Vitrification Technology Research Articles

Follicular fluid – perspective cryoprotective agent in the technology of porcine oocytes vitrification

Follicular fluid – perspective cryoprotective agent in the technology of porcine oocytes vitrification

Analysis of the morphology of Sus scrofa domesticus oocyte-cumulus complexes exposed to low and ultra-low temperatures

Relevance. The priority task of reproductive technologies in animal husbandry is the development of effective storage protocols for female gametes. One way to preserve gametes is vitrification, which reduces damage to intracellular organelles by increasing viscosity upon cooling and minimizing crystallization.Methods. Various additives to cryoprotectants are used for optimization. In our work, titanium tetrapolyethylene glycolate was used in a 10-fold molar excess of polyethylene glycol (TTPEG*10PEG), the nature of the effect of which on cells was studied during short-term storage at low (5 °С) and ultra-low temperatures (-196 °С).The purpose of the study is a comprehensive analysis of the morphology of gametes and somatic cells (cumulus) of ovarian follicles of pigs after exposure to low and ultra-low temperatures (vitrification) when included in the TTPEG*10PEG low-temperature storage and vitrification protocol.Results. Exposure to TTPEG*10PEG at 5 °С caused an increase in the level of oocytes with subsequent morphological degenerations in points with control (from 13% to 5%, р = 0.005). After vitrification with TTPEG*10PEG: the level of gametes with low cumulus cells expansion increased to 35% in terms of the proportion of control gametes (23%); the proportion of denudated gametes was reduced to 50% compared to the control 65% (р < 0.05); the level of morphologically degenerated gametes corresponded to that of native oocytes (8% each) and was lower than in the control (17%), р < 0.005. The obtained data indicate the protective and cryoprotective effects of TTPEG*10PEG at a concentration of 2%, which suggests the possibility of its use in the technology of intraovarian vitrification of female reproductive cells.

Multi‐Scale Ice Inhibition Platform Enables Full‐Cycle Cryogenic Protection for Mouse Oocyte

AbstractCryopreservation of metaphase II (MII) oocytes is crucial to preserve female fertility. However, the existing vitrification technology just provides partial cryogenic protection for oocytes and must use high concentrations of toxic penetrating cryoprotective agents (CPAs, up to 4.8 M). Here, a multi‐scale ice inhibition platform is proposed, which effectively suppresses ice crystal morphology, ice recrystallization (IR), temperature gradient, and devitrification. This platform achieves mouse oocyte survival rate of 98.6% using the low concentration of CPA (3.1 M) while also possessing batch oocyte cryopreservation ability. In contrast, recovered oocytes show a much more modest variation of genes compared with the conventional method (85 vs. 1396 genes), retaining normal fertilization, embryonic development, and birth to healthy offspring. Diverging from the conventional vitrification method with partial cryogenic protection capability, this platform introduces full‐cycle ice inhibition, offering promising prospects for achieving high‐quality and scalable fertility preservation.

Viable Vitreous Grafts of Whole Porcine Menisci for Transplant in the Knee and Temporomandibular Joints.

The shortage of suitable donor meniscus grafts from the knee and temporomandibular joint (TMJ) impedes treatments for millions of patients. Vitrification offers a promising solution by transitioning these tissues into a vitreous state at cryogenic temperatures, protecting them from ice crystal damage using high concentrations of cryoprotectant agents (CPAs). However, vitrification's success is hindered for larger tissues (>3 mL) due to challenges in CPA penetration. Dense avascular meniscus tissues require extended CPA exposure for adequate penetration; however, prolonged exposure becomes cytotoxic. Balancing penetration and reducing cell toxicity is required. To overcome this hurdle, a simulation-based optimization approach is developed by combining computational modeling with microcomputed tomography (µCT) imaging to predict 3D CPA distributions within tissues over time accurately. This approach minimizes CPA exposure time, resulting in 85% viability in 4-mL meniscal specimens, 70% in 10-mL whole knee menisci, and 85% in 15-mL whole TMJ menisci (i.e., TMJ disc) post-vitrification, outperforming slow-freezing methods (20%-40%), in a pig model. The extracellular matrix (ECM) structure and biomechanical strength of vitreous tissues remain largely intact. Vitreous meniscus grafts demonstrate clinical-level viability (≥70%), closely resembling the material properties of native tissues, with long-term availability for transplantation. The enhanced vitrification technology opens new possibilities for other avascular grafts.

