Cryopreservation of sugarcane somatic embryos

In this paper, we compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos. Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium. Droplet-vitrification was the most efficient technique. Optimal conditions included loading with a solution containing 1.5 M glycerol and 0.3 M sucrose for 30 min at 25 degree C, treatment with the PVS2 solution for 20-40 min at 0 degree C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution. Under such conditions, viability of cryopreserved somatic embryos reached 55 percent.     

Effect of cryopreservation protocols on the phenotypic stability of yeast

Eight cryopreservation protocols were assessed for their effects on the viability and phenotypic stability of the yeast Saccharomyces cerevisiae during a five-year study. It is found that viability and phenotypic features have remained largely unchanged when the yeast was preserved in glycerol, dimethyl sulphoxide, or sucrose at -80 degrees C or in liquid nitrogen. When sorbitol was used as a cryoprotectant, yeast cells frozen and stored at -80 degrees C manifested great decreases in viability after six months in storage and concomitantly large fluctuations in the rate of the trpl auxotrophic reversion. This phenotypic reversion was stable passage after passage. Such a degree of phenotypic fluctuations, however, was not observed for yeast cells preserved in the same sorbitol solution that went through a controlled freezing program and were subsequently stored in liquid nitrogen. These results indicate that some combinations of cryoprotective agent, freezing program, and storage temperature disturb biomaterials more profoundly during cryopreservation and imply a genetic basis of this phenotypic change.     

Mushroom cryopreservation and its effect on survival, yield and genetic stability

Mycelial stock cultures of Agaricus bisporus, A. Bitorquis, Pleurotus flabellatus, P. Sajor-caju, P. Ostreatus, P. Sapidus, Auricularia polytricha, Lentinula edodes, Morchella esculenta and Volvariella volvacea were maintained by frequent subculturing at an interval of two months and separately as wheat grain spawn in liquid nitrogen with 15 percent glycerol. Preservation of mushroom stock cultures as wheat grain spawn under liquid nitrogen proved to be the better method of maintenance. The percent recoveries of stored samples were unchanged from the first recovery after six months to the last recovery after 42 months in nine out of 11 stock cultures preserved under liquid nitrogen. However, a marginal decline in survival of 10 % was recorded in Auricularia polytricha and Volvariella volvacea. Yields before preservation of mushroom stock cultures and after 30 months of preservation exhibited static biological efficiency and fruitbody weight. The comparison of Random Amplified Polymorphic DNA (RAPD) and Internal Transcribed Spacers (ITS) PCR amplified products did not exhibit DNA fragment variation in banding patterns at the intraspecific level during preservation of stock cultures by either method. The modified cryopreservation protocol and experimental demonstration of genetic stability of stock cultures reported here validate the use of mushroom cryopreservation techniques and supports studies on genetic stability of preserved biological materials.     

Cryopreservation of shoot tips of blackberry and raspberry by encapsulation-dehydration and vitrification

Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.     

Cryopreservation of basal stem buds of in vitro-grown mat rush (Juncus spp.) by vitrification

An optimal protocol for the cryopreservation of in vitro-grown mat rush (igusa) buds by vitrification has been successfully developed. Established multiple stemmed cultures, which were induced in liquid MS medium containing 8.9 microM BA by roller culture, were cut into small clumps, plated on solid MS medium and cultured for three weeks at 25 degree C. Clumps that grew many buds were cold-hardened at 5 degrees C, with an 8 h photoperiod, for more than 30 d. The basal stem bud (1 to 2 mm long) was dissected from the clumps and precultured at 5 degrees C for 2 d on solid MS medium containing 0.3 M sucrose. The precultured buds were placed in 2 ml plastic cryotubes and osmoprotected with 1 ml loading solution containing 2 M glycerol and 0.6 M sucrose for 30 min at 25 degree C. Then they were dehydrated in 1 ml PVS2 solution at 25 degree C for 30 min and immersed in liquid nitrogen. Using this protocol, the survival level of cryopreserved igusa 'NZ219' buds reached 87 percent. This protocol was successfully applied to 42 different lines from three Juncus species, which had relatively high survival levels ranging from 30 to 90 percent and an average of 63 percent.     

