Abstract: The target of any cryopreservation procedure should be to ensure high survival rates of living cells after thawing. Two important parameters determine the success of any cryopreservation protocol: the manner in which cells regain equilibrium in response to cooling, and the speed of freezing (cooling rate). Slow-rate freezing protocols result in the formation of ice crystals during cooling and warming. Vitrification, in which high cooling rates in combination with a high concentration of cryoprotectant are used, does not produce any ice crystals during cooling and warming. However, there is a practical limit to the attainable cooling speed, and also a biological limit to the concentration of cryoprotectant tolerated by the cells during vitrification. Although post-warming survival depends on the species, the developmental stage and the quality of the embryos being vitrified, it seems clear that vitrification methods are increasingly successful and might be a better method than slow cooling procedures in the field of cryobiology. Many of the potential problems and benefits underlying vitrification as a method of choice for embryo cryopreservation in clinical embryology will be discussed in this review.

Abstract: Shoot tips of sweet potato were successfully cryopreserved using an encapsulation vitrification method. Encapsulated shoot tips were pre-incubated in liquid Murashige-Skoog medium containing 30 g/l sucrose for 24 h, then precultured in sucrose-enriched medium (0.3 M sucrose) for 16 h. Shoot tips were osmoprotected with a mixture of 2 M glycerol and 1.6 M sucrose for 3 h before being dehydrated with a highly concentrated vitrification solution (PVS2) for 1 h at 25°C. The encapsulated and dehydrated shoot tips were transferred to a 2 ml cryotube, suspended in 0.5 ml PVS2, and plunged directly into liquid nitrogen. Rapidly warmed shoot tips developed normal shoots and roots in 21 days without any morphological abnormalities after plating on a recovery medium. High levels (average of about 80%) of shoot formation were obtained for three cultivars of sweet potato. This encapsulation vitrification method appears promising for cryopreservation of sweet potato germplasm.

Abstract: A method for vitrification of a tissue or organ includes immersing the tissue or organ in increasing concentrations of cryoprotectant solution at a temperature greater than −15° C. to a cryoprotectant concentration sufficient for vitrification; cooling the tissue or organ at an average rate of from 2.5-100° C. per minute to a temperature between −80° C. and the glass transition temperature; and further cooling the tissue or organ at an average rate less than 30° C. per minute to a temperature below the glass transition temperature to vitrify the tissue or organ. After the vitrified tissue or organ has been stored, the tissue or organ may be removed from vitrification by warming the tissue or organ at an average rate of from 20-40° C. per minute to a temperature between −80° C. and the glass transition temperature; further warming the tissue or organ at a rate of from 200-300° C. per minute to a temperature above −75° C.; and reducing the concentration of the cryoprotectant. Tissues or organs treated in this manner exhibit near normal functions, for example, blood vessels exhibit near normal smooth muscle contractility and normal graft functions.

Abstract: The aim of the present study was to evaluate the effect of different vitrification protocols on reactive oxygen species (ROS) and apoptosis in human ovarian tissue. Human ovarian tissue pieces were exposed to different vitrification solutions. The intracellular redox state level was measured using the fluorescent dye dichlorodihydrofluorescein diacetate. Imaging of apoptotic cells was monitored by anti-caspase-3 immunolabelling after vitrification and warming. Following equilibration in either 40% ethylene glycol (EG) (v/v), 0.35 M sucrose + 10% egg yolk extract (v/v) or 40% EG (v/v), 18% Ficoll-70 (w/v) + 0.35 M sucrose for 6 min, ovarian pieces were cooled to -196 degrees C using four different protocols. Tissue that was cooled very rapidly (plunged directly into liquid nitrogen in straws or on grids or plunged directly into metal filings precooled to -196 degrees C) showed no statistically significant increase in either tissue ROS levels or the number of apoptotic cells after warming. In contrast, cooling using a less rapid method (nitrogen vapour at -120 degrees C) resulted in significantly elevated ROS levels and apoptosis after warming. There were no significant differences between the two vitrification solutions. This indicates that human ovarian tissue pieces should be vitrified using very rapid cooling rates.