Parameter relationship and product characteristics analysis of heavy-metal hazardous wastes vitrification under the combination of data science and experiment

Heavy-metal hazardous wastes (HMHW) have a large emission, high toxicity and strong resource properties. Its proper disposal and resource utilization is a key challenge. High-temperature melt vitrification technology is an effective solution to realize the detoxification and resource utilization of HMHW. This paper summarizes and analyzes the control parameters of the existing vitrification treatment of HMHW. The phase transformation process of temperature, slag content, and CAS ratio (CaO-Al2O3-SiO2) in the vitrification treatment of zinc leaching residue (ZLR) was investigated around the vitreous content. The common evolution characteristics of the formed vitrification products in infrared spectrum, UV–vis diffuse reflection spectrum, and microstructures were revealed. The idea of using the maximum UV–vis absorption intensity to qualitatively determine the vitreous content of the products is proposed. Moreover, the correlation between experimental parameters and vitreous content was quantified by combining the data science method, and the mapping of the whole process of ZLR vitrification treatment was constructed. This work provides reliable parameter guidance and method induction for the vitrification treatment of HMHW, especially for wastes with less glassy matrix such as Ca and Si.

Recent advances of oocyte/embryo vitrification in mammals from rodents and large animals.

Vitrification is a valuable technology that enables semipermanent preservation and long-distance or international transportation of genetically modified and native animals. In laboratory mice, vitrification maintains and transports embryos, and many institutions and companies sell vitrified embryos. In contrast, despite numerous papers reporting on vitrification in livestock over the past decade, practical implementation has yet to be achieved. However, with advances in genome editing technology, it is anticipated that the number of genetically modified domestic animals will increase, leading to a rise in demand for vitrification of oocytes and embryos. Here, we provide an objective overview of recent advancements in vitrification technology for livestock, drawing a comparison with the current developments in laboratory animals. Additionally, we explore the future prospects for vitrification in livestock, focusing on its potential benefits and drawbacks.

Dry LAMP: A point of care diagnostics for diagnosis of bovine tropical theileriosis.

Theileriosis is an important tick-bome hemoprotozoan disease of cattle which causes severe economic loss due to morbidity and mortality. A diagnostic test having high sensitivity, specificity and easy application at the field level is the need of the hour. In this regard Loop-mediated isothermal amplification (LAMP) is proven to be a sensitive, easy and time efficient method. One of the major obstacles for the application of LAMP is the difficulty in maintaining the cold chain to preserve reagents. Thus, the challenge is to develop a LAMP kit in a ready-to-use format with dried reagents useful for quick and simple application in field conditions. The optimized reaction of wet LAMP was followed for the standardization of dry LAMP with certain modifications which are needful. The major modification is vitrification technology of enzyme using trehalose. LAMP assay (dry and wet LAMP) was found to be more sensitive (100%) when compared to microscopy (69.5%) and PCR (86.9%). It was observed that the dry LAMP reaction tubes at room temperature as well as refrigeration temperature provided successful amplification till 7 weeks. The drying conditions of LAMP reagents were optimized, and finally managed to dry them in a single reaction tube without reducing the sensitivity. This technology enables us to transport LAMP kits to areas where the cold chain is not easily available.

Effect of vitrification on protein O-GlcNAcylation in mouse metaphase II oocytes.

