+/-0.15x10(-3) cm/min). Furthermore, at 22 degrees C the EG oolemma permeability coefficient was lower than that of PG and DMSO (1.17+/-0.60x10(-3) cm/min versus 2.15+/-0.70x10(-3) and 1.56+/-0.38x10(-3) cm/min, respectively). Our results also indicate, that human IVM MII oocytes tolerated exposure to solutions in the range of 39-2264 mOsmol/kg H2O as assessed by the oocytes' developmental competence after exposure. The results of the present study may contribute to a better understanding of the biology and cryobiology of human oocytes, and to the design of better and more robust cryopreservation (freezing or vitrification) protocols.

Clinical-pregnancy outcome after vitrification of blastocysts produced from in vitro maturation cycles

This study was conducted to report the clinical-pregnancy outcome after vitrification of the blastocysts produced from in vitro maturation cycles. The survival rate after thawing was 92.0% (92/100). The clinical-pregnancy and implantation rates were 43.8% and 23.6%, respectively. These results suggest that the blastocyst-stage embryos produced from in vitro maturation cycles can be safely cryopreserved through vitrification.

Vitrification of Boer Goat Morulae and Early Blastocysts by Straw and Open‐Pulled Straw Method

The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.

Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles

Effects of isolation and vitrification protocols on follicular survival after warming were examined. Mouse preantral follicles enzymatically or mechanically isolated from ovaries of 12-day-old mice were exposed either to 2 M ethylene glycol (EG) for 2 or 5 min, or to ascending concentrations (0.15 then 0.3 M) of raffinose for 2 or 5 min each (2-2 and 5-5 min). They were then exposed to a vitrification solution (VS) composed of 6 M EG and 0.3 M raffinose for 0.5, 1, or 2 min before vitrification. Mechanically isolated follicles showed higher survival than enzymatically isolated follicles, regardless of periods of exposure to EG or raffinose and subsequent exposure to VS. After 10 days of culture, follicular growth and maturational ability of oocytes derived from vitrified follicles exposed to 2 M EG for 5 min and to VS for 1 min were higher than those from follicles exposed to raffinose solutions for 2-2 min and to VS for 1 min. Histological evaluation revealed that exposure of preantral follicles to raffinose solutions caused cytoplasmic vacuolation in granulosa cells which could be due to cellular shrinkage during dehydration; whereas, exposure to 2 M EG induced morphological alterations in follicles only to a lesser extent.

Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births

Vitrification technology has shown great promise for cryopreservation of human embryos. The majority of this work has been with blastocyst-stage embryos. This report describes initial clinical results following vitrification of human embryos on day 3 of culture at the 6- to 8-cell stage. A total of 236 embryos were cryopreserved on cryoloops using a vitrification protocol. Warmed embryos were cultured until day 5 before transfer to the patient. The post-warming survival rate was 85%. The clinical pregnancy rate was 44% (34/77), and the implantation rate was 20% (40/201). In transfers where at least one warmed embryo reached the blastocyst stage by the day of transfer, the clinical pregnancy rate was 58% (28/48). The cryoloop was an excellent vessel for ultra-rapid cryopreservation of embryos. This study supports the effectiveness of a dimethylsulphoxide/ethylene glycol cryoprotectant combination for vitrification of human embryos at the 6- to 8-cell stage.

Modified vitrification method for cryopreservation of human ovarian tissues

Vitrification is a prospective technology in ovarian tissue cryopreservation, but it is still in an initial stage. This study was conducted to investigate a modified vitrification protocol for human ovarian tissue, which can be used as an alternative to preserve fertility for young women with cancer who have to undergo cytotoxic therapy and sterilization. Ovarian tissue samples were collected from 15 patients and randomly allocated to groups of fresh, vitrification, and conventional slow freezing. A modified carrierless vitrification method was applied. The proportion of morphologically intact follicles in fresh ovarian tissues was compared with that in warmed/thawed tissues. The initial growth of the follicles and the concentrations of estradiol and progesterone were detected to determine the viability and endocrine function of the cryopreserved tissues. The proportion of morphologically intact primordial follicles in the fresh group (97.6%) was significantly higher than that in the other two groups (vitrification group 80.3% and slow-freezing group 72.6%, P < 0.001). In both the vitrification and slow-freezing groups, estradiol and progesterone were secreted continuously during 2-week culture in vitro, the proportion of primary follicles were both significantly increased compared to the fresh group. No statistically significant differences existed between the two groups after cryopreservation in the proportion of both primordial and primary follicles, and the concentrations of estradiol and progesterone (P > 0.05). The modified vitrification method for cryopreservation of human ovarian tissues is effective, simple, and inexpensive.

Maintenance of the meiotic spindle during vitrification in human and mouse oocytes

Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37°C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21–22°C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37°C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37°C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37°C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37°C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.