Abstract: Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 10(5) degrees C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (-160 degrees C) with speed in the range 150-250 degrees C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.

Abstract: BACKGROUND: Manual puncture of the trophectoderm of human blastocysts with a needle before vitrification increases their survival rate, but the embryos take a long time to re-expand. This study examined whether causing human blastocysts to collapse by manual pipetting before vitrification would allow more rapid re-expansion and improve pregnancy rates. METHODS: After embryo transfer in IVF cycles, surplus embryos that developed to the expanded blastocyst stage were placed in cryoprotectant and then artificially shrunk by mechanical pipetting with a fine hand-drawn glass pipette slightly smaller in diameter than the blastocyst. The shrunken embryos were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. The blastocysts were thawed by warming and then dilution in 1 mol/l sucrose. RESULTS: Of 49 expanded vitrified blastocysts, 48 (98%) re-expanded within 3 h after warming. Following transfer (48 blastocysts in 28 cycles), 14 women (50%) became clinically pregnant, and the implantation rate was 33% (16/48). Eight healthy babies have been born in six deliveries, and the other eight pregnancies are ongoing. To date, there have been no spontaneous abortions. CONCLUSIONS: The results suggest that artificial shrinkage with pipetting is a simple and effective technique to assist successful cryopreservation of expanded blastocysts by vitrification.

Abstract: Vitrification is a method in which not only cells but also the whole solution is solidified without the crystallization of ice. For embryo cryopreservation, the vitrification method has advantages over the slow freezing method. For example, injuries related to ice is less likely to occur, embryo survival is more likely if the embryo treatment is optimized, and embryos can be cryopreserved by a simple method in a short period without a programmed freezer. However, solutions for vitrification must include a high concentration of permeating cryoprotectants, which may cause injury through the toxicity of the agents. Since the development of the first vitrification solution, which contained dimethylsulphoxide, acetamide, and propylene glycol, numerous solutions have been composed and reported to be effective. However, ethylene glycol is now most widely used as the permeating component. As supplements, a macromolecule and/or a small saccharide are frequently added. Embryos of various species, including humans, can be cryopreserved by conventional vitrification using insemination straws or by ultrarapid vitrification using minute tools such as electron microscopic grids, thin capillaries, minute loops, or minute sticks, or as microdrops. In the ultrarapid method, solutions with a lower concentration of permeating cryoprotectants, thus having a lower toxicity, can be used, because ultrarapid cooling/warming helps to prevent ice formation.

Abstract: Human embryonic stem cells (hESCs) promise to revolutionize reparative medicine through their potential in developing cell replacement therapies for diseases like diabetes and parkinsonism. Most of the existing hESC lines available for research, including all National Institutes of Health-registered lines, have been derived and maintained on mouse embryonic fibroblast feeders in the presence of xenoproteins. For future clinical application, many more hESC lines derived and grown in current good manufacturing practice, good tissue culture practice, and xeno-free conditions need to be developed. Concurrently, effective cryopreservation methods that prevent or limit the accidental contact of hESCs with nonsterile liquid nitrogen during periods of long-term storage have to be formulated. We describe a safe, xeno-free cryopreservation protocol for hESCs involving vitrification in closed sealed straws using human serum albumin as opposed to fetal calf serum as the main protein source in the cryoprotectant and long-term storage in the vapor phase of liquid nitrogen. After thaw, hESCs exhibited high thaw-survival rates and low differentiation rates, remained pluripotent, and maintained normal diploid karyotypes throughout extended passage. The cryopreservation technique we describe here should complement xeno-free culture conditions for hESCs already in refinement and will prove very useful for the setting up of hESC banks throughout the world.

Abstract: Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, an ice-free cryopreservation method by vitrification was tested. Osteochondral plugs from New Zealand White rabbits were preserved using either a freezing method or an ice-free vitrification method of cryopreservation. Preserved and fresh control plugs were implanted in the tibial plateau of allogeneic recipients. A modified O'Driscoll grading scale, based on gross pathology, histopathology, and histochemistry, was used to evaluate the explants.The histology of fresh and vitrified explants was essentially the same, while the frozen cryopreserved explants were devoid of chondrocytes and only fibroblastlike cells were observed. The O'Driscoll grading indicated that both fresh and vitrified plugs performed significantly better than frozen plugs (p≤ .05). The results demonstrate the feasibility of vitrification as a storage method for cartilaginous tissues.

Abstract: BACKGROUND: The efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method. METHODS: Human ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified. RESULTS: Eighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P < 0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers. CONCLUSIONS: Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.

