Abstract: Crystallization in glasses is usually considered to be a problem in the glass industry. However, controlled crystallization of glasses is an important prerequisite in the development of glass-ceramics with tailored useful properties. Similar boundary conditions apply when considering glass-ceramics for the immobilization of nuclear waste via vitrification. While uncontrolled crystallization in nuclear-waste glasses is problematic, chemically durable glass-ceramics with significantly high waste loadings can be produced with controlled crystallization of glasses. This article presents an overview of various aspects of nuclear-waste glasses where crystallization is either considered to be advantageous or problematic. The classification of glass-ceramic waste forms and strategies to design glass-ceramics for a given waste stream is discussed. Some open and relevant problems faced by researchers developing nuclear-waste glass-ceramics are also offered.

Abstract: Recent findings have reported the reason why some living beings are able to withstand the huge thermal amplitudes between winter and summer in their natural habitats. They are able to produce metabolites decreasing deeply the crystallization temperature of water, avoiding cell disrupture due to the presence of ice crystals and overcoming osmotic effects. In vitro, the possibility to cool living cells and tissues to cryogenic temperatures in the absence of ice can be achieved through a vitrification process. Vitrification has been suggested as an alternative approach to cryopreservation and could hereafter follow an interesting biomimetic perspective. The metabolites produced by these animals are mostly sugars, organic acids, choline derivatives, or urea. When combined at a particular composition, these compounds form a new liquid phase which has been defined as Natural Deep Eutectic Solvents (NADES). In this review, we relate the findings of different areas of knowledge from evolutive biology, cryobiology...

Abstract: Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cooling injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at sufficiently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and growth of ice during subsequent storage and rewarming if not appropriately handled.The commercial and clinical application of stem cells requires robust and reproducible cryopreservation protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical applications are available, current cryopreservation protocols, whether for vitrification or conventional slow freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreservation engenders, there is a danger that such processes will impose a selective pressure on the cells selecting out a nonrepresentative, freeze-resistant subpopulation. Optimizing this process requires knowledge of the fundamental processes that occur during the freezing of cellular systems, the mechanisms of damage and methods for avoiding them. This chapter draws together the knowledge of cryopreservation gained in other systems with the current state-of-the-art for embryonic and induced pluripotent stem cell preservation in an attempt to provide the background for future attempts to optimize cryopreservation protocols.

Abstract: In the present work, the (100 − x)(58SiO2-20Fe2O3-10B2O3-12Na2O) − xAs (x = 0, 2, 4, 6, 8 wt%) glasses, which are main constituents of the copper slag based glasses, were produced by conventional melting method to study the arsenic vitrification mechanism and stability. X-ray diffraction (XRD), Fourier transform infrared spectrum (FTIR) and X-photoelectron spectroscopy (XPS) were used to investigate the mechanism of arsenic vitrification. The XRD analysis for the x ≤ wt 8% samples shows that all the samples form homogeneous glasses and the arsenic is compatible with the copper slag based glass. The FTIR spectrum of the glasses have revealed that the arsenic exists in the form of AsO4 tetrahedra and the Qn structure of the copper slag based glass has been strengthened with the increase addition of arsenic. The changes of the binding energies of Si2p, O1s, As3d and Fe2p directly indicate the formation of Si O As and Fe O Si/As bonds in the glass, which strengthens the chemical stability of the glass. Lastly, USEPA toxicity characteristics leaching procedure (TCLP) and differential scanning calorimetry (DSC) tests were used to study the chemical durability and thermal stability of the glasses containing arsenic. The leaching concentrations of As, B, Fe, Na, and Si are under safety thresholds and the increase addition of arsenic into the glasses can decrease the leaching concentrations, indicating that the arsenic can improve the chemical stability of the glass. The arsenic added into the glass can improve the ΔT, which strengthens the thermal stability of the glasses.

Abstract: This review describes the 120-year history of technology for cryoprotectant-free cryopreservation of human spermatozoa by direct plunging into liquid nitrogen (vitrification). It explains why cryoprotectant-free vitrification of some human ejaculate samples is better than conventional freezing and vitrification with the presence of cryoprotectants. Special attention is given to the extremely high viability of viruses, bacteria, and mycoplasmas after cryoprotectant-free cryopreservation in culture medium and even in distilled water. This increases the potential risk of disease transmission through liquid nitrogen. The concept of asepticity is concretized as an obvious parameter for any medical assisted reproduction technology that includes cooling of cells in liquid nitrogen. The roles of nonpermeating compounds in media for cytoprotectant-free vitrification—carbohydrates, proteins, lipoproteins, antioxidants—are described. This review summarizes relevant data regarding two groups of different current technologies for cryoprotectant-free vitrification of human spermatozoa: those involving direct contact of spermatozoa with liquid nitrogen and those involving full isolation of these cells from liquid nitrogen (aseptic technologies).

