• 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:
    Device for the cryopreservation of a sample of ovules or embryos by vitrification comprising a support (1) having a single piece and comprising a slender fastening portion (2) configured to hold and label the sample and which joins with an intermediate portion (3) connecting with a tip (4), where said tip (4) has a distal end (5) of pointed shape and comprises a through hole (6) with an elliptical configuration comprising a maximum width of 0.6 mm intended to form a thin film of vitrification medium on which the ovules or embryos are deposited to vitrify by direct contact with liquid nitrogen. The device by its geometry, generates a tangential force taking advantage of the surface tension of the liquid to retain and envelop the cells, favoring even more to reach ultra-fast speeds of cooling and heating. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2713800A1
    申请号:ES201930351
    申请日:2019-04-16
    公开日:2019-05-23
    发明作者:Jiménez Francisco Marco；Antón José Salvador Vicente；Valero Luis García；Domínguez Ximo García
    申请人:Universidad Politecnica de Valencia；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] OBJECT OF THE INVENTION
    [0005]
    [0006] The present invention discloses a device for the cryopreservation of ovules and embryos based on the vitrification technique. During the vitrification, ovules and embryos are handled within the vitrification medium, whose volume has to be reduced to the maximum to guarantee the success of the technique. Thus, the device presented, by its geometrics, generates a tangential force on the deposited sample that allows to generate a thin pellicle of vitrification medium for the storage of ovules or embryos in biobanks. Likewise, the device allows storage both individually and in groups, as well as the visualization of the process under an optical microscope to verify the deposition and morphology of the cells.
    [0007]
    [0008] BACKGROUND OF THE INVENTION
    [0009]
    [0010] The preservation of ovules, embryos and tissues is a technique required in various fields. In reproductive medicine, since the birth of the first human being conceived through in vitro fertilization in 1978, more than 6.5 million babies have been born with the use of assisted reproduction techniques (ART).
    [0011]
    [0012] In recent years, and thanks to the improvement in the efficiency of the vitrification technique, there has been a notable increase in ART cycles that include vitrification, thus maximizing the efficiency of the ovarian stimulation cycles, by allowing the storage of excess ovules or embryos for later use.
    [0013]
    [0014] In the treatment of human fertility, both ovules and embryos are vitrified and stored until the most appropriate time for transplantation. Among other things, the use of cryopreservation allows avoiding the so-called "suboptimal" endometrium that occurs after the application of ovarian stimulation treatments as a consequence of the supra-physiological hormone levels, thus improving the implantation and pregnancy rates. this, the cryopreservation of human embryos is currently more important than ever for the pregnancy rate accumulated in TRA. specifically in cattle, more than 900,000 embryos are obtained annually, being mostly commercialized after their cryopreservation. In pigs, and unlike cattle, so far no commercial device that allows the commercial use of embryos is commercialized. The straw-type supports of different diameter and capacity, handle or tongue have been widely disclosed in the state of the art. These were developed in order to obtain ultra-fast speeds in the temperature exchange that favored the vitrification of ovules and embryos. This was achieved by minimizing the volume of the vitrification medium, passing from the classic use of straws of 250-125 microliters to volumes of 1 microliter. Thus, in 1985, Rall and Fahy managed to vitrify mouse embryos for the first time, since vitrification became the optimal procedure for the cryopreservation of ovules and embryos. It was not long after, when Dr. Arav and his colleagues introduced the idea of using the vitrification technique, but in a small drop that they ended up defining it as the "minimum size of essential drop" (Arav, 1989: 1992: 2014; et al., 1987: 2002). Currently all vitrification devices are based on this concept (Cobo et al., 2008). Currently, and under our knowledge, this same concept is used by other commercial devices such as Cryotop, Cryolock, Cryotech, endorsed by the increase in their survival results, but do not use the technical solution of a handle. Also, to date the devices known by the applicant that allow operating with minimum volumes have a storage capacity limited to a reduced number of ovules or embryos.
    [0015]
    [0016] Thus, for example, Mavrides & Morroll mention a device of this type manufactured for the cryopreservation of bovine ovules, describing a 1mm diameter loop with a circular shape. Other modifications allow to increase the number of cells to be deposited in the support, as occurs with the device disclosed by Cardona-Costa and Garcia-Ximenez (although the volume used is 250 microliters), or result in the reduction of manufacturing costs by providing at the same time, a greater ease of handling or labeling of the sample, as shown in the Chinese utility model CN205196832U.
    [0017]
    [0018] A vitrification support with minimal essential volume is unknown in a closed system that also allows to store a considerable number of ovules and embryos, for use in polltocas species such as porcine or rabbit. Furthermore, in the existing devices to date, the reduction of the volume of vitrification to said "minimum essential volume" requires a high technical capacity on the part of the manipulator.
