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1.
Methods Mol Biol ; 2180: 317-330, 2021.
Article in English | MEDLINE | ID: mdl-32797418

ABSTRACT

Cryoprotectants are essential to prevent ice formation during tissue cryopreservation procedures. However, the control of their concentration and spatial distribution in the tissue is necessary to avoid toxicity and other damages associated with the cryopreservation procedures, especially for bulky samples such as tissues and organs. X-ray computed tomography measures the attenuation of an X-ray beam when it passes through a substance, depending on the material properties of the samples. The high electronic density of the sulfur atom of the dimethyl sulfoxide makes it an excellent cryoprotectant to be assessed by X-ray CT, and its concentration is proportional to the X-ray attenuation either at room or cryogenic temperatures. In addition, this imaging technique also allows to detect the formation of ice and eventual fractures within tissues during the cooling and warming processes. Therefore, X-ray CT technology is an excellent tool to assess and develop new cryopreservation procedures for tissues and organs.


Subject(s)
Cryopreservation/methods , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Image Processing, Computer-Assisted/methods , Ovary/cytology , Tomography, X-Ray Computed/methods , Female , Humans , Ovary/diagnostic imaging , Ovary/drug effects , Phase Transition , Specimen Handling
2.
J Colloid Interface Sci ; 573: 278-286, 2020 Aug 01.
Article in English | MEDLINE | ID: mdl-32283416

ABSTRACT

The combination of different bioimaging techniques, mainly in the field of oncology, allows circumventing the defects associated with the individual imaging modalities, thus providing a more reliable diagnosis. The development of multimodal endogenous probes that are simultaneously suitable for various imaging modalities, such as magnetic resonance imaging (MRI), X-ray computed tomography (CT) and luminescent imaging (LI) is, therefore, highly recommended. Such probes should operate in the conditions imposed by the newest imaging equipment, such as MRI operating at high magnetic fields and dual-energy CT. They should show, as well, high photoluminescence emission intensity for their use in optical imaging and present good biocompatibility. In this context, we have designed a single nanoprobe, based on a core-shell architecture, composed of a luminescent Eu3+:Ba0.3Lu0.7F2.7 core surrounded by an external HoF3 shell that confers the probe with very high magnetic transverse relaxivity at high field. An intermediate, optically inert Ba0.3Lu0.7F2.7 layer was interposed between the core and the shell to hinder Eu3+-Ho3+ cross-relaxation and avoid luminescence quenching. The presence of Ba and Lu, with different K-edges, allows for good X-ray attenuation at high and low voltages. The core-shell nanoparticles synthesized are good potential candidates as trimodal bioprobes for MRI at high field, dual-energy CT and luminescent imaging.

3.
Nanomaterials (Basel) ; 10(1)2020 Jan 14.
Article in English | MEDLINE | ID: mdl-31947630

ABSTRACT

Uniform Nd3+-doped LuVO4 nanophosphors have been synthesized for the first time in literature by using a poliol-based method at 120 °C from Nd3+ and vanadate precursors. After optimizing the Nd doping level, these phosphors present intense luminescence in the near-infrared biological windows. The X-ray attenuation capacity of the optimum nanophosphor has been found to be higher than that of a commercial X-ray computed tomography contrast agent. After surface coating with polyacrylic acid, such nanoparticles present high colloidal stability in physiological pH medium and high cell viability. Because of these properties, the developed Nd3+-doped LuVO4 nanoparticles have potential applications as a bimodal probe for NIR luminescent bioimaging and X-ray computed tomography.

4.
Sci Rep ; 9(1): 20008, 2019 12 27.
Article in English | MEDLINE | ID: mdl-31882972

ABSTRACT

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants.