Improved cryopreservation of chrysanthemum (Chrysanthemum morifolium) using droplet-vitrification

A droplet-vitrification protocol has been established for cryopreserving Chrysanthemum morifolium cv. Peak using axillary shoot tips and apical shoots of in vitro plants. In the optimized procedure, explants were submitted to a step-wise preculture in liquid sucrose-enriched medium (0.3, 0.5 and 0.7 M for 31,17 and 7 h, respectively). Precultured explants were treated for 40 min with C4 loading solution comprising (w/v) 17.5 percent glycerol + 17.5 percent sucrose, then dehydrated with PVS3 vitrification solution (w/v, 50 percent glycerol + 50 percent sucrose) for 60 min (axillary shoot tips) or 90 min (apical shoots). Explants were cryopreserved by direct immersion in liquid nitrogen in minute drops of PVS3 attached to aluminum foil strips. The optimal age of donor plants was 4-5.5 weeks for apical shoots and 7 weeks for axillary shoot tips, producing post-cryopreservation regeneration percentages of 81.9 percent and 84.9 percernt, respectively. Plants regenerated from cryopreserved samples showed no phenotypical abnormalities and similar profiles of relative DNA content were recorded for control and cryopreserved plants. Our results suggest that the modified droplet-vitrification protocol described in this paper is highly effective and may prove user-friendlier than the cryopreservation protocols already published for chrysanthemum.     

Studies on the toxicity of dimethyl sulfoxide, ethylene glycol, methanol and glycerol to loach (Misgurnus fossilis) sperm and the effect on subsequent embryo development

The process of sperm cryopreservation consists of several steps: equilibration of sperm in cryoprotectant medium, freezing of sperm to subzero temperatures, low temperature storage and thawing of the sperm suspension. It has been shown that cryopreservation can cause some damage to the genetic material of cells although the mechanism and significance of these changes are still unknown. The aim of this work was to study the effect of cryoprotectant equilibration process on genetic damage of Loach (Misgurnus fossilis) sperm, using embryo survival as an indicator. Decrease in embryo survival after the 20th stage is generally believed to result from the failure in the genome function of embryos. In the first set of the experiments, Loach sperm were equilibrated in cryoprotectants Me2SO, ethylene glycol, methanol and glycerol (0.6, 1.2, 2.5 M) for 60 min at 10 degree C. The effect of cryoprotectant equilibration on sperm was evaluated based on the survival of embryos derived from cryoprotectant treated sperm. Embryo survival was evaluated at the following stages: 7th, 14th, 17th, 20th, 23rd, 26th, 31st, 34th, 35th, 36th and 37th. Cryoprotectants at concentrations greater than 1.2 M had significant effect on the survival of the embryos after the 20th stage. The effect of glycerol was the most significant with 64.8 +/- 2.4% of embryos survival compared to 77.0 +/- 2.4% for control. Me2SO treatment also effects embryo survival significantly. Possible mechanisms of the genetic instability of cryoprotectants are discussed.     

Cryopreservation of in vitro shoot apices of Oxalis tuberosa Mol

Oca (Oxalis tuberosa Mol.) is an under-utilized tuber crop from the Andean region. Cryopreservation would allow the safe and long-term preservation of the genetic resources of this crop. A protocol for the cryopreservation of in vitro grown shoots has been developed using the vitrification solution PVS2. Two genotypes were studied (G1 and G27). Nodal segments were cultured on MS medium and incubated at 10 degree C with 16 h photoperiod and 10 mol per square meter per second irradiance, for two weeks. Apices were then excised and cultured on MS+0.15 M sucrose for 3 days at 5 degree C in darkness. Subsequently, apices were immersed in a loading solution (liquid MS medium+2 M glycerol+0.4 M sucrose), and then treated with the vitrification solution PVS2 for 0 to 40 minutes. Cryovials were then immersed in liquid nitrogen. Four weeks after rewarming and culture on recovery medium, genotype G1 showed approximately 60 percent recovery (normal growth) with 20 min PVS2 treatment. Genotype G27 showed lower recovery (30 percent). Differential scanning calorimetry yielded a Tg midpoint for PSV2 solution of ca. -120 degree C. Calorimetric studies on apices at different stages of the cryopreservation protocol showed a change in calorimetric parameters consistent with a decrease in the amount of frozen water as the protocol advanced.     