Abstract: Toxic lead-rich solid industrial wastes were stabilized by the vitrification method. Vitrification was attained by the addition of SiO 2 and Na 2 O as vitrifying and melting agent, respectively. The non-toxic, homogeneous, vitreous products studied in the present work, contain 60 wt.% of solid waste. Products with such a high content of solid waste comprise an economically realistic suggestion, but are easily devitrified in conditions of large-scale production due to the difficulty to achieve rapid cooling conditions in the whole volume of a large piece of stabilized product. Thus, it must be ascertained that the loss of homogeneity is not accompanied with the loss of chemical stability. Differential Thermal Analysis (DTA) was applied in order to inspect the prospect to crystal phase separation. The separated crystal phases were characterized by X-ray diffraction and transmission electron microscopy. Possible devitrification processes are investigated in order to interconnect the microstructure with the chemical stability of the devitrified products.

Abstract: The efficacy of cryopreservation by direct plunging into liquid nitrogen (vitrification) of human pronuclear oocytes using open pulled straws with a super-finely pulled tip, as well as the ultrastructural changes caused by cooling and vitrification, were evaluated. Clinical and electron microscopic studies of cooled and vitrified oocytes were performed. Oocytes were cooled to 4°C in the presence and absence of cryoprotectants, vitrified, warmed, cultured and transferred. Abnormally fertilized oocytes were examined by electron microscopy. Vitrified and warmed 2-pronuclear oocytes showed 71.1% survival rate and 83.3% developmental rate. One- and 3-pronuclear oocytes, after cooling without cryoprotectants (presumably non-viable), showed progressive swelling of mitochondrial smooth endoplasmic reticulum (SER). After vitrification, oocytes (presumably viable) showed the formation of large SER vesicles associated with mitochondria. The described protocol of vitrification of human pronuclear oocytes was shown to be effective in producing pregnancy. Normal ultrastructure after undergoing the described vitrification protocol was confirmed.

Abstract: While human oocytes have been successfully cryopreserved using traditional slow-rate freezing protocols, inconsistent results post-thaw have limited the routine clinical application of oocyte cryopreservation. Despite interest in the potential benefits of vitrification as an alternative laboratory approach to long-term oocyte preservation in assisted reproduction, there is little agreement on how best to configure such cryopreservation protocols to optimize oocyte viability. To comparepost-thaw oocyte survival rates,we performed cryoloop vitrification of human oocytes utilizing two different cryoprotectant mixtures that included polymer macromolecules. Human oocytes (n = 1120) were obtained from consenting patients undergoing in vitro fertilization, but only failed-matured (uninseminated) or failed-fertilized (inseminated but without 2pn development) were included in this investigation. Protocol A consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.4 M sucrose and 20% synthetic serum substitute. Protocol B consisted of 20% ethylene glycol and 20% dimethyl sulphoxide + 0.65 M sucrose, 1 mg/ml polyethylene glycol, 10 mg/ml Ficoll and 20% synthetic serum substitute. Following cryostorage for 10-14 d at -196 degrees C, the survival rate for oocytes vitrified with protocol A was 80.9%, whereas the post-thaw viability among protocol B oocytes was 80.6% (p > 0.005). Our results indicate that an ethylene glycol + dimethyl sulphoxide mixture (with or without polymer macromolecules) can be an effective cryoprotectant strategy for human oocyte vitrification; either approach can be employed without any observed compromise in post-warming survival and/or morphology.

