• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Procedure for preparing oocytes and/or magnetic embryos by means of nanoparticles for assisted reproduction techniques and for use in techniques for assisted reproduction of oocytes and/or non-human embryos prepared with said procedure, said procedure comprising: a stage where magnetic nanoparticles are conjugated with OVGP1r recombinant oviductin protein; a step in which it is verified that the nanoparticles-OVGP1r conjugate binds to the oocyte/embryo ZP after their joint incubation; and a stage in which the number of oocytes/embryos that have nanoparticles distributed around the ZP without being endocitated is checked, and it is evaluated whether the amount of the magnetic nanoparticles bound to the ZP of oocytes/embryos are sufficient to be attracted by a magnetic field (Machine-translation by Google Translate, not legally binding)
    公开号:ES2724873A1
    申请号:ES201830239
    申请日:2018-03-11
    公开日:2019-09-17
    发明作者:Vazquez Francisco Alberto Garcia；Movilla Maria Jimenez；Sastre Maria Caballero
    申请人:Universidad de Murcia；
    IPC主号:
    专利说明:

    [0001]
    [0002] PROCEDURE FOR PREPARATION OF OVOCITS AND / OR MAGNETIC EMBRYOS THROUGH NANOPARTICLES FOR ASSISTED REPRODUCTION TECHNIQUES AND USE IN ASSISTED REPRODUCTION TECHNIQUES OF OVOCITS AND / OR NON-HUMAN EMBRYOS PREPARED WITH SUCH PROCEDURE
    [0003]
    [0004] OBJECT OF THE INVENTION
    [0005]
    [0006] The invention, as stated in the present specification, refers to a process for preparing oocytes and / or magnetic embryos by means of nanoparticles for assisted reproduction techniques and for use in techniques for assisted reproduction of oocytes and / or non-embryos. humans prepared with this procedure, all of which is a remarkable novelty in the current state of the art within its field of application.
    [0007]
    [0008] More particularly, the object of the invention focuses, on the one hand, on a process for magnetizing oocytes and / or embryos based on the use of magnetic nanoparticles and a protein binding technology between the nanoparticles and the external part of the oocytes / embryos, called the zona pellucida (ZP), since said magnetization of oocytes / embryos can become a very useful property in the use of such oocytes / embryos in different assisted reproduction techniques in different animal species.
    [0009]
    [0010] Essentially, the process comprises a first stage in which the magnetic nanoparticles are conjugated with the recombinant protein of the oviductal fluid, oviductin (OVGP1r); a second stage in which said conjugation of nanoparticles-OVGP1r, in turn, binds to the ZP of oocytes / embryos by their joint incubation or co-incubation; and a third stage in which, by means of a magnetic field, it is evaluated that the amount of magnetic nanoparticles attached to the oocyte / embryo ZP is sufficient to be attracted by a magnetic field.
    [0011]
    [0012] Also, a second aspect of the invention relates to the specific use in assisted reproduction techniques of oocytes and / or non-human embryos prepared with said process.
    [0013]
    [0014] FIELD OF APPLICATION OF THE INVENTION
    [0015]
    [0016] The field of application of the present invention is part of the biochemistry sector and the nano-biotechnology or nano-medicine sector, covering more specifically the field of assisted reproduction techniques.
    [0017]
    [0018] BACKGROUND OF THE INVENTION
    [0019]
    [0020] The use of magnetic nanoparticles is widespread today in the field of biotechnology and biomedicine as it is a non-invasive procedure for diagnosis and therapy of some diseases, such as cancer, Alzheimer's or stem cell transplantation in diseases such as myocardial infarction. In the branch of assisted reproduction, these magnetic nanoparticles are currently used in sperm cells, on the one hand, for the detection of damaged sperm by conjugating said nanoparticles with different antibodies and lectins that bind to said gametes, and on the other hand for their use in sperm-mediated transgenesis.
    [0021]
    [0022] However, the present invention concerns the use of magnetic nanoparticles in oocytes and / or embryos through a novel protein binding technology between nanoparticles conjugated to the recombinant protein OVGP1r and the outer part of the oocytes / embryos, called the pellucid zone ( ZP), which makes said union very specific. Naturally, the OVGP1 protein present in the oviductal fluid binds to the ZP of oocytes and embryos.