Abstract: BACKGROUND Vitrification of human blastocysts has been successfully applied using grids, straws and cryoloops. We assessed the survival rate of human compacted morulae and early blastocysts vitrified in pipette tips with a smaller inner diameter and solution volume than the previously described open pulled straw (OPS) method. METHODS Excess day 5 human embryos (n = 63) were experimentally vitrified in vessels. Embryos were incubated at 37 degrees C with sperm preparation medium (SPM) for 1 min, SPM + 7.5% ethylene glycol (EG)/dimethylsulphoxide (DMSO) for 3 min, and SPM + 16.5% EG + 16.5% DMSO + 0.67 mol/l sucrose for 25 s. They were then aspirated (0.5 microl) into a plastic micropipette tip (0.36 mm inner diameter), exposed to liquid nitrogen (LN(2)) vapour for 2 min before being placed into a pre-cooled cryotube, which was then closed and plunged into LN(2). Embryos were warmed and diluted using 0.33 mol/l and 0.2 mol/l sucrose. RESULTS The survival rate for compacted morulae was 73% (22/30) and 82% (27/33) for early blastocysts. CONCLUSIONS The survival rates of human compacted morulae and early blastocysts after vitrification with this simple technique are similar to those reported in the literature achieved by slow cooling and other vitrification protocols.

Abstract: Using vitrification and encapsulation-vitrification protocols, we successfully cryopreserved shoot apices from in-vitro plants of different Gentiana cultivars (lines). Although both protocols gave high survival percentages after storage in liquid nitrogen, the encapsulation-vitrification protocol had several distinct advantages over the vitrification protocol: (i) survival was higher under optimal conditions, (ii) the range of optimal exposure periods to the plant vitrification solution 2 (PVS2) was broader, and (iii) regrowth of cryopreserved shoot apices was apparently more vigorous and faster. Shoot apices from ten cultivars/lines of three Gentiana species (G. scabra, G. triflora, and G. pneumonanthe) were successfully cryopreserved using the two protocols with average survival of 49.0 percent and 73.7 percent for vitrification and encapsulation-vitrification, respectively. These results indicate that the two protocols optimized in the present study are promising for cryopreservation of a wide range of Gentiana genetic resources.

Abstract: A commercial citrus scion cultivar, '439' tangor [C. suavissima Hort. et Tanaka × C. sinensis (L.) Osbesk cv. Gailiangcheng] was used to investigate whether GSH (reduced form of glutathione) could improve survival of a vitrification procedure. The optimal pre-growth treatment was a 3-day pre-culture on basal pre-culture medium (BPM: MT basal medium containing 0.5 mol/L glycerol and 5% sucrose at pH 5.8). GSH of 40 mg/L in the pre-culture medium improved shoot tip survival after cryopreservation. GSH in the recovery medium also improved survival, with 10 mg/L giving the best result. GSH of 40 mg/L in the loading and vitrification solutions also improved survival. The optimal cryopreservation protocol was successfully applied to 12 other citrus cultivars. This is the first report on the successful cryopreservation of shoot-tips from commercial citrus scion cultivars.

Abstract: Immobilization of special categories of radioactive wastes containing both actinide and chloride species is of increasing interest. In the present contribution we report the results of investigations into the feasibility of immobilizing these wastes by vitrification and solid state reaction between zeolite and calcium phosphate and the waste. We also report on the reactions between the ceramic wasteforms and various glass binders used to convert the powder wasteforms into a monolithic form as these can adversely effect the properties of the monolith, e.g. leach resistance. These reactions can result in a loss of chloride from the product due to the formation of a volatile chloride or the formation of the soluble phase halite. Methods for overcoming these undesirable reactions are discussed. It was shown that vitrification was not a suitable process for these wastes but that they could be incorporated into either zeolite 4A or calcium phosphate to produce powder wasteforms with chloride leach rates below 0.2 mg m−2 day−1.

Abstract: The growing need for improved methods of viable tissue cryopreservation has stimulated debate regarding the relative merits of traditional freezing methods versus ice-free vitrification methods. Articular cartilage has proved refractory to satisfactory cryopreservation using conventional freezing methods. Therefore, full-thickness rabbit femoral head articular cartilage was used to compare a freezing method of cryopreservation and an ice-free, vitrification method of cryopreservation with fresh controls in vitro. Chondrocyte viability was determined in vitro using vital fluorescent staining with calcein-acetoxymethyl ester and an oxidation-reduction assay with alamarBlue™ (Accumed International, Westlake, Ohio). Cryosubstitution with 1% osmium tetroxide in 100% methanol was used to detect ice during cryopreserved storage. Cryosubstitution studies of frozen and vitrified articular cartilage pieces revealed negligible ice in the vitrified specimens and extensive ice formation in frozen specimens with the ex...

Abstract: Purpose: In human frozen immature oocytes, there has been little successful delivery. We examined the feasibility of vitrification solution including Taxol, cytoskeltal stabilizer. Methods: We set four experimental groups that immature oocytes has cumulus cells or not, or including Taxol or not in the vitrification solution. Frozen–thawed oocytes have been performed IVM, ICSI, and IVC. Results: There were no significant differences in survival, maturation, and fertilization rate, respectively. However, in the group enveloped by cumulus cells and including Taxol in the vitrification solution, one embryo was developed to blastocyst. Conclusions: Our results showed that using vitrification solution with Taxol proved so effective.