Oocyte vitrification leads to DNA hypomethylation, which results in defect in early embryo development. This study reveals that oocyte vitrification impairs the DNA methylation pattern by influencing protein O-GlcNAcylation. Oocyte vitrification leads to decreased DNA methylation levels, which impairs the quality and the developmental potential of oocytes. However, the underlying molecular mechanism still need to be further revealed. In this study, mouse metaphase II (M II) oocytes were frozen by vitrification technology, while fresh oocytes were used as the control group. The effect of oocyte vitrification on protein O-GlcNAcylation and its impact on the developmental potential of oocytes were elucidated. We found that the protein O-GlcNAcylation levels were significantly increased in vitrified oocytes. Increase of protein O-GlcNAcylation levels in control oocytes by PUGNAc (an O-GlcNAcase inhibitor) decreases blastocyst rate after parthenogenetic activation (20.82% in PUGNAc-treated group; 53.82% in control group, P < 0.05). We also discovered that DNA methylation was disrupted in two-cell embryos derived from vitrified oocytes, resulting in decreased 5mC and increased 5hmC, showing similar phenotypes to that derived from PUGNAc-treated oocytes. In vitrified and PUGNAc-treated oocytes, O-GlcNAcylated TET3 was significantly increased. Notably, by inhibiting protein O-GlcNAcylation in vitrified oocytes using OSMI1 (an O-GlcNAc transferase inhibitor) we restored the DNA methylation in two-cell embryos and ameliorated the developmental defects in early embryo. Thus, elevated protein O-GlcNAcylation in vitrified oocytes is an essential contributor to their declining embryonic developmental potential. Modulation of protein O-GlcNAcylation improves the developmental potential of vitrified oocytes.

Melatonin improves the vitrification of sheep morulae by modulating transcriptome.

Embryo vitrification technology is widely used in livestock production, but freezing injury has been a key factor hindering the efficiency of embryo production. There is an urgent need to further analyze the molecular mechanism of embryo damage by the vitrification process. In the study, morulae were collected from Hu sheep uterine horns after superovulation and sperm transfusion. Morulae were Cryotop vitrified and warmed. Nine morulae were in the vitrified control group (frozen), and seven morulae were vitrified and warmed with 10-5 M melatonin (melatonin). Eleven non-frozen morulae were used as controls (fresh). After warming, each embryo was sequenced separately for library construction and gene expression analysis. p < 0.05 was used to differentiate differentially expressed genes (DEG). The results showed that differentiated differentially expressed genes (DEG) in vitrified morulae were mainly enriched in protein kinase activity, adhesion processes, calcium signaling pathways and Wnt, PI3K/AKT, Ras, ErbB, and MAPK signaling pathways compared to controls. Importantly, melatonin treatment upregulated the expression of key pathways that increase the resistance of morulae against vitrification induced damage. These pathways include kinase activity pathway, ErbB, and PI3K/Akt signaling pathway. It is worth mentioning that melatonin upregulates the expression of XPA, which is a key transcription factor for DNA repair. In conclusion, vitrification affected the transcriptome of in vivo-derived Hu sheep morulae, and melatonin had a protective effect on the vitrification process. For the first time, the transcriptome profiles caused by vitrification and melatonin in sheep morulae were analyzed in single embryo level. These data obtained from the single embryo level provide an important molecular mechanism for further optimizing the cryopreservation of embryos or other cells.

Robotic denudation of zygotes

The vitrification technology is used in the embryo freezing process. To ensure intracellular water removal and avoid ice crystal formation, the cumulus cells surrounding zygotes need to be removed. As operators have varying skill and long-time fatigue, it is challenging to completely remove the cumulus cells surrounding zygotes and reduce zygote loss inside the micropipette. The objective of this work is to develop a robotic zygote denudation system for the vitrification procedure. The robotic system enables accurate segmentation of cumulus cells for estimating the mass of cumulus zygote complexes (CZCs) inside the micropipette via the Res-Unet neural network. A mathematical model was built to describe the dynamic motion of CZCs inside the micropipette, and a model-based optimal controller was developed to aspirate and deposit CZCs inside the micropipette. To denude zygote completely, the Res-Attention neural network was used to detect cumulus cells for predicting the quality of zygote denudation. In the mouse zygotes experiments, the yield rate is 98.2% ± 1.6% and the denudation efficacy is 96.9% ± 0.2% for the robotic system. Compared with manual denudation, the survival rate and development rate of embryos cultured from zygotes denuded by the robotic system were both higher in subsequent vitrification procedure.