Abstract: BACKGROUND: The purpose of this study was to test the effectiveness of one two-step (A) and two one-step (B1 and B2) vitrification procedures on denuded expanded or hatching rabbit blastocysts held in standard sealed plastic straws as a possible model for human blastocysts. The effect of blastocyst size was also studied on the basis of three size categories (I: diameter <200 μm; II: diameter 200-299 μm; III: diameter ≥300 μm). METHODS: Rabbit expanded or hatching blastocysts were vitrified at day 4 or 5. Before vitrification, the zona pellucida was removed using acidic phosphate buffered saline. For the two-step procedure, prior to vitrification, blastocysts were pre-equilibrated in a solution containing 10% dimethyl sulphoxide (DMSO) and 10% ethylene glycol (EG) for 1 min. Different final vitrification solutions were compared: 20% DMSO and 20% EG with (A and B1) or without (B2) 0.5 mol/l sucrose. RESULTS: Of 198 vitrified blastocysts, 181 (91%) survived, regardless of the vitrification procedure applied. Vitrification procedure A showed significantly higher re-expansion (88%), attachment (86%) and trophectoderm outgrowth (80%) rates than the two one-step vitrification procedures, B1 and B2 (46 and 21%, 20 and 33%, and 18 and 23%, respectively). After warming, blastocysts of greater size (II and III) showed significantly higher attachment (54 and 64%) and trophectoderm outgrowth (44 and 58%) rates than smaller blastocysts (I, attachment: 29%; trophectoderm outgrowth: 25%). CONCLUSIONS: These result demonstrate that denuded expanded or hatching rabbit blastocysts of greater size can be satisfactorily vitrified by use of a two-step procedure. The similarity of vitrification solutions used in humans could make it feasible to test such a procedure on human denuded blastocysts of different sizes.

Abstract: The immobilisation of hazardous (toxic and radioactive) waste using self sustaining vitrification processes is reviewed. Self sustaining vitrification processes utilise the energy released during exothermic chemical reactions in a mixture of waste and powder metal fuel (PMF) to form a glass-like material without requiring an external power supply. The self sustaining vitrification process is controlled by the composition of the initial mixture of waste and PMF. A number of wastes have been successfully vitrified into durable glass-like waste forms. Since they do not require complex equipment or energy supplies, self sustaining vitrification processes are particularly attractive for immobilising relatively small amounts of problematic wastes. Heavy metals (Pb, Hg, Cd, Zn) are known to have adverse effects on human health and are stable and persistent environmental contaminants that cannot be degraded or destroyed. (1) Important waste streams containing heavy metals as contaminants are ashes, foundry and smelter residues, combustion by-products, furnace slag, electro-galvanic residues, and cullet from cathode ray tubes. Radioactive waste is generated from reprocessing of spent nuclear fuel, various applications of radionuclides in hospitals, research activities at universities and institutes and from industrial use of radionuclides. Both chemically toxic and radioactive wastes need a reliable immobilisation route to limit contamination of the environment. Immobilisation of nonradioactive contaminants may enable them to be reused in some industrial applications, e.g. developments in using Zn containing slags as cement additives. However immobilisation of radioactive waste via vitrification is intended to make it safe for long term storage and disposal. Vitrification is one of the most developed of the immobilisation technologies. Vitrification of waste comprises melting or sintering of waste materials with the necessary glass-forming additives so that the final vitreous product incorporates the waste contaminants in its microstructure. Vitrification is particularly attractive because of the high chemical durability of the vitrified glass product. This characteristic has been used by industry for centuries. The chemical resistance of glass can allow it to remain in a corrosive environment for many thousands and even millions of years. Several glasses are found in nature such as obsidians (volcanic glasses), fulgarites (formed by lightning strikes), tektites found on land in Australasia and associated microtektites from the bottom of the Indian Ocean, moldavites from central Europe, and Libyan Desert glass from western Egypt. Some of these glasses have been in the natural environment for up to 300 million years with a very low alteration of only hundredths of a centimetre per million years. (2,3) In addition to its good durability glass has the ability to incorporate a vast range of elements into its structure. Vitrification is especially well suited to wastes that contain heavy metals or radioactive constituents enabling chemical binding of waste metals into the glass. Hazardous constituents from the waste can be immobilised into a vitreous product by two mechanisms: either by direct incorporation into the glass network or encapsulation. In the first case the waste constituents are dissolved in the glass melt and on cooling some of them are incorporated in the glass network, while others are confined as modifiers. Immobilisation by encapsulation is applied to elements and compounds that have low reduced solubility in the glass and thus are unable to be dissolved in the glass network. These are refractory oxides with very high liquidus temperatures such as spinels, sulphates, chlorides and molybdates. They are immobilised by the glass matrix by encapsulation into its structure in the form of a dispersed phase. Encapsulation can be carried out either by sintering or dispersion of insoluble compounds into the glass melt. Vitreous products in the form of both homogeneous glasses (e.g. having a relative small volume fraction of inhomogeneities) and