Abstract: The formation of ice crystals is known to be lethal to biological cells. The presence of cryoprotectants at high cooling rates suppresses crystallization and promotes vitrification, where vitrification is solidification by rapid elevation of the viscosity (vitreous, in Latin, means glass). All materials have a tendency to change volume with a change in temperature, where the rate at which the volume changes with respect to the temperature is defined as the thermophysical property of thermal expansion. In the presence of a non-uniform temperature distribution in a bulky specimen, when different regions of the material tend to expand differently, mechanical stress may develop. It has been demonstrated that this mechanical stress can easily lead to macro structural damage to the cryopreserved specimen. As part of an ongoing effort to characterize the mechanical behavior of biological tissues and solutions in the cryogenic temperature range, the current study focuses on mapping the thermal expansion effect of...

Abstract: A prospective randomized trial was performed to compare post-thaw development of murine blastocysts following programmable rate freezing and two methods of vitrification. Frozen 2-cell murine embryos (n = 429) thawed and cultured for 48 h, were randomly allocated by stage of development into four groups: control (not refrozen), programmable rate freezing (PR) in 0.25 ml straws, vitrification in flexible micropipettes by immersion in super-cooled (VSC) liquid nitrogen (LN2), and vitrification in flexible micropipettes by immersion in LN2 (VLN). Survival, developmental stage progression, presence or absence of an inner cell mass (ICM), and cell counts were recorded 24 h post-thaw. All measured outcomes were different between embryos from the control group and all freezing methods. Controlled-rate freezing resulted in the lowest total cell counts and fewest embryos with a distinct ICM. A higher percentage of embryos survived 24 h post-thaw, progressed to more advanced developmental stages and had higher total cell counts after VLN compared with PR. Moreover, fewer embryos, frozen by either PR or VSC, contained a detectable ICM compared with VLN. These data demonstrate that vitrification may be a better method for freezing murine blastocysts than PR, and may prove to be a superior method for freezing human blastocysts.

Abstract: An improved method for vitrification of biological cells, especially blastocyst-derived stem cells (BS cells). The method is very mild for the cells that remain viable after they have been thawed. The method comprises, i) transfer of the cells to a first solution (solution A), ii) optionally incubation of the cells in the first solution, iii) transfer the cells obtained in step i) or ii) to a second solution (solution B), iv) optionally incubation of the cells in the second solution, v) transfer of the cells obtained from step iii) or iv) into one or more closed straws with dimensions that allow a volume of at least 20 µl to be contained in them vi) sealing the one or more closed straws, and vii) vitrification of the one or more closed straws. An important feature of the present invention is the use of closed straw and that relatively large volumes can be efficiently vitrified and subsequently thawed.

Abstract: A low-k organic dielectric material having stable nano-sized porous is provided as well as a method of fabricating the same. The porous low-k organic dielectric material is made from a composition of matter having a vitrification temperature (Tv-comp) which includes a b-staged thermosetting resin having a vitrification temperate (Tv-resin), a pore generating material, and a reactive additive. The reactive additive lowers Tv-comp below Tv-resin.

Abstract: Several modifications to the cryogenic protocols previously described for pineapple apices were performed using vitrification and encapsulation-vitrification. Pregrowth of apices in sucrose-proline before loading significantly reduced the exposure duration to PVS2 and PVS3 required for successful cryopreservation. Encapsulation and treatments with PVS3 at 0 degree C gave the highest survival before and after cooling. Optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3 percent) followed by a 2-d preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h and then transfer to 0.3 M sucrose + 0.3 M proline for an additional 24 h. After preculture, samples were loaded in 0.75 M sucrose + 1 M glycerol solution at room temperature (25 min) and dehydrated with PVS3 at 0 degree C for 60 min before immersion into liquid nitrogen. Following this procedure 54 percent and 83 percent of apices from MD-2 and Puerto Rico varieties respectively survived.

Abstract: A novel application of millimeter-wave radiometry has been made for the first time to non-contact detection and monitoring of molten salt layer formation on a nuclear waste glass melt (without the nuclear waste) in a joule-heated melter, which could eventually be implemented for on-line monitoring in nuclear waste vitrification facilities. The experiments were carried out at a frequency of 137 GHz in the EV-16 melter at Clemson Environmental Technology Laboratory (CETL) with 245 lbs. (111 kG) of glass and a total of 4.2 lbs. (1.9 kG) of added salt. The dynamics of salt layer build up were observed from the initial formation of small drops of about 5 mm diameter or less to larger pools >28 mm cross-section that were coincident with the increase in millimeter-wave surface level fluctuations causing the salt to flow back and forth until a continuous layer was formed. The millimeter-wave emissivity at 137 GHz of DWPF black frit glass melt and molten sodium sulfate salt at 950 °C was determined to be 0.64 ± 0.05 and 0.44 ± 0.05, respectively.