Subject(s)
Cryopreservation/methods , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Ovarian Follicle/drug effects , Vitrification , Adult , Female , Humans
5.
Nanomaterials (Basel) ; 9(8)2019 Aug 12.
Article in English | MEDLINE | ID: mdl-31408936

ABSTRACT

Due to the high atomic number of lutetium and the low phonon energy of the fluoride matrix, Lu-based fluoride nanoparticles doped with active lanthanide ions are potential candidates as bioprobes in both X-ray computed tomography and luminescent imaging. This paper shows a method for the fabrication of uniform, water-dispersible Eu3+:(H3O)Lu3F10 nanoparticles doped with different Eu contents. Their luminescent properties were studied by means of excitation and emission spectra as well as decay curves. The X-ray attenuation capacity of the phosphor showing the highest emission intensity was subsequently analyzed and compared with a commercial contrast agent. The results indicated that the 10% Eu3+-doped (H3O)Lu3F10 nanoparticles fabricated with the proposed polyol-based method are good candidates to be used as dual probes for luminescent imaging and X-ray computed tomography.

6.
J Colloid Interface Sci ; 554: 520-530, 2019 Oct 15.
Article in English | MEDLINE | ID: mdl-31330425

ABSTRACT

A one-pot simple procedure for the synthesis of uniform, ellipsoidal Eu3+-doped sodium lanthanum tungstate and molybdate (NaLa(XO4)2, X  = W, Mo) nanophosphors, functionalized with carboxylate groups, is described. The method is based on a homogeneous precipitation process at 120 °C from appropriate Na+, Ln3+ and tungstate or molybdate precursors dissolved in ethylene glycol/water mixtures containing polyacrylic acid. A comparative study of the luminescent properties of both luminescent materials as a function of the Eu3+ doping level has been performed to find the optimum nanophosphor, whose efficiency as X-ray computed tomography contrast agent is also evaluated and compared with that of a commercial probe. Finally, the cell viability and colloidal stability in physiological pH medium of the optimum samples have also been studied to assess their suitability for biomedical applications.


Subject(s)
Contrast Media/chemistry , Europium/chemistry , Lanthanum/chemistry , Luminescent Agents/chemistry , Molybdenum/chemistry , Tungsten Compounds/chemistry , Animals , Chlorocebus aethiops , Contrast Media/chemical synthesis , Luminescent Agents/chemical synthesis , Tomography, X-Ray Computed , Tungsten Compounds/chemical synthesis , Vero Cells
7.
Biopreserv Biobank ; 17(2): 119-128, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30489143

ABSTRACT

One of the main problems in the cryopreservation of biological samples is the formation of ice and the consequent mechanical damage to cells and tissues, due to the crystalline structure of ice and its associated mechanical damage. It is necessary to detect this deleterious formation of ice, especially in tissues and organs, because of their large volume and the complexity of their vascular system in the case of bulky organs. In this work, we propose the use of X-ray Computed Tomography (CT) to detect this ice formation inside tissues and organs. To achieve this aim, rabbit kidneys were loaded with cryoprotectant solutions containing Me2SO at low temperatures (below -140°C). Drops of water with a volume between 2 and 8 µL were then introduced inside the organs. Finally, the rabbit kidneys were cooled to -196°C. Volumes of ice of up to 1 µL were detected in our CT device, with a resolution of up to 50 µm, validating the proposed technology. On the contrary, we analyzed bovine ovarian tissues cryopreserved with a controlled-rate slow-cooling protocol. CT images showed the different structure on the extracellular ice formation according to the procedure, and even the intracellular ice that can be formed in the tissues. These positive results have a straightforward application in the control of the formation of ice, of significant importance for the creation of biobanks.