Droplet Generation, Vitrification, and Warming for Cell Cryopreservation: A Review.

Cryopreservation is currently a key step in translational medicine that could provide new ideas for clinical applications in reproductive medicine, regenerative medicine, and cell therapy. With the advantages of a low concentration of cryoprotectant, fast cooling rate, and easy operation, droplet-based printing for vitrification has received wide attention in the field of cryopreservation. This review summarizes the droplet generation, vitrification, and warming method. Droplet generation techniques such as inkjet printing, microvalve printing, and acoustic printing have been applied in the field of cryopreservation. Droplet vitrification includes direct contact with liquid nitrogen vitrification and droplet solid surface vitrification. The limitations of droplet vitrification (liquid nitrogen contamination, droplet evaporation, gas film inhibition of heat transfer, frosting) and solutions are discussed. Furthermore, a comparison of the external physical field warming method with the conventional water bath method revealed that better applications can be achieved in automated rapid warming of microdroplets. The combination of droplet vitrification technology and external physical field warming technology is expected to enable high-throughput and automated cryopreservation, which has a promising future in biomedicine and regenerative medicine.

Cryopreservation of oocytes: history, achievements and future

There have been increasing requirements for women’s fertility preservation due to oncological and nononcological reasons in recent years, and meeting these demands will be a hot topic in the coming years. Oocyte cryopreservation is a workable option for preserving women’s fertility, and great advances have already been made and much progress has been made in mammalian gene banking and human oocyte banks. In this paper, we systematically introduce the history of oocyte cryopreservation and vitrification technology and highlight the vitrification carrier. Furthermore, we summarize the fundamentals of oocyte vitrification and discuss the effects of vitrification on oocyte quality. Strategies to improve the effect of oocyte cryopreservation are also proposed. At the end of this review, we conclude oocyte cryopreservation and outline future perspectives.

Development of an Open Microfluidic Platform for Oocyte One-Stop Vitrification with Cryotop Method.

Oocyte vitrification technology is widely used for assisted reproduction and fertility preservation, which requires precise washing sequences and timings of cryoprotectant agents (CPAs) treatment to relieve the osmotic shock to cells. The gold standard Cryotop method is extensively used in oocyte vitrification and is currently the most commonly used method in reproductive centers. However, the Cryotop method requires precise and complex manual manipulation by an embryologist, whose proficiency directly determines the effect of vitrification. Therefore, in this study, an automatic microfluidic system consisting of a novel open microfluidic chip and a set of automatic devices was established as a standardized operating protocol to facilitate the conventional manual Cryotop method and minimize the osmotic shock applied to the oocyte. The proposed open microfluidic system could smoothly change the CPA concentration around the oocyte during vitrification pretreatment, and transferred the treated oocyte to the Cryotop with a tiny droplet. The system better conformed to the operating habits of embryologists, whereas the integration of commercialized Cryotop facilitates the subsequent freezing and thawing processes. With standardized operating procedures, our system provides consistent treatment effects for each operation, leading to comparable survival rate, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) level of oocytes to the manual Cryotop operations. The vitrification platform is the first reported microfluidic system integrating the function of cells transfer from the processing chip, which avoids the risk of cell loss or damage in a manual operation and ensures the sufficient cooling rate during liquid nitrogen () freezing. Our study demonstrates significant potential of the automatic microfluidic approach to serve as a facile and universal solution for the vitrification of various precious cells.

Can delayed grafting of frozen teeth achieve periodontal ligament healing?