    [0019] DESCRIPTION OF THE INVENTION
    [0020]
    [0021] The present invention provides an easy-to-use device for the cryopreservation of ovules and embryos by vitrification which, in addition, increases the number of ovules or embryos that can be stored. That is, the surface tension generated by the vitrification solution that contains the cells when depositing them in the device object of the patent guarantees a minimum volume around them, thus avoiding the need to do more operations to eliminate the excess solution of vitrification (for example, direct suction in a micropipette, or by contact with absorbent elements). The device also allows an easy, reliable and efficient devitrification of the cells. More specifically, the device allows a minimum layer of vitrification medium around the ovule and the embryo, taking advantage of the forces due to the surface tension of the liquid, to retain and wrap the cells by a minimum layer of vitrification medium, favoring even more to reach ultra-fast speeds of cooling and heating, which represents a remarkable technical improvement over the solutions in the state of the art.
    [0022]
    [0023] The present invention consists of a support having a single piece and comprising a portion of slender fastening configured to hold and label the sample, which with the intermediate portion connecting with a tip, a distal end with a pointed shape comprising a hole with an elliptical configuration whose internal axes have measures of 0.6 mm and 8.8 mm. This geometry is what allows to form a thin film of vitrification medium, which includes the ovules or embryos to be vitrified by direct contact with liquid nitrogen.
    [0024]
    [0025] Preferably, the device further comprises a protective sheath intended to seal the support hermetically, thus protecting the sample from microbiological contamination during storage. The pointed end distal end of the object facilitates the introduction of the support into the sheath when operating under liquid nitrogen.
    [0026]
    [0027] The device provides as a fundamental advantage the capacity of the support to house multiple ovules and embryos with a minimum volume of vitrification solution without the need to carry out elimination operations of the excess of said solution, which allows to obtain high cooling and heating rates . In addition, it is easy to use and allows a simple labeling of the sample in the area of attachment.
    [0028] Preferably the through hole of elliptical configuration whose external axes have measures of 1.8 mm and 10 mm. The support measures longitudinally between 60 and 140 mm, where the intermediate portion measures approximately 30-50% of the length of the support and the portion of slender hold measures approximately between 30-40% of the length of the support.
    [0029]
    [0030] Preferably, the slender fastening portion is rectangular with a length / width ratio of a magnitude 10. The width can be 3 mm and the length 30 mm. The intermediate portion can be truncated.
    [0031]
    [0032] Preferably the elliptical configuration through hole in the tip has a width of 0.6 mm and a length of 10 mm, with a coating on the outside with a width of 1.8 mm, the trunk truncated portion measures 65 mm and the holding portion 30 mm .
    [0033]
    [0034] In addition, the support comprises a protective sheath for sealing the device to prevent microbial and viral infection / contamination during storage. In addition, the termination of the arrow-shaped support facilitates the introduction of the protective cover under the liquid nitrogen.
    [0035]
    [0036] The device is easy to use and its geometry allows the ovules and embryos to be surrounded by the minimum volume (minimum essential volume), which contributes to generate high speeds to reach the vitreous state. This also entails a great ease of use by the manipulator, since, once the sample is deposited in the support, no additional steps are required to reduce the volume of cryoprotective solution. Likewise, the device allows individualized storage as well as a high number of ovules or embryos. The supporting portion of the support allows a labeling with the relevant information of easy visualization. In addition, the geometry of the fastening portion guarantees a tight seal with the protective cover. In this way, the direct contact of the biological sample with the liquid nitrogen of the biobank during its storage is avoided, thus avoiding! its potential microbiological / viral contamination.
    [0037]
    [0038] EXAMPLE
    [0039]
    [0040] The support has been validated with rabbit embryos, demonstrating its functionality both in the in vitro development of these, and in the number of animals born. To do this, 15 donor females were used that after receiving a superovulation treatment (3 pg of Corifollitropin alfa) were artificially inseminated (AI). At 3 days, the embryos were recovered and cryopreserved by vitrification according to the protocol developed by Vicente et al. (1999), using both the device object of the patent, and 2 commercial devices: Cryotop (Kitazato Co., Fuji, Japan) as the most widely used system in humans and 0.125 mL mini-straw (French ministraw, IMV, L ' Aigle, France) as a conventional system used in rabbit. Briefly, the vitrification was carried out in two steps. In the first step, the embryos are deposited in a solution containing 12.5% (v / v) of dimethylsulfoxide (DMSO) and 12.5% (v / v) of ethylene glycol (EG) as cryoprotective agents for 2 minutes. In the second step, the embryos are deposited in a solution containing a cryoprotectant concentration of 20% DMSO and 20% EG, these being recovered at 45 seconds to be deposited in the vitrification devices, and then submerged directly in liquid nitrogen, ensuring that the incubation with the vitrification solution does not exceed 1 minute. After storage in the liquid nitrogen tanks, the embryos were devitrified, evaluating the effect of the device in both in vitro (embryonic development) and in vivo conditions (individuals born). The vitrification medium was removed by washing the embryos in a Dulbecco's phosphate buffered saline solution (DPBS) with sucrose (0.33 M) for 5 minutes, followed by a bath in a single solution of DPBS for another 5 minutes.