Cryopreservation of embryogenic cultures from mature Quercus suber trees using vitrification

Recent progress in somatic embryogenesis from selected mature trees of Quercus suber, has led to a demand for maintenance of a large number of selected embryogenic lines. To facilitate the management of this material a protocol for the long-term storage of this germplasm should be defined. This study reports on the use of a simple vitrification procedure for the successful cryopreservation of three cork oak embryogenic lines. High embryo recovery levels (88-93 percent) were obtained by first preculturing 2-4 mg clumps of two or three globular embryos on semisolid medium containing 0.3 M sucrose for three days, followed by incubation in PVS2 vitrification solution at 0 degree C for 60 min before direct immersion in liquid nitrogen. The mean number of embryos produced per explant was significantly greater for cryostored embryos than for untreated stock cultures, but the productivity of the latter was recovered in subsequent subcultures of the material produced by cryostored embryos. The germination and plant regeneration rates achieved by cultures derived from cryostored embryos, around 60 percent, were similar to those of non-cryopreserved stock cultures.     

Studies on cryoprotectant toxicity to zebrafish (Danio rerio) ovarian tissue fragments

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but it has not been studied in fish. Selection of cryoprotectant is an important step in designing cryopreservation protocols. In order to identify the optimum cryoprotectant (CPA) in a suitable concentration for zebrafish ovarian tissue cryopreservation, studies on toxicities of five commonly used cryoprotectants methanol, ethanol, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were carried out. Experiments were conducted on ovarian tissue fragments consisting of stage I and stage II ovarian follicles. Ovarian tissue fragments were incubated in 90% L-15 medium (pH 9) containing 1-4M cryoprotectants for 30min at 22°C. Three different tests were used to assess ovarian tissue fragment viability: trypan blue (TB) staining, fluorescein diacetate (FDA) combined with propidium iodide (PI) staining and adenosine 5′- triphosphate (ATP) assay. Results from these tests showed that ATP assay was more sensitive than FDA+PI or TB staining for assessing cryoprotectant toxicity to follicles in tissue fragments. Methanol and ethanol were the least toxic cryoprotectants tested. Cryoprotectant toxicity increased in the order of methanol/ethanol, DMSO, PG and EG. Ethanol was used for zebrafish ovarian tissue for the first time and the results showed that the effect of methanol and ethanol on ovarian tissue fragments were comparable. As methanol has been shown to be the most effective cryoprotectant for zebrafish ovarian follicles in our laboratory, the use of ethanol will also be considered in assisting future freezing protocol design. The present study also showed that stage II ovarian follicles are more sensitive to cryoprotectant treatment than stage I follicles in tissue fragments. The results obtained in this study provided useful information for ovarian tissue fragment cryopreservation protocol design in the future.     

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells (UCB-derived MSCs) is crucial step for its clinical applications in cell transplantation therapy. In the cryopreservation of MSCs, dimethyl sulfoxide has been widely used as a cryoprotectant (CPA). However, it has been proved that DMSO has toxic side effects to human body. In this study, DMSO-free CPA solutions which contained ethylene glycol (EG), 1, 2-propylene glycol (PG) and sucrose as basic CPAs, supplemented with polyvinyl alcohol (PVA) as an additive, were developed for the cryopreservation of UCB-derived MSCs. The cryopreservation of UCB-derived MSCs was achieved by vitrification via plunging into liquid nitrogen and by programmed freezing via an optical-DSC system respectively. The viability of thawed UCB-derived MSCs was tested by trypan blue exclusion assay. Results showed that the viability of thawed UCB-derived MSCs was enhanced from 71.2% to 95.4% in the presence of PVA for vitrification, but only < 10% to 45% of viability was found for programmed freezing. These results indicate that PVA exerts a beneficial effect on the cryopreservation of UCB-derived MSCs and suggest the vitrification in combination with the dimethyl sulfoxide free CPA solutions supplemented with PVA would be an efficient protocol for the cryopreservation of UCB-derived MSCs.     