Abstract: The performance of the vitrification process currently used in the La Hague commercial reprocessing plants has been continuously improved during more than ten years of operation. In parallel COGEMA (industrial Operator), the French Atomic Energy Commission (CEA) and SGN (respectively COGEMA’s R&D provider and Engineering) have developed the cold crucible melter vitrification technology to obtain greater operating flexibility, increased plant availability and further reduction of secondary waste generated during operations. The cold crucible is a compact water-cooled melter in which the radioactive waste and the glass additives are melted by direct high frequency induction. The cooling of the melter produces a soldified glass layer that protects the melter’s inner wall from corrosion. Because the heat is transferred directly to the melt, high operating temperatures can be achieved with no impact on the melter itself. COGEMA plans to implement the cold crucible technology to vitrify high level liquid waste from reprocessed spent U-Mo-Sn-Al fuel (used in gas cooled reactor). The cold crucible was selected for the vitrification of this particularly hard-to-process waste stream because it could not be reasonably processed in the standard hot induction melters currently used at the La Hague vitrification facilities: the waste has a high molybdenum content which makes it very corrosive and also requires a special high temperature glass formulation to obtain sufficiently high waste loading factors (12% in molybednum). A special glass formulation has been developed by the CEA and has been qualified through lab and pilot testing to meet standard waste acceptance criteria for final disposal of the U-Mo waste. The process and the associated technologies have been also being qualified on a full-scale prototype at the CEA pilot facility in Marcoule. Engineering study has been integrated in parallel in order to take into account that the Cold Crucible should be installed remotely in one of the R7 vitrification cell. This paper will present the results obtained in the framework of these qualification programs.Copyright © 2003 by ASME

Abstract: The final disposal of ash from an incinerator is of special concern because of the possibility of its releasing toxic substances. Melting/vitrification has been regarded as a prospective technology of ash treatment. The object of this investigation was to evaluate the effect of silica (SiO2) addition on the immobilization of hazardous metals and the encapsulation of a glass network during the vitrification process. Four specimens with SiO2/fly ash mixing ratios of 0, 0.1, 0.2, and 0.3, respectively, were tested. The mobility of metals in slag was then estimated by a sequential extraction procedure. X-ray diffraction analysis indicates that SiO2 leads to the polymerization of silicates. The encapsulation of aluminum, calcium, and magnesium would not be observed unless adequate amount of SiO2 was added. It was also found that SiO2 addition enhances the formation of a compact and interconnected glass network structure and, thus, contributes to the chemical stability of metals in slag. After vitrific...

Abstract: To improve cryopreservtion of sea perch ( Lateolabrax japonicus ) embryos, we selected five vitrification solutions that could vitrify consistently. The cryoprotectants concentration in the five vitrification solutions were low to reduce toxicity. The vitrification probability of the five solutions was 48 1%-100% during freezing and 44 4%-63 0% during thawing at 35-43℃ water bath, respectively. VSD2 was more stable during thawing than the other four vitrification solutions and was used to cryopreserve sea perch embryos in the experiment. The durable time of sea perch neurula stage, 20 pair muscle stage, tail bud stage, heart beating stage and prehatching stage in VSD2 was compared and the most suitable stage to cryopreserve by vitrification was selected. We found that the neurula stage sustained the least time in VSD2 and the heart beating stage was the most suitable stage to cryopreserve by vitrification. The pre hatching stage was also feasible to cryopreserve by vitrification. The effect of different elution times in 0 5 mol/L sucrose was compared and elution for 10-20min in 0 5 mol/L sucrose was found to be efficient. We used VSD2 as vitrification solution to cryopreserve(-196℃) sea perch embryos of different stages and got 2 1%-27 9% transparent embryos after thawing. Furthermore, two frozen thawed embryos at heart beating survived 42-50 h after thawing. Another frozen thawed embryo at pre hatching stage hatched.