Abstract: STUDY QUESTION Is permeable cryoprotectant-free vitrification of native sperm samples a good alternative to conventional slow freezing? SUMMARY ANSWER The permeable cryoprotectant-free sperm vitrification protocol tested in this study renders considerably better recovery rates of good quality sperm compared to slow freezing. WHAT IS KNOWN ALREADY Slow freezing is currently the most commonly used technique for sperm cryopreservation, though this method has been repeatedly shown to have negative effects on both structural and functional sperm features. New alternative methods such as vitrification have been established as a successful alternative in other reproductive cell types, but vitrification of spermatozoa is still a rather unexplored methodology, with limited studies showing its efficacy in male gametes. STUDY DESIGN SIZE, DURATION This study included 18 normozoospermic sperm samples from patients seeking ART treatment between 2014 and 2015. The effects of a new vitrification protocol on functional and structural sperm quality parameters in comparison to fresh and slow-frozen samples were assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS All samples were divided into three aliquots: fresh (F), slow freezing-thawing (S) and vitrification-warming (V). Sperm concentration, motility, morphology, vitality, DNA fragmentation, cytoskeleton integrity and spontaneous acrosome reaction were assessed and compared between the groups. MAIN RESULTS AND THE ROLE OF CHANCE Results showed improved preservation of sperm features after vitrification compared to conventional freezing. Permeable cryoprotectant-free vitrification presented a significantly higher percentage of live spermatozoa, than slow freezing, better preservation of acrosomes was achieved in vitrified samples and DNA fragmentation was reduced approximately one-third on average compared to slow freezing. Regarding tubulin assay, three different labelling patterns were observed. The frequency of these labelling patterns was similar in F and V groups but this was not the case of the S group. The multivariate analysis of all sperm quality parameters studied revealed that the V group presented features that are closer to the F group than the S group, indicating that samples are better preserved through vitrification than slow freezing. LIMITATIONS REASONS FOR CAUTION This validation has been undertaken only on normozoospermic sperm samples. It would be necessary to compare these results in pathological samples and also to evaluate the influence of the application of this methodology on clinical outcomes. WIDER IMPLICATIONS OF THE FINDINGS The sperm vitrification protocol here described warrants better maintenance of sperm quality parameters than traditional freezing methods and may be a good alternative to preserve sperm samples from patients seeking IVF treatment. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by IVF-Spain Foundation. The authors have no conflicts of interest to declare.

Abstract: Hazardous residues from ferronickel smelting plant are mixed with glass cullet and are vitrified for 2 h at 1400 °C. The obtained glass is characterized with a very high crystallization trend. As a result, it is inappropriate for sinter-crystallization, but a fine crystalline glass-ceramic can be obtained by bulk crystallization after short heat-treatment at low temperatures. Due to the presence of 1.5 wt.% chromium oxide in the parent glass, together with high amounts of iron oxides and magnesium oxides, the crystallization process is peculiar. It starts during the melt cooling with the precipitation of preliminary Fe-Mg-Cr spinel crystals, which then act as centers for further epitaxial growth of pyroxene phase. It is also highlighted that, due to liquid–liquid immiscibility, the main amorphous phase is characterized by a nonhomogeneous binodal structure, which becomes finer after the nucleation treatment. As a result, pyroxenes with sizes below 1 μm are formed as main crystal phase after the crystallization step. Notwithstanding of the high amount of hazardous wastes in the batch the obtained glass is characterized with high chemical durability. At the same time, the obtained glass-ceramics is characterized by a suitable coefficient of thermal expansion and attractive mechanical characteristics.