Optimising cryopreservation protocols for haematopoietic progenitor cells: a methodological approach for umbilical cord blood

Current cryopreservation protocols for haematopoietic cells have developed largely empirically and there is no consensus on an optimal method of preservation. These protocols, though providing sufficient cells to permit engraftment, can lead to cell loss of the order of 50 percent. In the context of umbilical cord blood such losses are unacceptable. Whilst an empirical approach can provide an acceptable level of recovery, the cryopreservation process can only be optimised by adopting a methodological approach. This paper provides an overview of just such an approach as illustrated by a study on CD34 cells from umbilical cord blood. It involves firstly the determination of membrane permeability parameters that can then be used to model safe addition and elution protocols for the chosen cryoprotectant, in this case dimethyl sulphoxide. This in turn permits cryoprotectant toxicity to be evaluated free from the confounding effect of osmotic damage caused by inappropriate addition and elution protocols. Finally, non-toxic concentrations of cryoprotectant may be investigated in a cooling rate study to provide an optimal cryopreservation protocol. Using the model, the effect on CD34 cells of current addition and elution protocols was also examined.     

Recovery and characterisation of hybrid firs (Abies alba x A. cephalonica, Abies albax A. numidica) embryogenic tissues after cryopreservation

Embryogenic tissues of hybrid firs were cryopreserved using a slow freezing protocol. The procedure involved preculture of tissues for 24, 48 or 72 h in media with different sorbitol concentrations (0.4 or 0.8 M) and addition of 5% (v/v) DMSO as cryoprotectant. The cell lines tested withstood cryopreservation, even though tissue regrowth after thawing was dependent on treatment and cell line. For cell line AN72, regrowth was 100% for all experimental conditions tested. With cell line AC78, regrowth was 100% except after shorter pretreatment durations, which produced 83% and 86% regrowth for 0.4 M and 0.8 M sorbitol pretreatment, respectively. Cell lines AC1 and AC4 were more sensitive to cryopreservation with 37.5 to 100% regrowth, respectively. Growth parameters evaluated 3 months after cryopreservation showed cell line and treatments effects. In most cases, cryopreservation had no negative effect on growth of tissues. Statistically significant differences in fresh mass accumulation were found for four samples out of 24 investigated, although growth increase of these tissues still reached 79.4-84.6%, compared with non-cryopreserved ones (100% increase). Maturation capacity and genetic fidelity were studied in tissues whose growth was not negatively influenced by cryopreservation. Maturation capacity of embryogenic tissues cryopreserved using the optimal protocol was comparable to that of non-frozen controls. RAPD analysis of 88 genomic regions per cell line did not reveal any changes in genetic fidelity of cryopreserved tissues compared to non-cryopreserved controls.     

Cryopreservation of adult bovine testicular tissue for spermatogonia enrichment

To develop a procedure for cryopreservation of adult bovine testis tissue, the effects of dimethyl sulphoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), and their concentrations (v/v), as well as different thawing temperatures, on the cell viability of bovine testis tissue after freezing/thawing were examined. The highest testicular cell viabilities came from the media containing DMSO (85.3 ± 1.2 percent), PG (82 ± 1.0 percent) and EG (83.4 ± 1.0 percent) at 10 percent concentration respectively. Using 10 percent DMSO gave significantly higher spermatogonia percentage (61.1 ± 1.2 percent, P < 0.001) than processing with 10 percent PG (54.3 ± 0.6 percent) or 10 percent EG (55 ± 1.8 percent) after differential plating. Thawing in water bath of 37 or 97-100 degree C also provided significantly higher viabilities (85.1 ± 1.0, 85 ± 1.0 percent, P < 0.01, respectively) and spermatogonia percentages (56.6 ± 2.0, 56.6 ± 2.6 percent, P < 0.01, respectively) than that thawing at 4C (23.4 ± 0.8 percent for total viability, 8.97 ± 1.0 percent for spermatogonia percentage). Collectively, 10 percent DMSO and thawing in 37-100 degree C water baths were appropriate for the cryopreservation of bovine testicular tissue and subsequent spermatogonia enrichment.     

Implementation of garlic cryopreservation techniques in the national plant germplasm system