Subject(s)
Cryopreservation , Ice , Kidney/diagnostic imaging , Ovary/diagnostic imaging , X-Ray Microtomography , Animals , Cattle , Female , Rabbits
8.
Reprod Biomed Online ; 37(6): 653-665, 2018 Dec.
Article in English | MEDLINE | ID: mdl-30391164

ABSTRACT

RESEARCH QUESTION: Could a modification in the ovarian tissue freezing protocol improve follicle survival after cryopreservation and xenotransplantation? DESIGN: Ovarian tissue was used from 13 adult patients, frozen either with our original protocol, or a modified version involving a higher concentration of dimethyl sulphoxide (DMSO), larger volume of cryopreservation solution and lower seeding temperature. After thawing, the ovarian fragments were xenotransplanted to six mice with severe combined immunodeficiency (SCID) for 3 weeks. RESULTS: The proportion of primordial follicles decreased, and the proportion of growing follicles increased significantly (all P < 0.01) after cryopreservation and xenografting compared with fresh controls for both protocols. Follicle density, development, ultrastructure and function were similar between treatments. CONCLUSIONS: This study showed that, although the higher DMSO concentration did not improve survival of preantral follicles, it did not seem to induce any major toxicity in the follicle population either.


Subject(s)
Cryopreservation/methods , Ovary/pathology , Animals , Dimethyl Sulfoxide , Female , Humans , Immunohistochemistry , Mice , Mice, SCID , Ovarian Follicle , Ovary/blood supply , Ovary/ultrastructure , Tissue Preservation , Transplantation, Heterologous
9.
Theriogenology ; 119: 183-188, 2018 Oct 01.
Article in English | MEDLINE | ID: mdl-30015147

ABSTRACT

Cryopreservation and subsequent transplantation of ovarian tissue is the only option to preserve fertility in certain patients facing gonadotoxic treatment. So far, cryopreservation of ovarian tissue has been carried out mostly by a controlled rate slow cooling process, typically known as slow freezing. Even though there are still some concerns about the iatrogenic damage on the follicle population, this technique has been used in the more than 100 live births reported to date. It is well known that the control of the cryoprotectant loading in the tissue is crucial to in a cryopreservation procedure. We have used the technology of X-ray computed tomography to assess the concentration and distribution of dimethyl sulfoxide (one of the cryoprotectants most used in fertility preservation) inside pieces of bovine ovarian tissue after its cryopreservation. The low voltage used in our device (75 kV) and the high electronic density of this cryoprotectant makes the X-ray attenuation proportional to its concentration. By assessing and comparing the permeation and homogeneity of the cryoprotectant inside ovarian tissue fragments subjected to a controlled rate slow cooling process, we have characterized the effect of variations in the main parameters involved in the process, with the goal of achieving an optimized protocol with higher permeation of the cryoprotectant in the tissue. The most promissory results were obtained by increasing the initial concentration of dimethyl sulfoxide in the vehicle solution from 10 to 20%v/v.


Subject(s)
Cattle , Cryopreservation/veterinary , Ovary , Tissue Preservation/veterinary , Tomography, X-Ray Computed/veterinary , Animals , Cold Temperature , Cryopreservation/methods , Female , Methanol/pharmacology , Time Factors , Tomography, X-Ray Computed/methods
10.
Dalton Trans ; 47(25): 8382-8391, 2018 Jun 25.
Article in English | MEDLINE | ID: mdl-29897075

ABSTRACT

Uniform Ba0.18Ce0.82F2.82 nanospheres have been obtained after aging a solution of barium and cerium nitrates and sodium tetrafluoroborate in a mixture of ethylene glycol and water at 120 °C for 20 hours. The diameter of the spheres could be tailored from 65 nm to 80 nm by varying the NaBF4 concentration while maintaining their colloidal stability in aqueous suspension. Increasing the aging temperature led to a phase transformation from hexagonal to cubic symmetry and to a concomitant increase of the Ba/Ce ratio, which reached a value close to the nominal one (50/50) at 240 °C. The same method was successful in obtaining Tb3+-doped nanospheres with homogeneous cation distribution and the same morphological features as the undoped material. An intense green emission was observed after the excitation of the Tb3+-doped samples through the Ce3+-Tb3+ energy transfer (ET) band. The ET efficiency increased with increasing Tb content, the maximum emission being observed for the 10% Tb-doped nanospheres. Aqueous suspensions of the latter sample showed excellent X-ray attenuation values that were superior to those of an iodine-based clinically approved contrast agent. Their fluorescence and X-ray attenuation properties make this material a potential dual bioprobe for luminescence bioimaging and X-ray computed tomography.