Abstract: To examine the maturational competence, embryo development and expression of genes involved in oocyte maturation and cumulus expansion (GDF9, BMP15, HAS2, TNFAIP6, FGF17 and FSHr) following two standard methods of bovine COCs vitrification. Bovine cumulus-oocyte complexes (COCs) were aspirated from slaughtered ovaries and then distributed into three groups: non-vitrified COCs (control), vitrification 1 group (V1); vitrification was performed by 15% ethylene glycol (EG) and 15% DMSO in holding media (TCM-199 with 20% FCS); and vitrification 2 group (V2); vitrification was performed by 40% EG in holding media. After vitrification, COCs were warmed in two steps and cultured and then evaluated for nuclear maturation, embryo development and gene expressions. The mean (±SD) percentages of nuclear maturation and blastocyst/cleaved were higher in control group (79.5 ± 8.0 and 31.0 ± 5.1%) than the V1 (34.8 ± 9.1 and 4.4 ± 5.1%) and V2 (47.8 ± 11.7 and 7.1 ± 5.8%) groups (P < 0.05), respectively. Further, COCs in V2 group showed higher mean (±SD) percentages of cleavage compared to V1 group (31.8 ± 1.0 vs 21.7 ± 2.8%; P < 0.05). GDF9 and BMP15 expression levels were higher in COCs in the control than of the vitrification groups (P < 0.05). In addition, expression level of GDF9 and BMP15 was higher in V2 group than in V1group (P < 0.05). The expression of HAS2 and FGF17 in V1 group was lower (P < 0.05) than that of the V2 groups. Expression of oocyte maturation genes was affected by vitrification procedure and conditions. Using EG alone for vitrification of bovine immature COCs, resulted in higher expression of GDF9, BMP15 and production of more in vitro matured and cleaved oocytes.

Abstract: There have been 60 births after transplantation of cryopreserved ovarian tissue: 58 using the slow freezing method, and two using the vitrification method. DMSO and EG are widely used as cryoprotectants. However DMSO is a known epimutagen, and EG has been reported to be toxic in high concentrations. In this study, we measured residual DMSO and EG in ovarian tissue after vitrification and slow freezing. Cryoprotectants remained at a high concentration in the vitrified/warmed ovarian tissue just before transplantation (DMSO: 9.8 mg/g, EG: 9.8 mg/g). We must consider the impact of the cryoprotectants on the mother and the baby.

Abstract: The current paper reviews the concept of the production of high-added value construction materials produced as part of a zero waste enhanced landfill mining process. The calorific fraction of the excavated waste is concentrated to produce a solid recovered fuel, which is introduced to a gasification/vitrification process to be converted to a synthetic gas, a slag and a metal alloy. The slag is subsequently cooled to produce a glass. The glass is milled and blended with an alkaline silicate solution to produce an inorganic polymer binder. The binder can be used as an alternative for ordinary Portland cement (OPC) in concrete to produce precast construction materials, such as pavers, tiles and wall elements. Pilot industrial production and testing of the durability, environmental footprint and economic feasibility of the process are currently being performed. Traditional OPC based production lines can be used, and when comparing with OPC based concrete, materials with similar to improved properties (e.g. higher hardening rate and higher final strength) can be produced.

Abstract: Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated e-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.

Abstract: Contents Ovarian tissue cryopreservation followed by tissue culture is a promising approach to preserving the fertility of biomedical models and endangered species. The objective of this study was to investigate the impact of exposure time to vitrification solution and presence of sucrose using different exposure temperatures and base media on intra-ovarian follicle integrity. Peripubertal ovarian cortical pieces were obtained by isolating the cortex and dissecting it into 1 × 1 × 0.2 mm3 pieces. The cortical pieces were then exposed to equilibration solution and then vitrification solutions (VS) in one of the conditions mentioned above, plunged directly into liquid nitrogen and stored for ≥24 hr in liquid nitrogen. After thawing, the cortical pieces were cultured in vitro for 0, 1 or 7 days to determine the follicle integrity (through histological assessment) and the ability of the tissue to recover from cryoinjury. Fresh controls maintained a constant level of normal morphology (>60% of the total follicles) throughout the culture period. Cortical pieces exposed to VS with sucrose for 10 min had the highest percentage of normal follicles (approximately 20% after 7 days of culture) throughout the culture period. Other conditions using different base medium, lower exposure temperatures or different thawing methods did not improve the follicle integrity. This protocol provides a solid foundation on which to optimize ovarian tissue cryopreservation in the domestic cat and to investigate the molecular effects of vitrification.

