Bulk Vitrification Research Articles

Bulk vitrification of in vitro produced bovine zygotes without reducing developmental competence to the blastocyst stage

Cryopreservation of mammalian zygotes can be advantageous since it enables their flexile use in time and space for alternative purposes such as genome editing. Here we report a simple, quick and inexpensive vitrification protocol for in vitro produced bovine zygotes which enables their bulk preservation. Slaughterhouse-derived oocytes were subjected to in vitro maturation and fertilization (IVF). Ten h after IVF, cumulus-enclosed zygotes were equilibrated in 2% (v/v) ethylene glycol + 2% (v/v) propylene glycol for 13–15 min then vitrified in groups of 52–100 in 2 μL microdrops of 17.5% (v/v) ethylene glycol + 17.5% (v/v) propylene glycol supplemented with 0.3 M sucrose and 50 mg/mL polyvinylpyrrolidone. The presence of cumulus cells is important for the success of the process. Therefore, we applied a modified IVF protocol using a short (30 min) co-incubation interval which allowed zygote culture with attached cumulus cells until vitrification and even reduced polyspermy rates without affecting the total fertilization rate. Vitrified zygotes were similar to their non-vitrified counterparts in terms of survival, post-warming development to the blastocyst stage and blastocyst quality measured by cell numbers and cryo-survival. In conclusion, our vitrification protocol integrated with the modified IVF system enabled the quick cryopreservation of bovine zygotes in large groups without reducing their developmental competence to the blastocyst stage.

Vitrification and increase of basicity in between ice Ih crystals in rapidly frozen dilute NaCl aqueous solutions.

The freezing of ionic aqueous solutions is common in both nature and human-conducted cryopreservation. The cooling rate and the dimensions constraining the solution are known to fundamentally influence the physicochemical characteristics of the sample, including the extent of vitrification, morphology, and distribution of ions. The presence of some salts in an aqueous solution often suppresses the ice crystallization, allowing bulk vitrification during relatively slow cooling. Such a process, however, does not occur in NaCl solutions, previously observed to vitrify only under hyperquenching and/or in sub-micrometric confinements. This work demonstrates that, at freezing rates of ≥100 K min-1, crystallized ice Ih expels the freeze-concentrated solution onto the surfaces of the crystals, forming lamellae and veins to produce glass, besides eutectic crystallization. The vitrification covers (6.8% ± 0.6%) and (17.9% ± 1.5%) of the total eutectic content in 0.06M and 3.4 mM solutions, respectively. The vitrified solution shows a glass-to-liquid transition succeeded by cold crystallization of NaCl · 2H2O during heating via differential scanning calorimetry. We establish that ice crystallization is accompanied by increased basicity in freeze-concentrated solutions, reflecting preferential incorporation of chloride anions over sodium cations into the ice. After the sample is heated above the glass transition temperature, the acidity gradually returns towards the original value. The morphology of the samples is visualized with an environmental scanning electron microscope. Generally, the method of vitrifying the freeze-concentrated solution in between the ice Ih crystals via fast cooling can be considered a facile route towards information on vitrified solutions.

Cryopreservation of Human Embryonic Stem Cells with a New Bulk Vitrification Method1

To increase the manipulation efficiency and storage capability of vitrified human embryonic stem cells, a new bulk vitrification method was established using transformed cryovials. This method vitrified a large number of cell clumps, as opposed to those cryopreserved by a slow-freezing method with conventional cryovials at one time (round). After warming, vitrified human embryonic stem cells exhibited a much higher survival rate than the slow-freezing cells. The vitrified stem cells continued to express markers of pluripotency and formed teratomas in mice with severe combined immunodeficiency, confirming the pluripotency of vitrified-warmed human embryonic stem cell clumps. The new bulk vitrification method is superior to and more practical than the open pulled straw vitrification method and the slow-freezing method for the cryopreservation of human embryonic stem cells.

Vitrification of human embryonic stem cells with cryovials

OBJECTIVE: The traditionally used vitrification method is no longer suitable for the vigorously proliferating human embryonic stem cells (HES) because of mini vitrification carriers. Recently we have reported a bulk vitrification (BV) method with cell strainer, which could cryopreserve a large quantity of HES cell clumps at one time. Since the cell strainer is not specially designed for cell and tissue cryopreservation, it is still not convenient to store cell strainers. Moreover, a cell strainer is unnecessarily big for vitrification.

Pretreatment of Hanford Medium-Curie Wastes by Fractional Crystallization

Acceleration of the schedule for decontamination of the Hanford site using bulk vitrification requires implementation of a pretreatment operation. Medium-curie waste must be separated into two fractions: one is to go to a waste treatment and immobilization plant and a second, which is low-activity waste, is to be processed by bulk vitrification. The work described here reports research on using fractional crystallization for that pretreatment. Sodium salts are crystallized by evaporation of water from solutions simulating those removed from single-shell tanks, while leaving cesium in solution. The crystalline products are then recovered and qualified as low-activity waste, which is suitable upon redissolution for processing by bulk vitrification. The experimental program used semibatch operation in which a feed solution was continuously added to maintain a constant level in the crystallizer while evaporating water. The slurry recovered at the end of a run was filtered to recover product crystals, which were then analyzed to determine their composition. The results demonstrated that targets on cesium separation from the solids, fractional recovery of sodium salts, and sulfate content of the recovered salts can be achieved by the process tested.