    [0023]
    [0024] As a reference to the current state of the art, it should be noted that patents and documents related to the object of the present invention are known, the most relevant being the following:
    [0025]
    [0026] EP2237039A1, which refers to a coded microparticle of a biocompatible material for labeling and / or tracking an oocyte or an isolated embryo. Said microparticle is fixed to the cell's ZP and can be tracked, by optical microscopy, by its external form which constitutes a particular identification code.
    [0027] US2016091410A1, which discloses a method for processing animal sperm to improve its motility, viability and fertility comprising compositions with magnetic particles.
    [0028]
    [0029] The document “ Barcode tagging of human oocytes and embryos to prevent mix-ups in assisted reproduction technologies” published in 2014 by Novo Sergi; Nogues Carme; Penon Oriol; Leonardo neighborhoods; Santalo Josep; Gomez-Martinez Rodrigo; Esteve Jaume; Errachid Abdelhamid; Antonio Plaza Jose; Perez-Garcia Lluisa; Ibanez Elena, which describes the application in the technologies of assisted reproduction of oocytes and human embryos labeled in the zona pellucida with biofunctional polysilicon barcodes.
    [0030]
    [0031] The document “Direct embryo tagging and identification system by attachment of biofunctionalized polysilicon barcodes to the pellucida area of mouse embryos”, published in 2013 by Novo Sergi; Penon Oriol; Leonardo neighborhoods; Nogues Carme; Santalo Josep; Duran Sara; Gomez-Matinez Rodrigo; Samitier Josep; Antonio Plaza Jose; Perez-Garcia Luisa; Ibanez Elena, which describes a direct system of labeling and identification of embryos by coupling biofunctional polysilicon barcodes to the mouse embryonic pellucid zone.
    [0032]
    [0033] The document “Biomolecule screening for efficient attachment of biofunctionalized microparticles to the pellucida area of mammalian oocytes and embryos”, published in 2013 by Novo S; Ibañez E; L neighborhoods; Castell O; Nogues C, which describes the selection of biomolecules for the efficient fixation of biofunctional microparticles to the ZP of oocytes and mammalian embryos. Individual labeling of oocytes and embryos by fixing polysilicon barcodes to the ZP is a technique that is applied in human assisted reproduction and animal production programs. To provide barcodes with the ability to attach to the ZP, these must first be subjected to a biofunctionalization process by conjugating a biomolecule capable of attaching to the ZP of oocytes and embryos. The biomolecules analyzed are an anti-ZP2 antibody, the two wheat germ lectins agglutinin (WGA) and phytohemagglutinin-1.
    [0034]
    [0035] The document “Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa”, published in 2016 by Vasquez ES; Feugang JM; Willard ST; Ryan PL; Walters KB, which describes bioluminescent magnetic nanoparticles as potential agents for the bioimaging of mammalian sperm. Bioluminescent compounds comprising magnetic nanoparticles and the firefly luciferase (Photinus pyralis) are analyzed as potential light emitting agents for imaging, detection and monitoring of mammalian sperm.
    [0036]
    [0037] The document “Lectin-Functionalized Magnetic Imn Oxide Nanoparticles for Reproductive Improvement ', published in 2015 by Feugang JM; Shengfa FL; Crenshaw MA; Clement H; Willard ST; Ryan PL, describes the use of biofunctionalized iron oxide magnetic nanoparticles by lectins as a new strategy for purification and selection of sperm cells to increase semen fertility in assisted reproduction methods.
    [0038]
    [0039] The document “The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters”, published in 2016 by Algarra B; Han L; Soriano-Ubeda C; Aviles M; Coy P; Jovine L; Jimenez-Movilla M. analyzes the function of the C-terminal region of the OVGP1 protein ('Oviduct-specific glycoprotein 1') in the process of binding OVGP1 to the extracellular area of the ovocyte and in the activity of the protein during fertilization.
    [0040]
    [0041] However, it is not observed that any of the foregoing patents or documents, taken separately or in combination, describe the present invention, as claimed.