Abstract: The Southern Flounder Paralichthys lethostigma is a high-value species and a promising aquaculture candidate. Because sperm volume can be limited in this species ( 1,000°C/min). The goal of this work was to develop a standardized approach for vitrification of Southern Flounder sperm. The specific objectives were to (1) evaluate thawing methods and vitrification solutions, (2) evaluate the postthaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the ability of vitrified sperm to fertilize eggs. From the vitrification solutions tested, the highest postthaw motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%) was observed for 20% ethylene glycol plus 20% glycerol. There was no sign...

Abstract: Embryo vitrification is a modern technique for cryopreservation in assisted reproductive programs. From all the embryos, blastocysts are the most challenging during cryostorage due to their size, multicellular structure and the presence of blastocoelic fluid. The aim of this study was to evaluate the suitability for vitrification of various developmental stages of feline embryos and the influence of the artificial shrinkage (AS) of expanded blastocyst on post-vitrification survival rates. The AS procedure is the manual puncture of the trophectoderm allowing for the reduction of blastocoelic fluid prior to vitrification and thus preventing the ice crystal formation. The vitrified embryos were divided into groups of 2-cell, 4- to 8-cell, >8-cell, morulae, compacted and expanded blastocyst, based on morphological assessment and vitrified in groups of 1-3 embryos per Cryotop. The post-warming survival was similar regardless the embryo developmental stage prior vitrification; however, development to blastocysts was only noted in 4- to 8-cell and >8-cell vitrified embryos (13% and 27%, respectively). Following warming, the significantly more viable blastocysts were noted in vitrified compacted versus expanded blastocyst and in expanded blastocyst subjected to AS procedure versus expanded blastocyst without AS (total survival: 58.3% vs. 33.3% and 64.3% vs. 38.5%, respectively; re-expansion rate within 2 hr post-warming: 41.7 vs. 6.7% and 50% vs. 7.7%, respectively). One-fifth of vitrified expanded blastocyst showed morphological damage immediately after warming procedure, whereas no visible damage was noted in compacted blastocyst and artificially collapsed expanded ones. The obtained results suggest that the most suitable for vitrification are feline embryos containing four to 16 blastomeres and compacted blastocyst. In addition, the reduction of blastocoel cavity in expanding blastocyst by artificial collapse improved the blastocyst vitrification outcome.

Abstract: This study compared the efficacy of docetaxel (DT) and paclitaxel (PT) in reducing spindle damage during vitrification and maintaining the developmental competence of in vitro-matured (IVM) bovine oocytes after vitrification and warming. Pretreatment of IVM oocytes with 0.05µM DT for 30min before vitrification resulted in significantly higher (P<0.05) rates of oocyte survival and cleavage after IVF, as well as subsequent blastocyst rates on Days 7-9 and hatching on Days 8-9, compared with oocytes pretreated with 1.0µM PT before vitrification or those vitrified without pretreatment. When nuclear status and spindle morphology of vitrified oocytes were assess after warming by immunostaining, DT pretreatment before vitrification resulted in a significantly higher (P<0.05) percentage of oocytes at the MII stage with a normal, intact spindle compared with PT pretreatment or no pretreatment, but the percentage of MII oocytes was still significantly lower (P<0.05) than in the control group. Pretreatment of IVM bovine oocytes with 0.05µM DT or 1.0µM PT for 30min before vitrification reduces spindle damage to the same extent, without side effects on fertilisation and development. Pretreatment with 0.05µM DT improved the developmental competence of vitrified-warmed oocytes to a greater degree than 1.0µM PT pretreatment.

Abstract: Aseptic vitrification of semen samples packed in straws has been successfully developed in human but not in donkeys. The aim of this study was to compare the effect of two extenders for donkey sperm vitrification using straws. Ejaculates from four Andalusian donkeys were collected, and samples were extended in INRA-96 (I) or Gent (G) supplemented with sucrose 0.25 M and 1% bovine serum albumin (BSA). Extended samples were cooled for one hour at 5°C. For vitrification, samples were filled in covered 0.25 ml straws and then plunged directly into liquid nitrogen. For warming, straws were immersed in INRA-96 at 43°C. Results showed no significant differences between I and G treatments for TM (34.2% ± 8.7 vs. 30.7% ± 9.6) and PM (26.8% ± 7.3 vs. 24.6% ± 7.9), respectively. In conclusion, donkey sperm could be vitrified in straws either with INRA-96 or with Gent in combination with sucrose and BSA.