Autologous teeth transplanted technology is putting healthy teeth into empty alveolar socket and waiting to be fixed by newly formed alveolar bone and periodontal membrane, to perform chewing function. Vigorous donor teeth are usually impacted teeth or orthodontic excess teeth that are often extracted and discarded during adolescence. However, they should have more potential function that repairing dentition defects due to caries, apical periodontitis, cysts, and other causes after being an adult. Therefore, once the preserved technical is skilled, utilization of dental tissues extracted early in life would create new possibilities. The problem is whether cryopreservation reduces tissue regeneration. Scholars who emphasize periodontal ligament (PDL) healing firmly believe it can avoid pathological reaction and ensure the success of transplantation. Nowadays, the ideal success rate can reach 80.0 % – 91.1%, but the classic transplantation procedure is time-dependent, which requires the donor tooth to be extracted and transplanted immediately. Freezing is a recognized effective way to make physiological and biochemical activities quiescent, and to maintain the potential of cells to relive after rewarming in the same medium. During cells cryopreservation, the challenge is in the inappropriate freezing and thawing condition, intracellular freezing would break cells’ physical structure, spoil normal PDL and increase resorption of root. So even though delay in freezing autotransplantation would greatly expand procedure indications, it has not yet become a mainstream treatment. Slow freezing and vitrification technology are main cryopreservation ways certified effective that essentially prevent ice crystals from damaging cells. To some extent, none of them is perfect considering cell cytotoxicity from cryoprotectants or controlling of freezing rate. Now, with the modified freezing method, we firstly put forward Antifreeze proteins (AFPs) to tooth cryopreservation, and assume that low concentration AFPs combined with vitrification may yield better results in PDL healing of delayed autotransplanted tooth. Based on analyzing the freezing mechanism, cell studies, animal experiments and individual case reports, we propose a procedural freezing method and summarized how to reduce cryogenic injury of cryopreserved tooth for potential use in tooth transplantation. However, more studies and data are needed about masticatory function and long-term prognosis observation.

Cesium in Gaseous Emissions from the Vitrification Technology of Radioactive Waste (Review of Scientific and Technical Information)

Cesium in Gaseous Emissions from the Vitrification Technology of Radioactive Waste (Review of Scientific and Technical Information)

Self-reduction with escaped sulfur and immobilization of toxic chromium in Co-vitrification for synergistic recovery of chromium sludge and blast furnace slag

Vitrification technology has been widely used to treat hazardous solid waste to form molten glasses. But the heavy metals with active valence in the glass matrix are unstable and easy to dissolve, easily leading to potential environmental risks. Based on the laboratory simulated preheating feed mode, this study first explored the feasibility of using high-temperature melting metallurgical blast furnace slag to synergistic melt chromium sludge to form the stabilized glass matrix. The results found that the stabilization of chromium in the molten glass was highly enhanced by the self-reduction effect of escaping sulfur when chromium sludge and blast furnace slag were co-melted by preheating feed. The leaching concentration of chromium decreased to 0.03 mg/L, which met the limit requirements of resource utilization for molten glasses. TG-MS and XPS supported that SO2 generated from chromium sludge uninterruptedly made more chromium kept in low valence during the co-melting process. As a result, 22.13% of Cr(III) species existed in molten glass produced from preheating feed larger than 6.3% of the heating feed. The polarizing microscope also showed that the increase of the Cr(III) species promoted the formation of micro-crystals, thereby improving the stability of chromium in molten glass. Therefore, this study highlights the new perspective on realizing high value-added utilization of chromium sludge with low energy consumption and higher stability.

Fracture behavior of concrete containing MSWI vitrified bottom ash

The incorporation of waste materials into concrete allows responding to some of the most significant issues of our society: waste management and climate change. Experimental studies carried out in last decades have shown that municipal solid waste incineration (MSWI) ash, and particularly bottom ash, which constitutes the major solid by-product of incineration process, can be adopted to produce building materials. However, several issues are related to the safety and the environmental impact of MSWI ash utilization for concrete production, mainly linked with the leaching of heavy metals and toxic organic components. To solve these problems, several treatments for MSWI ash can be adopted and, among them, in this work the attention was focused on vitrification technology, which enables to convert the ash in a glassy inert solid material. The aim of the present paper is to study the feasibility of developing a “green concrete” that incorporates vitrified MSWI bottom ash as partial cement replacement, so reducing the cement content and consequently the carbon dioxide emissions as well as the raw materials consumption related to its production. The vitrified MSWI bottom ash, ground at micrometer size, was inserted into the admixtures by considering two percentages of cement substitution (10% and 20% by weight of cement). The flexural behavior of concrete containing vitrified MSWI ash was investigated through three-point bending tests under crack mouth opening displacement control. The crack path evolution was further explored by adopting the Digital Image Correlation technique. By analyzing the obtained results, it can be concluded that the use into concrete of vitrified MSWI bottom ash as cement replacement up to a percentage of 20% by weight of cement, allows reaching comparable flexural resistances with respect to the reference concrete. So, the proposed approach can represent a viable solution for the development of environmental-friendly concretes able to reduce the environmental impact of the concrete industry, which is mostly related to cement production, as known.