    [0042]
    [0043] EXPLANATION OF THE INVENTION
    [0044]
    [0045] The procedure for preparing oocytes and / or magnetic embryos by means of nanoparticles for assisted reproduction techniques and for the use in techniques for assisted reproduction of oocytes and / or non-human embryos prepared with said procedure that the invention proposes is therefore configured as a remarkable novelty within its scope, the characterizing details that distinguish them conveniently included in the final claims that accompany the present description.
    [0046]
    [0047] As noted above, what the invention proposes is a procedure for magnetizing oocytes and / or embryos of the different animal species based on the use of magnetic nanoparticles conjugated with OVGP1 protein present in the oviductal fluid and in this case expressed recombinantly (OVGPIr) that has affinity for the external part of the oocytes / embryos, called the pellucid zone (ZP), which provides a very useful property in the management of such oocytes / embryos in the Different assisted reproduction techniques.
    [0048]
    [0049] Specifically, the procedure proposed by the invention comprises at least the following steps:
    [0050]
    [0051] - A first stage in which magnetic nanoparticles are conjugated (binding process) with the recombinant oviductin protein (OVGP1r). Said protein is used for its ability to interact with the extracellular matrix, ZP, of the oocyte / embryo naturally when oocyte and embryo transit through the oviduct occurs.
    [0052]
    [0053] Specifically, a truncated OVGP1r recombinant protein is used, that is, it lacks the D-region of the C-terminal end, region responsible for the endocytosis of this protein, since this truncated form of OVGP1r binds better to the outer zone of the ZP of the oocyte / embryo that completes the protein and is also not endocrited.
    [0054]
    [0055] Through different experiments, it is verified that said conjugation (union between the nanoparticles and the OVGP1r protein) is a strong and stable union over time.
    [0056]
    [0057] - A second stage in which it is checked whether said nanoparticle-OVGP1r conjugate, in turn, binds to the oocyte / embryo ZP after its joint incubation.
    [0058]
    [0059] For this, the oocytes / embryos are co-incubated with the nanoparticles-OVGP1r at different concentrations over time (up to 24 h of co-incubation).
    [0060]
    [0061] - And, after the corresponding co-incubation time has elapsed, a third stage in which, after checking the number of oocytes / embryos that have the nanoparticles attached and that they are distributed, more or less homogeneously, around the ZP without being endocitated, it is evaluated whether the amount of magnetic nanoparticles attached to the oocyte / embryo ZP is sufficient for said oocytes / embryos to be attracted by a magnetic field.
    [0062] For this, a magnetic device is used where it is verified that approximately 80% of oocytes / embryos with the attached nanoparticles are attracted by the magnetic field.
    [0063]
    [0064] It should be mentioned that, preferably, the diameter of the nanoparticles is between 1 and 500 nanometers and that, also preferably, the conjugation temperature between the nanoparticles and the O VG PIr is between 4 ° C and 38 ° C.
    [0065]
    [0066] In any case, this magnetic capacity represents a great advance in the manipulation of oocytes and embryos, either for their movement to specific places through the application of a mobile magnetic field or to keep them immobile through a fixed magnetic field. The use of this technology is of great interest in assisted reproduction techniques in different animal species. One of the major limitations in reproduction techniques is the handling and manipulation of oocytes and embryos, since they require conditions of maximum control to preserve their fertilizing quality in the case of the oocyte or development in the case of the embryo. Processes such as in vitro maturation of oocytes, artificial insemination, in vitro fertilization , embryo culture and development or vitrification require the manipulation of oocytes and embryos, both to displace them and provide the modifications of media and reagents or to immobilize them in supports that allow their transfer. On the other hand, the determination of the best embryonic quality for the choice of the embryo that is going to be implanted requires the observation of its development during several hours by means of visualization techniques ( time-lapse) where the immobilization of the embryo is fundamental for its focus during long time. Finally, the magnetic nature of the particles adhered to the ZP could be used for the location of the embryo in the female genital tract by magnetic resonance imaging or other imaging techniques.