Abstract: The results of investigations of the vitrification of chloride wastes produced during test reprocessing of spent BOR-60 fuel by the pyroelectrochemical method are presented. A method is proposed for converting chlorides into metaphosphates beforehand, which makes it possible to introduce up to 46% wastes into the glass matrix. The optimal composition of the glass matrix is determined, and the properties of the model and real samples obtained are presented. The hydrolytic and thermal resistance of the samples is investigated.

Abstract: The invention relates to methods of vitrifying waste and for lowering the melting point of glass forming systems by including lithia formers in the glass forming composition in significant amounts, typically from about 0.16 wt % to about 11 wt %, based on the total glass forming oxides. The lithia is typically included as a replacement for alkali oxide glass formers that would normally be present in a particular glass forming system. Replacement can occur on a mole percent or weight percent basis, and typically results in a composition wherein lithia forms about 10 wt % to about 100 wt % of the alkali oxide glass formers present in the composition. The present invention also relates to the high lithia glass compositions formed by these methods. The invention is useful for stabilization of numerous types of waste materials, including aqueous waste streams, sludge solids, mixtures of aqueous supernate and sludge solids, combinations of spent filter aids from waste water treatment and waste sludges, supernate alone, incinerator ash, incinerator offgas blowdown, or combinations thereof, geological mine tailings and sludges, asbestos, inorganic filter media, cement waste forms in need of remediation, spent or partially spent ion exchange resins or zeolites, contaminated soils, lead paint, etc. The decrease in melting point achieved by the present invention desirably prevents volatilization of hazardous or radioactive species during vitrification.

Abstract: Improving the efficiency of nuclear waste immobilization is constantly desired by all nuclear waste management programs world-wide. For high-level and other waste to be vitrified in traditional ceramic Joule-heated melters operated at temperatures up to 1150 C, process flexibilities including waste loadings are often restricted by this temperature limit as well as the need to consider wasteform corrosion of refractory linings and electrodes. New melter technologies, such as the cold-crucible melter (CCM), enable processing up to significantly higher temperatures free of many of the limitations of conventional melters. Higher processing temperatures open up the way for wider composition and processing envelopes to be considered for the vitrification process, including the possibility for higher waste loadings. In many instances the presence of crystals in the final cooled wasteform is not considered desirable within presently existing glass specifications. For some feed compositions in creased waste loadings can lead to the formation of large amounts of crystals, and thus to a significant departure from the ''glass'' state. Nevertheless it is recognized that, in general, increasing the acceptable volume fractions of crystals in the glass offers the best opportunity to increase waste loading, all other factors being equal. In addition, the deliberate promotion of specific crystalline phases by design may enhance the quality of the wasteform, for example by partitioning a long-lived radionuclide into a very stable crystalline phase, or by depleting the glass in detrimental elements. In order to explore the potential improvements by harnessing the higher achievable processing temperatures and immunity to refractory corrosion available with the cold-crucible melter, and after promising indications for synroc-based matrices, it was decided to investigate the feasibility of designing and producing via melting new high temperature ''glass-ceramic'' wasteforms for high level was te immobilization. The INEEL calcines were selected as example feed compositions. These calcines have a wide range of problematic compositions. They either have high amounts of crystal-forming components, and/or components that lead to corrosive melts, and for good measure, the components in some waste types are quite refractory for vitrification as well. The recent DOE High-Level Waste Melter Review Report concluded that, for the INEEL calcine wastes in particular, the CCM could have sufficient advantages over the Joule-heated ceramic melter to justify its evaluation for direct vitrification of these wastes. Based on the extensive ceramic design experience of ANSTO, in collaboration with the CEA and COGEMA for a CCM implementation, a preliminary set of waste forms has been developed that immobilize long-lived waste actinides into highly chemically durable crystalline phases by design, using refractory crystal-forming components already in the wastes to advantage, while at the same tim e maintaining a very good overall leach resistance for the glass-ceramics even after ''canister centerline cooling'' (CCC) heat treatments. This paper presents the results of a 50 kg technological scale test in the CCM of a glass-ceramic formulation for the average Bin Set 2 formulation, at a conservative waste loading of 50%.