Abstract: Vitrification has become the preferred method for cryopreserving in vitro-produced bovine embryos (IVP). Here we introduce a technique for vitrification developed at CryoLogic (the CLV Method), in conjunction with a study comparing the post-thaw viability of IVP embryos frozen by the widely used open pulled straw method (OPS—Vajta et al., 1997 Cryo-Letters 18, 191) and the new CLV Method. Vitrification on thin metal surfaces has been explored and demonstrated previously (Le Gal & Massip 1999, Cryobiology 38, 290), and Dinnyes presented a Solid Surface Vitrification (SSV) (Dinnyes et al., 2000, Biol. Reprod. 63, 513). The CLV Method utilizes vitrification on the surface of a solid metal block. This surface has been custom shaped and treated to enhance vitreous formation. The method also includes handling, storage and thawing protocols designed to avoid damage from crystallization of the unstable glass. Briefly, the block is precooled in LN2 to −196°C. Up to 10 embryos are collected into a droplet of medium (3 μL), on the end of a fibre carrier attached to a handle. The droplet is presented to the vitrification surface, where it is converted into a glass bead by cooling rates comparable to that of plunging into solid/liquid phase nitrogen (−210°C) (Arav et al., 2001 Theriogenology 55, 313). The glass bead, fibre and handle are transferred quickly into a half-sealed, precooled straw, and the handle seals the open end. A bead is thawed very rapidly by removing the handle, fibre and bead from the straw and transferring the bead into washing medium. COCs collected from bovine ovaries obtained from abattoirs were matured, fertilized and cultured for 6 days (Fry et al., 2003 Theriogenology 59, 446). Embryos reaching the blastocyst or expanding blastocyst stage of development were graded (Grades 1, 2, and 3), equilibrated for 5 min in HEPES-199 medium with 20% FCS (HFm), placed in HFm with 10% EG, and 10% DMSO (VS1) for 2 min, and then transferred to HFm with 20% EG, 20% DMSO (VS2) for 30 s (Vajta). Between 5 and 10 IVP embryos were processed and collected for vitrification, either in an OPS plunged into LN2, or in a 3 μL droplet vitrified by the CLV Method. The two sets of specimens were stored in LN2, and later thawed. Both OPS tips and beads were thawed in 0.5 mL of HFm with 0.2 M sucrose at 39°C. Embryos were maintained at 39°C, examined after 5 min for contraction, and again after 6 h for re-expansion. They were then transferred to culture medium, incubated and examined at 24 and 48 h to assess hatching. As shown in Table 1, the CLV method appears to be satisfactory for maintaining membrane integrity (expansion) and developmental potential (hatching) for even poorer grade embryos, that might be more sensitive to the stresses of cryopreservation. Table 1 Re-expansion and hatching rates of graded thawed bovine embryos vitrified by OPS or CLV methods

Abstract: ................................................................................................................................. ix CHAPTER I. REVIEW OF LITERATURE ..............................................................................

Abstract: PURPOSE: An apparatus and a process for vitrification of low and intermediate-level radioactive wastes are provided to reduce the environmental pollution by processing efficiently radioactive wastes generated from a nuclear power plant. CONSTITUTION: An apparatus for vitrification of low and intermediate-level radioactive wastes includes an input unit, a glass input unit(1), and a melt furnace(7). The input unit is used for inputting spent resins and radioactive solid wastes. The glass input unit(1) is used for inputting glass. The melt furnace(7) is used for melting the glass by using the inductive current. A glass coating layer is formed on a surface of an inner wall of the melt furnace(7). A cooling water path(11) is formed in the melt furnace(7) in order to prevent the abrasion due to a contact between the melt glass and the surface of the inner wall of the melt furnace(7).

Abstract: In the developed world, incineration of wastes is widely and increasingly practiced. The incineration produces residues, mainly bottom ash and air pollution control fly ash. Fly ash is especially problematic because of its high heavy-metal content. Thermal processes, based mainly on electrical arc processes, show great promise: the residues are melted at high temperature and converted in a relatively inert glass. The major toxic elements in the fly ash are lead, zinc, cadmium, etc., with different volatilities at very high temperature. Consequently, it is necessary to control the volatility of the heavy metals during plasma mineral waste melting and vitrification. A twin-torch plasma system, mounted above a crucible, filled with a known amount of synthetic glass and of toxic elements, has been used as experimental setup to reach basic data about metals volatility under the plasma column of an electrical arc transferred on the melt. Vapors above the melt have been probed by optical emission spectroscopy. M...