Nuclear Waste Glasses: Continuous Melting and Bulk Vitrification

This contribution addresses various aspects of nuclear waste vitrification. Nuclear wastes have a variety of components and composition ranges. For each waste composition, the glass must be formulated to possess acceptable processing and product behavior defined in terms of physical and chemical properties that guarantee that the glass can be easily made and resist environmental degradation. Glass formulation is facilitated by developing property-composition models, and the strategy of model development and application is reviewed. However, the large variability of waste compositions presents numerous additional challenges: insoluble solids and molten salts may segregate; foam may hinder heat transfer and slow down the process; molten salts may accumulate in container refractory walls; on cooling, the glass may precipitate crystalline phases. These problems need targeted exploratory research. Examples of specific problems and their possible solutions are discussed.

Bulk vitrification of human embryonic stem cells

The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method. HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing. Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency. This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.

Preparation and Properties of Hydroceramic Waste Forms Made with Simulated Hanford Low‐Activity Waste

Approximately 85 million gallons of high‐level waste (HLW) is currently stored in underground tanks at the Hanford Reservation and the Savannah River Site (SRS). The waste consists of a hydroxide‐rich precipitate (sludge) and a sodium‐rich supernate. The supernate is a NaOH rich solution containing lesser amounts of NaNO3 and NaNO2 and small amounts of soluble fission products, cladding materials, and organics (volatile organics and semi‐volatile organics known as VOCs and SVOCs). The Department of Energy (DOE) has chosen glass as its waste form for both sludge and sodium‐rich supernate. However, because of the volume of the supernate, alternatives to vitrification are being sought for some of this waste. One alternative is to remove 137Cs and 90Sr from the supernate. Decontaminating the waste in this way allows the waste to be designated as low‐activity waste (LAW) and as such the waste now becomes eligible for solidification and disposal on site. SRS is solidifying its LAW with a blended Portland cement forming Saltstone. Hanford has been considering a bulk vitrification process in which the LAW will be mixed with Hanford soil and vitrified in place in a disposable carbon‐arc powered glass melter/waste container. Both waste forms can then be buried on site in appropriate vaults or low‐level waste land fills. A hydroceramic is an alternative waste form designed to solidify and stabilize LAW that is made from metakaolin plus NaOH and/or NaOH rich LAW supernate. In addition to NaOH, LAW can contain a wide range of sodium nitrate and sodium nitrite concentrations. Although a hydroceramic waste form can be made directly from some types of decontaminated waste, e.g., those that are highly alkaline (8–12M NaOH) and contain less than 25 mol% of NOx (NOx is used as the short‐note for nitrates and nitrites in this article.) relative to the total Na in the waste, by simply mixing the LAW with metakaolin and curing the resulting paste at 90°C, the remaining LAW, especially that stored at Hanford must be pretreated in some way before it can be similarly solidified; the relative molar proportion of NOx/Na must be reduced to 25% or less. In this paper calcination is evaluated as a potential pretreatment method for Hanford AN‐107 (AN‐107 is a waste storage tank on Hanford site) LAW, but in choosing this method it is necessary to divide the preparation of the hydroceramic waste form into two steps: denitration/denitrition of the liquid waste stream to produce a granular calcine followed by solidification using a metakaolin plus 4M NaOH binder. A simulated Hanford AN‐107 LAW was calcined at 375°, 450°, 525°, 600°, and 675°C in the presence of sucrose and metakaolin added as a calcination aid. It was shown that the leachability of the calcines decreased as calcination temperature increased, i.e., the waste form became more crystalline. In the second step, each of the granular calcines was mixed with additional metakaolin and just enough 4M NaOH to form a thick paste. The paste was precured at 40°C and then autoclaved at 90°C to form a monolith. X‐ray diffraction and scanning electron microscopy characterization showed that the calcines themselves contained an amorphous phase and crystalline hydroxysodalite, and that the hydroceramics made from these calcines plus additional metakaolin/NaOH binder consisted predominantly of zeolite A and hydroxysodalite. The temperature used to prepare the calcines not only affected the properties of the calcines, but those of the monolithic hydroceramics as well. Experimental results demonstrated that 525°C represented the optimal temperature for producing the most suitable calcine for subsequent solidification with metakaolin and 4M NaOH. The resulting hydroceramic nuclear waste form was strong and had the lowest overall leachability. The leachability of the hydroceramic is normally lower than that of the corresponding calcines up to ∼600°C. The product consistency test (PCT) determined normalized release rate NRNa for the hydroceramic (0.14 g/m2·day) was comparable to similar leach rates determined for Hanford's low‐activity waste reference material glass (0.08 g/m2·day) and a steam reformed calcine made with Hanford's AN‐107 tank waste (0.25 g/m2·day).