Abstract: Factors affecting sensitivity of preimplantation embryos and follicular oocytes to cryopreservation were analyzed in the equine and bovine species. (1) Survival of equine blastocysts after two-step freezing in the presence of glycerol as the cryoprotective agent (CPA) was influenced by development of the embryonic capsule. The use of ethylene glycol (EG) with sucrose as CPAs improved the post-thaw survival of blastocysts and made it possible to transfer the embryos into recipient mares without removing the CPAs. In addition, early blastocysts cryopreserved by vitrification could develop both in vitro and in vivo when the embryos were exposed to vitrification solution in a stepwise manner. The vitrification procedure was also applied to the relatively large expanded blastocysts. (2) Bovine embryos produced in vitro have been considered to be highly sensitive to the process of cryopreservation. To solve this problem, Day-7 blastocysts produced in a serum-free system were cooled at 0.3 C/min rather than 0.6 C/min before being plunged into liquid nitrogen, resulting in no loss of the post-thaw viability. The supplementation of LAA in IVM/IVF media or IVC medium was effective in producing pronuclear-stage zygotes or morula-stage embryos relatively tolerable to freezing, respectively. (3) Transmission electron microscopic observation of immature equine oocytes showed that cellular injury occurred near the sites of gap-junctions between cumulus cells and the oocyte. In cattle, higher fertilization rates of oocytes were obtained when the oocytes were subjected to cryopreservation at an intermediate stage during IVM (GVBD for freezing, Met-I for vitrification). Vitrification of bovine Met-II oocytes in open-pulled glass capillaries, characterized by an ultra-rapid cooling rate (3,000-5,000 C/min), was found to avoid any harmful influence of vitrification and warming.

Abstract: Liver sinusoidal endothelial cells (LSECs) can optimally be imaged by whole mount transmission electron microscopy (TEM). However, TEM allows only investigation of vacuum-resistant specimens and this usually implies the study of chemically fixed and dried specimens. Cryo-electron microscopy (cryo-EM) can be used as a good alternative for imaging samples as whole mounts. Cryo-EM offers the opportunity to study intact, living cells while avoiding fixation, dehydration and drying, at the same time preserving all solubles and water as vitrified ice. Therefore, we compared the different results obtained when LSECs were vitrified using different vitrification conditions. We collected evidence that manual blotting at ambient conditions and vitrification by the guided drop method results in the production of artefacts in LSECs, such as the loss of fenestrae, formation of gaps and lack of structural details in the cytoplasm. We attribute these artefacts to temperature and osmotic effects during sample preparation just prior to vitrification. By contrast, by using an environmentally controlled glove box and a vitrification robot (37 degrees C and 100% relative humidity), these specific structural artefacts were nearly absent, illustrating the importance of controlled sample preparation. Moreover, data on glutaraldehyde-fixed cells and obtained by using different vitrification methods suggested that chemical prefixation is not essential when vitrification is performed under controlled conditions. Conditioned vitrification therefore equals chemical fixation in preserving and imaging cellular fine structure. Unfixed, vitrified LSECs show fenestrae and fenestrae-associated cytoskeleton rings, indicating that these structures are not artefacts resulting from chemical fixation.