    [0067]
    [0068] It is important to highlight the differentiating fact of the technique of adhesion of nanoparticles to oocytes and embryos. Unlike methodologies used in oocyte labeling in which a lectin (exogenous proteins from plants) or antibody is used as a binding element, in this case we use an endogenous recombinant protein typical of the species and which is found in natural conditions attached to the outer part of oocytes and embryos in vivo. The fact that it is a recombinant protein makes the methodology easily transferable to any species, since only the protein should be used recombinant OR VG PIr characteristic of the species. On the other hand, this same fact, makes this protein can be easily generated together with fluorescent proteins which will allow easy detection.
    [0069]
    [0070] Therefore, a second aspect of the invention relates to the use of oocytes and / or non-human magnetic embryos, prepared according to the procedure described above, in assisted reproduction techniques in the different animal species, such as gamete manipulation or maturation In vitro oocytes, artificial insemination, in vitro fertilization in fertility treatments, vitrification or culture, monitoring and embryonic development, due to the magnetic capacity acquired by oocytes and / or embryos with this procedure, allows their movement to specific places through the application of a mobile magnetic field or to keep them immobile by means of a fixed magnetic field, their immobilization for visualization by means of microscopy and image techniques ( time-lapse), by the ability to retain the magnetic field of oocytes and / or magnetized embryos , as well as its detection, monitoring and visualization through of the female genital tract by magnetic resonance, by the ferric and magnetic nature of the nanoparticles.
    [0071]
    [0072] Given the above, it is found that the procedure for preparing oocytes and / or magnetic embryos using nanoparticles for assisted reproduction techniques and the use of said oocytes and / or non-human embryos in assisted reproduction techniques represent an innovation of unknown characteristics up to now for the purpose it is intended, reasons that together with its practical utility, provide it with sufficient grounds to obtain the privilege of exclusivity that is requested.
    [0073]
    [0074] PREFERRED EMBODIMENT OF THE INVENTION
    [0075]
    [0076] Specific examples of the process of the invention applied in porcine oocytes are described below.
    [0077]
    [0078] - Conjugation of the OVGP1r with magnetic nanoparticles (MNPs):
    [0079]
    [0080] In this experiment, a Small Carboxyl-Modified Paramagnetic Microspheres (-COOH) (Estapor®) MNPs model was used. These ferric MNPs have a high capacity magnetic, with a diameter of 0.365 jm and a concentration of 1 mg / ml. For its handling a magnetic rack was used (GE Healthcare, Little Chalfont, United Kingdom).
    [0081]
    [0082] For the conjugation of the VG PIrcon with the MNPs, 10 | jl (1 mg MNPs / ml) of MNPs were used. First, the MNPs were washed twice in 500 ml of milli-Q water and resuspended in 240 ml of activation buffer (100 mM Sodium Phosphate, pH 6.2), 30 ml of conjugate buffer 1- (3-dimethylaminopropyl) - 3-ethylcarbodiimide HCl or EDC (50 mg / ml diluted in water) and 30 jl of Sulfo NHS conjugation buffer (50 mg / ml diluted in water). Then, 20 minutes were kept under stirring at room temperature. After this time, the MNPs were washed twice in 500 jl of coupling buffer (0.1 M sodium bicarbonate, pH 8) and incubated with 15 jl (corresponding to 6 jg) of OVGP1r (0.4 mg / ml) in 300 jl of Coupling buffer (0.1 M sodium bicarbonate, pH 8) overnight at 4 ° C or at room temperature for 2 hours.
    [0083]
    [0084] The next day (or after 2 hours) the MNPs were washed twice in PBS (20 mM sodium phosphate buffer). All experiments were performed with a battery of buffers to improve the conjugation and storage conditions of the MNPs-OVGP1r complex.
    [0085]
    [0086] - Electrophoresis and Western Blot:
    [0087]
    [0088] In order to verify that the conjugation of the OVGP1 r with the MNPs had been carried out successfully, an electrophoresis and a Western Blot were performed.
    [0089]
    [0090] First, the MNPs were incubated with the loading buffer 10 minutes at 100 ° C. Next, electrophoresis was performed at 400 milliamps and 200 volts for 40 minutes. A commercialized polymerized acrylamide-bisacrylamide gel was used: NovexTM WedgeWellTM 4-20% Tris-glycine 1.0mm x 100 Well gel (InvitrogenTM).