The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 200 Allium sativum (garlic) accessions at the Western Regional Plant Introduction Station in Pullman, WA. All accessions must be grown out in the field annually since garlic plants from these accessions do not reliably produce seeds and bulbs do not store well. Shoot tips excised from garlic cloves can be successfully cryopreserved using either Plant Vitrification Solution 2 (PVS2; 15 percent v/v DMSO, 15 percent v/v ethylene glycol, 30 percent v/w glycerol, 0.4 M sucrose) or Plant Vitrification Solution 3 (PVS3; 50 percent v/w sucrose, 50 percent v/w glycerol). We compared regrowth of shoot tips representing diverse garlic germplasm after exposure to either PVS2 or PVS3 during the cryopreservation procedure. At the USDA-ARS National Center for Genetic Resources Preservation, a component of the NPGS, we consider accessions successfully preserved if a minimum of 40 percent of explants exhibit regrowth after liquid nitrogen exposure and at least 60 viable shoot tips remain in long-term storage. Ten of twelve diverse garlic accessions were successfully cryopreserved using either PVS2 or PVS3 as cryoprotectants. Five genotypes had the best post liquid nitrogen regrowth after exposure to PVS2, four genotypes had the best regrowth after exposure to PVS3, and three genotypes performed equally well using either cryoprotectant solution. This project is part of an ongoing program to cryopreserve accessions of NPGS clonal crop collections.     

High viability of dormant Malus buds after 10 years of storage in liquid nitrogen vapour

Three hundred and sixty two Malus accessions from the Canadian Clonal Genebank of Plant Gene Resources of Canada were cryopreserved as dormant buds at the USDA-ARS National Center for Genetic Resources Preservation in 1996. According to grafting data collected on 165 of these accessions in 1999, 80 percent of the accessions had at least 40 percent viability. A subsample of these accessions was processed for cryopreservation by either adjusting the moisture content of the budwood sections containing dormant buds to 32 or 37 percent moisture (fresh weight basis) or by not drying the budwood sections (46 percent moisture fresh weight basis) prior to cooling. Budwood sections were then slow-cooled at 1 degree C per hour to -3 degree C, held for 24 h at -30 degree C and then rapidly transferred to the vapour phase of liquid nitrogen. Cryopreserved buds from 13 accessions that were dried using the various techniques were warmed and grafted in both 1999 and 2006 to determine viability. Overall, bud viability was high at both storage times. At the 10 year time point, some accessions had higher bud growth when they were desiccated prior to slow-cooling when compared to those that were not.     

Improvement of cryopreservation results in garlic using low temperature preculture and high-quality in vitro plantlets

The efficiency of garlic cryopreservation is, amongst other factors, depending on the origin of the donor explants. So far, in vitro grown material has always been the least responding one with respect of the regrowth rates. On the other side, the possibility to produce virus-free material via meristem culture and to keep these clones then under isolated conditions in a clean culture induced studies to increase the efficiency of cryopreservation using this kind of material. Experiments have been performed to use various materials and cultivation temperatures for a vitrification protocol. Best results (up to 70 percent regrowth) were obtained with cultures grown for only 10 months under in vitro conditions including a cold preculture of two months either at alternating or at permanently low temperatures. The conclusion was drawn that the quality of the explants and temperature conditions play a major role for the efficiency of cryopreservation using in vitro plantlets.     

Optimization of cryopreservation of stem cells cultured as neurospheres: comparison between vitrification, slow-cooling and rapid cooling freezing protocols

We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a straw-in-straw technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (>96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.     

Cryopreservation of Vanda coerulea protocorms by encapsulation-dehydration

Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2 percent Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 +/- 3 degree C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 +/- 3 degree C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40 degree C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40 percent was observed with cryopreserved beads with 35 percent water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.     

Cryopreservation of in vitro-grown shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree

Cryopreservation of in vitro axillary shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree, was investigated. Axillary buds (c. 1mm in length) excised from 4-week-old in vitro cultures, were pre-cultured on liquid MS medium supplemented with 0.4 M sucrose for 16 h. These were incubated in 2 M glycerol+0.4 M sucrose for 20 min at 25 degree C before being dehydrated with PVS2 solution for 40 min The dehydrated shoot tips were directly immersed in LN. Following cryopreservation and after rapid warming at 40 degree C, shoot tips were quickly washed with MS+1.2 M sucrose solution for 20 min and then plated on top of filter paper placed on MS medium supplemented with 0.1 mg l-1 BAP, kept in darkness for one day followed by placement of shoots directly on the medium and incubation in darkness for a day more, before transfer of cultures to light. Average survival in terms of normal shoot formation after 4 weeks of plating was 56.6 percent. The rescued shoot tips were bulked up by subsequent nodal cultures and when put onto 0.02 mg l-1 NAA showed a rhizogenic response. Thus, in vitro-grown shoot tips of Crateva nurvala were successfully cryopreserved following the optimization of the PVS2-vitrification protocol.