In Situ Vitrification of Lung Cancer Organoids on a Microwell Array.

Three-dimensional cultured patient-derived cancer organoids (PDOs) represent a powerful tool for anti-cancer drug development due to their similarity to the in vivo tumor tissues. However, the culture and manipulation of PDOs is more difficult than 2D cultured cell lines due to the presence of the culture matrix and the 3D feature of the organoids. In our other study, we established a method for lung cancer organoid (LCO)-based drug sensitivity tests on the superhydrophobic microwell array chip (SMAR-chip). Here, we describe a novel in situ cryopreservation technology on the SMAR-chip to preserve the viability of the organoids for future drug sensitivity tests. We compared two cryopreservation approaches (slow freezing and vitrification) and demonstrated that vitrification performed better at preserving the viability of LCOs. Next, we developed a simple procedure for in situ cryopreservation and thawing of the LCOs on the SMAR-chip. We proved that the on-chip cryopreserved organoids can be recovered successfully and, more importantly, showing similar responses to anti-cancer drugs as the unfrozen controls. This in situ vitrification technology eliminated the harvesting and centrifugation steps in conventional cryopreservation, making the whole freeze–thaw process easier to perform and the preserved LCOs ready to be used for the subsequent drug sensitivity test.

Thermal treatment of nuclear fuel-containing Magnox sludge radioactive waste

Magnesium aluminosilicate and magnesium borosilicate glass formulations were developed and evaluated for the immobilisation of the radioactive waste known as Magnox sludge. Glass compositions were synthesised using two simplified bounding waste simulants, including corroded and metallic uranium and magnesium at waste loadings of up to 50 wt.%. The glasses immobilising corroded simulant waste formed generally homogenous and fully amorphous glasses, while those immobilising metallic wastes contained crystallites of UO 2 and U 3 O 8 . Uranium speciation within the glass was investigated by micro-focus X-ray absorption near edge spectroscopy and it was shown that the borosilicate glass compositions were characterised by a slightly lower mean uranium oxidation state than the aluminosilicate counterparts. This had an impact upon the durability, and uranium within glasses of higher mean oxidation states was dissolved more readily. All material showed dissolution rates that were comparable with simulant high level radioactive waste glasses, while the borosilicate-based formulations melted at a temperature suitable for modern vitrification technologies used in radioactive waste applications. These data highlight the potential for vitrification of hazardous radioactive Magnox sludge waste in borosilicate or aluminosilicate glass formulations, with the potential to achieve >95 % reduction in conditioned waste volume over the current baseline plan. • Magnesium aluminosilicate and magnesium borosilicate glass systems were synthesised to incorporate metallic uranium and magnesium, U 3 O 8 and Mg(OH) 2 • Uranium speciation within the glass was investigated by micro-focus X-ray absorption near edge spectroscopy • Borosilicate glasses possessed greater chemically durable than their aluminosilicate counterparts • All glass compositions demonstrated durability that is comparable to UK and French high level waste glass

Investigation of Borosilicate Glasses with Simulated HLW Components and Determination of Their Chemical Durability

The paper discusses the current status of the HLW vitrification technology applied at the radiochemical plant of the Mayak PA. Conceptual and technical solutions proposed to develop the technology of vitrification of various types of liquid HLW at the Mayak PA are presented. Compositions of borosilicate glasses with HLW components are described, temperatures of their melting and easy pouring are determined. Chemical durability parameters obtained for the borosilicate glasses are provided.