    [0091]
    [0092] After electrophoresis, the proteins in the gel were transferred to a polyvinylnedifloride (PVDF) membrane with a pore size of 0.45 jm (Millipore Corporation, Bedford) previously treated according to the manufacturer's instructions. The transfer was carried out in XCell II TM Blot Module (Invitrogen TM) at 30 volts and 400 milliamps for 1 h and 6 minutes.
    [0093] Once the transfer was complete, the membrane was washed three times with TBS-T (50 mM Tris, pH 7.4, 150 mM NaCl and 0.1% Tween 20). Subsequently, the membrane was blocked with TBS-T with 1% bovine serum albumin (BSA, Sigma-Aldrich) for 1 hour under slow stirring at room temperature or at 4 ° C overnight. The membrane was then washed with TBS-T three times for 10 minutes each wash and incubated with the anti-rabbit antibody-HRP (Santa Cruz Biotechnology) at a concentration of 1: 40,000 for 1 hour at room temperature.
    [0094]
    [0095] Finally, the membrane was washed three times for 10 minutes with TBS-T and developed using the Pierce ECL-Plus reagent (Thermo Scientific). To detect the protein, the ImageQuant ™ LAS 500® image analyzer was used.
    [0096]
    [0097] - Obtaining and in vitro maturation of porcine oocytes:
    [0098]
    [0099] The oocytes used were obtained from prepubertal sows from slaughterhouse. The ovaries were transported to the laboratory in a sterile saline solution at 38.5 ° C within the first hour after the animals were slaughtered. Once in the laboratory, the ovaries were washed with bromide hexadecyltrimethylammonium (CTAB) and saline at 38.5 ° C.
    [0100]
    [0101] To obtain oocytes, follicles with a size of 3 to 6 mm were aspirated using 10 ml syringes and sterile needles of 18x40 mm. Follicular fluid was deposited in 15 ml conical tubes placed on a thermal plate at 38.5 ° C.
    [0102]
    [0103] After 5 min, the supernatant was removed, the pellet was washed with PBS at 38.5 ° C and deposited on a Petri dish. The cluster-oocyte complexes (COCs) were selected by aspiration, using a stereoscope (Motic SMZ-168) and a Pasteur pipette made of heat-elongated glass and connected to a silicone cannula. The oocytes were selected with a homogenous cytoplasm and at least three layers of cluster cells.
    [0104]
    [0105] The selected COCs were washed twice in warm PBS before being transferred to the NCSU-37 culture medium, previously equilibrated at 38.5 ° C in the incubator with 5% CO 2 and a saturated atmosphere of humidity for at least 3 hours. The COCs were grown in groups of 50-55 in a volume of culture medium of 0.5 ml. The first 20-22 hours of cultivation, the COCs were grown in the presence of pregnant gonadotropin from pregnant mare (PMSG), human chorionic gonadotropin (HCG) and dibutyryl cAMP. After these initial hours, the COCs were transferred to a NCSU-37 medium without hormones, where they were first washed once and then grown for another 20-22 hours. When the 40-44 hours of COC culture were finished, they were mechanically decumulated by gentle pipetting until the cluster cells separated. Then, only the oocytes without cluster cells were selected and washed in Tyroid's albumin-lactate-pyruvate (TALP) medium.
    [0106]
    [0107] - Experiment 1. Conjugation of the OVGP1r with MNPs
    [0108] In the first experiment, the MNPs were conjugated with OVGP1r for 2 hours at room temperature or at 4 ° C overnight. For the management of MNPs, it is important to mix the contents well by stirring.
    [0109]
    [0110] Next, 10 µl of MNPs were taken and mixed with 500 µl of milli-Q water in an Eppendorf. Using a magnetic rack (GE Healthcare), the MNPs were separated from the medium. After conjugation, the total volume of conjugated MNPs (MNPs-OVGP1r) was 300 µl at a final concentration of 0.033 mg of MNPs / ml.