11.
J Colloid Interface Sci ; 520: 134-144, 2018 Jun 15.
Article in English | MEDLINE | ID: mdl-29533853

ABSTRACT

The singular properties of lanthanide-based inorganic nanoparticles (NPs) has raised the attention of the scientific community in biotechnological applications. In particular, those systems with two or more functionalities are especially interesting. In this work, an effective and commercially attractive procedure has been developed that renders uniform, water-dispersible Ln3+:CeF3 (Ln = Tb, Nd) NPs with different shapes and size. The method consists of the homogeneous precipitation, in a mixture of polyol and water, of cations and anions using precursors that allow the controlled release of the latter. The advantages of the reported method are related to the absence of surfactants, dispersing agents or corrosive precursors as well as to the room temperature of the process. The obtained Tb:CeF3 NPs produce an intense emission after excitation through the Ce-Tb energy transfer band located in the UV spectral region, thus being potentially useful as phosphors for in-vitro imaging purposes. On the other hand, the synthesized Nd:CeF3 NPs are good candidates for in-vivo imaging because their excitation and emission wavelengths lie in the biological windows. Finally, the excellent X-ray attenuation efficacy of the Nd:CeF3 NPs is shown, which confers double functionality to this material as both luminescence bioprobe and contrast agent for X-ray computed-tomography.

12.
Cryobiology ; 81: 17-26, 2018 04.
Article in English | MEDLINE | ID: mdl-29518373

ABSTRACT

Ovarian tissue cryopreservation is, in most cases, the only fertility preservation option available for female patients soon to undergo gonadotoxic treatment. To date, cryopreservation of ovarian tissue has been carried out by both traditional slow freezing method and vitrification, but even with the best techniques, there is still a considerable loss of follicle viability. In this report, we investigated a stepped cryopreservation procedure which combines features of slow cooling and vitrification (hereafter called stepped vitrification). Bovine ovarian tissue was used as a tissue model. Stepwise increments of the Me2SO concentration coupled with stepwise drops-in temperature in a device specifically designed for this purpose and X-ray computed tomography were combined to investigate loading times at each step, by monitoring the attenuation of the radiation proportional to Me2SO permeation. Viability analysis was performed in warmed tissues by immunohistochemistry. Although further viability tests should be conducted after transplantation, preliminary results are very promising. Four protocols were explored. Two of them showed a poor permeation of the vitrification solution (P1 and P2). The other two (P3 and P4), with higher permeation, were studied in deeper detail. Out of these two protocols, P4, with a longer permeation time at -40 °C, showed the same histological integrity after warming as fresh controls.


Subject(s)
Cryopreservation/methods , Fertility Preservation/methods , Ovarian Follicle , Vitrification , Animals , Cattle , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Female , Freezing , Ovarian Follicle/drug effects , Tomography, X-Ray Computed
13.
Cryobiology ; 71(3): 419-31, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26434735

ABSTRACT

Cryoprotection of bulky organs is crucial for their storage and for subsequent transplantation. In this work we demonstrate the capability of the X-ray computed tomography (CT) as a non-invasive method to measure the cryoprotectant (cpa) concentration inside a tissue or an organ, specifically for the case of dymethil sulfoxide (Me2SO). It is remarkable that the use of Me2SO has been leader in techniques of cells and tissues cryopreservation. Although CT technologies are mainly based in density differences, and many cpas are alcohols with densities similar to water, the use of very low energies as acceleration voltage (∼70 kV) and the sulfur atom in the molecule of Me2SO makes possible the visualization of this cpa inside tissues. As result we obtain a CT signal proportional to the Me2SO concentration with a spatial resolution up to 50 µm in the case of our device.


Subject(s)
Cryopreservation/methods , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide/pharmacology , Tomography, X-Ray Computed
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