    [0111]
    [0112] Once the conjugation was finished, it was checked by electrophoresis and Western Blot. The fractions analyzed were: (A) 25 ^ l of water with 6 ^ g of OVGPIr, (B) 25 ^ l of the medium that was collected after conjugation (to evaluate the amount of protein that had not been bound), (C1 and C2) 25 ^ l of the washes that were performed after conjugation (to ensure that the protein was not being lost in these washes) and (D) 25 ^ l of the medium with the O VG PIr that had joined the MNPs. The loading buffer (8.33 ^ l) in each fraction was added and incubated 10 minutes at 100 ° C. Before loading the electrophoresis gel, the fractions were washed with the nanoparticles two to four times to ensure that the medium was clean.
    [0113]
    [0114] It was also evaluated if the O VG PIr remained bound to the MNPs after 72 hours stored at 4 ° C in 20 mM sodium phosphate buffer (PBS).
    [0115]
    [0116] - Experiment 2. Co-incubation of MNPs or VG PIr with porcine oocytes
    [0117] Once the MNPs were properly conjugated with the OVGPIr protein, the in vitro maturation of porcine oocytes was carried out.
    [0118] The oocytes were divided into three groups and incubated as follows:
    [0119] • Control group: the oocytes were incubated with 20 μl (concentration of 1.33 jg MNPs / ml) of MNPs without the conjugated OVGP1.
    [0120] • Group of 10 | jl: the oocytes were incubated with 10 | jl (0.67 | jg MNPs / ml) of MNPs-OVGPIr.
    [0121] • Group of 20 ml: the oocytes were incubated with 20 ml of MNPs-OVGPIr.
    [0122]
    [0123] Each group was placed in a well with TALP medium and stored in the incubator at 38.5 ° C and 5% CO 2 .
    [0124]
    [0125] The union between the oocytes and the MNPs was evaluated at different times: 0.5, 1, 6 and 24 h of co-incubation. After these times, some oocytes were separated from each group, washed in TALP medium and transferred to a well with 0.5 ml of TALP medium.
    [0126]
    [0127] Next, the oocytes were subjected to a magnetic field in order to evaluate the number of magnetized oocytes. For this, the 0.5 ml of TALP medium with the oocytes was transferred to an Eppendorf tube that was placed in a magnetic rack. Then, the medium was collected slowly (with a 500 micron automatic micropipette) without removing the Eppendorf from the magnetic rack, and it was placed back in the well. The number of oocytes in the well was counted and noted as these represent the number of oocytes that had not been retained by the magnet. Finally, the magnet was removed and the TALP medium (with the oocytes) was passed again through the Eppendorf tube to recover the oocytes that had joined in the presence of the magnet (referred to as the percentage of magnetized oocytes).
    [0128]
    [0129] Describing sufficiently the nature of the present invention, as well as the way of putting it into practice, it is not considered necessary to make its explanation more extensive so that any person skilled in the art understands its scope and the advantages that derive from it, stating that, within its essentiality, it may be carried out in other embodiments that differ in detail from that indicated by way of example, and to which it will also achieve the protection that is sought provided that it does not alter, change or modify its fundamental principle .
    权利要求:
    Claims (12)
    [1]
    1. - Procedure for preparing oocytes and / or magnetic embryos using nanoparticles for assisted reproduction techniques in different animal species, it is characterized by comprising the following stages:
    - A first stage in which, by binding process, magnetic nanoparticles with oviductin protein (OVGP1) are conjugated; specifically, with a recombinant protein OVGP1r.
    - A second stage in which, after their joint incubation, it is found that the nanoparticles-OVGP1r conjugate, in turn, binds to the oocyte / embryo ZP.
    - And, after the co-incubation time has elapsed, a third stage in which the number of oocytes / embryos that have the nanoparticles together and that these are distributed, more or less homogeneously, around the ZP is checked without being endocitated, and that the amount of magnetic nanoparticles attached to the oocyte / embryo ZP is sufficient for the oocytes / embryos to be attracted by a magnetic field.
    [2]
    2. - Procedure for preparing oocytes and / or magnetic embryos, according to claim 1, characterized in that, to verify that the nanoparticles-OVGP1r conjugate binds to the oocyte / embryo ZP, the oocytes / embryos are co-incubated with the OVGP1r nanoparticles at different concentrations over a period of up to 24 hours of co-incubation.
    [3]
    3. - Procedure for preparing oocytes and / or magnetic embryos, according to claim 1 or 2, characterized in that, to evaluate whether the amount of magnetic nanoparticles attached to the ZP of oocytes / embryos are sufficient for the oocytes / embryos to be attracted , these are subjected to a magnetic field.
    [4]
    4. - Procedure for preparing oocytes and / or magnetic embryos, according to claim 3, characterized in that the amount of magnetic nanoparticles attached to the ZP of oocytes / embryos is sufficient for the oocytes / embryos to be attracted when 80% of oocytes / embryos with attached nanoparticles are attracted to the magnetic field.
    [5]
    5. - Procedure for preparing oocytes and / or magnetic embryos, according to any of claims 1 to 4, characterized in that the diameter of the nanoparticles is between 1 and 500 nanometers.
    [6]
    6. - Procedure for preparing oocytes and / or magnetic embryos, according to any of claims 1 to 5, characterized in that the conjugation temperature between the nanoparticles and the O VG PIr is comprised between 4 ° C and 38 ° C.
    [7]
    7. - Use in techniques of assisted reproduction of oocytes and / or non-human embryos prepared with a procedure as described in any of claims 1 to 6.
    [8]
    8. - Use in techniques of assisted reproduction of oocytes and / or non-human embryos, according to claim 7, characterized in that it includes gamete manipulation.
    [9]
    9. - Use in assisted reproduction techniques oocytes and / or embryos according to claim 7, characterized by comprising the in vitro oocyte maturation,
    [10]
    10. - Use in assisted reproduction techniques of oocytes and / or non-human embryos, according to claim 7, characterized in that it comprises artificial insemination.
    [11]
    11. - Use in assisted reproduction techniques of oocytes and / or non-human embryos, according to claim 7, characterized in that it comprises in vitro fertilization in fertility, vitrification or culture treatments.
    [12]
    12. - Use in techniques of assisted reproduction of oocytes and / or non-human embryos, according to claim 7, characterized by understanding the monitoring and embryonic development.
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    同族专利:
    公开号 | 公开日
    ES2724873B2|2021-05-18|
    EP3766542A1|2021-01-20|
    US20210047658A1|2021-02-18|
    WO2019175449A1|2019-09-19|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP2237039A1|2009-04-03|2010-10-06|Universitat Autònoma De Barcelona|Encoded microparticles|
    US20160091410A1|2012-08-29|2016-03-31|Inguran, Llc|Cell processing using magnetic particles|
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    优先权:
    申请号 | 申请日 | 专利标题
    ES201830239A|ES2724873B2|2018-03-11|2018-03-11|PROCEDURE FOR THE PREPARATION OF OOCYTES AND / OR MAGNETIC EMBRYOS USING NANOPARTICLES FOR ASSISTED REPRODUCTION TECHNIQUES AND USE IN ASSISTED REPRODUCTION TECHNIQUES OF NON-HUMAN OOCYTES AND / OR EMBRYOS PREPARED WITH SAID PROCEDURE|ES201830239A| ES2724873B2|2018-03-11|2018-03-11|PROCEDURE FOR THE PREPARATION OF OOCYTES AND / OR MAGNETIC EMBRYOS USING NANOPARTICLES FOR ASSISTED REPRODUCTION TECHNIQUES AND USE IN ASSISTED REPRODUCTION TECHNIQUES OF NON-HUMAN OOCYTES AND / OR EMBRYOS PREPARED WITH SAID PROCEDURE|
    EP19717959.1A| EP3766542A1|2018-03-11|2019-02-26|Magnetic nanoparticles for use in assisted reproduction|
    PCT/ES2019/070108| WO2019175449A1|2018-03-11|2019-02-26|Magnetic nanoparticles for use in assisted reproduction|
    US16/980,364| US20210047658A1|2018-03-11|2019-02-26|Magnetic Nanoparticles for use in Assisted Reproduction|
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