    [0041]
    [0042] Next, the effects of the device on the in vitro development during experimentation are detailed. The number of embryos that were loaded in each of the devices was 25 for the device object of the patent, 6 for each Cryotop® and 25 in each mini-straw. Thus, a total of 216 vitrified embryos were cultured (65 patent device, 68 Cryotop® and 61 mini-straw) and 22 non-vitrified embryos (control) through 7 experimental sessions during 48 h in TCM199 culture medium. contains 10% fetal bovine serum (vv) (FBS) and 1% (vol / vol) of antibiotics (sodium penicillin G 300000 IU / L, procaine penicillin G 700 000 IU / L and dihydrostreptomycin sulfate 1250 mg / L; Divasa Farmavic, Barcelona, Spain) at 38.5 ° C and 5% CO2 in a humidified atmosphere. The development capacity until the blastocyst stage was hatched was recorded for analysis.
    [0043]
    [0044] Next, the effects of the device on the percentage of animals born during experimentation are detailed. A total of 288 vitrified embryos (102 device subject of the patent, 100 Cryotop® and 86 mini-straw) and 96 fresh embryos were transferred to 25 nullparas females. The embryos were transferred via oviductal by laparoscopy (Besenfelder and Brem, 1993). During laparoscopy, the recipients were anesthetized with an intramuscular injection of xylazine (5 mg / kg), followed at 5 minutes by an intravenous injection of ketamine hydrochloride (35 mg / kg). During laparoscopy, it was administered by intramuscularly a dose of morphine hydrochloride (3 mg / kg). At the end of the surgery, the animals were treated with antibiotics (4 mg / kg of gentamicin every 24 h) and analgesics (0.03 mg / kg of buprenorphine hydrochloride every 12 h and 0.2 mg / kg of meloxicam every 24 h) for 3 days.
    [0045]
    [0046] Embryo development rates up to the stage of blastocysts hatched after 48 h of in vitro culture were similar between the experimental groups (89.2%, 88.2%, 90.9% and 93.4%, for the device object of the patent, Cryotop®, mini -pajuela and control group, respectively, P <0.05).
    [0047]
    [0048] Regarding the offspring rate, this was similar between the groups of vitrification devices, but as expected, significantly lower than the control group (61 ± 4.8%, 65 ± 4.8%, 52 ± 4.5% vs 74 ± 5.2% , for the device object of the patent, Cryotop®, mini-straw vs. control embryos, respectively, P <0.05).
    [0049]
    [0050] DESCRIPTION OF THE DRAWINGS
    [0051]
    [0052] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to a preferred example of practical realization thereof, a set of drawings is included as an integral part of said description. where with illustrative and non-limiting character, the following has been represented:
    [0053]
    [0054] Figure 1 shows a perspective view of the support of the device for the cryopreservation of a sample of ovules or embryos by vitrification.
    [0055]
    [0056] Figure 2.- Shows a perspective view of the support and the protective sheath of the device for the cryopreservation of a sample of ovules or embryos by vitrification.
    [0057]
    [0058] Figure 3 shows a detailed view of the deposition zone of the cells at the tip of the support in the elliptical configuration through hole.
    [0059]
    [0060] PREFERRED EMBODIMENT OF THE INVENTION
    [0061]
    [0062] Figure 1 shows a perspective view of the support (2) of the device for the cryopreservation of a sample of ovules or embryos by vitrification according to an embodiment preferred where it is clearly seen that the support is a single piece of plastic injected or printed in 3D, and comprising a portion of slender fastener (2) configured to hold and label the sample that joins with an intermediate portion (3) that connects with a tip (4).
    [0063]
    [0064] It is further shown that the tip (4) has a distal end (5) of pointed shape and comprises a through hole (6) that has an elliptical configuration comprising a maximum width in one of its 0.6 mm axes intended to form a thin film of vitrification medium on which the ovules or embryos are deposited to vitrify by direct contact with liquid nitrogen.
    [0065]
    [0066] According to the preferred embodiment described in figure 2, the through hole (6) of elliptical configuration (4) has a length of 10 mm, with an outer covering (8) with a width of 1.8 mm and also defining an elliptical configuration. The intermediate portion is frustoconical and measures substantially 40 mm. The slender fastening portion (2) is rectangular and has a length of 30 mm and a width of 3 mm. The total length of the support is 70 mm.
    [0067]
    [0068] Figure 2 shows a perspective view of the support (1) and the protective cover (7) of the device where it is observed that the protective cover (7) is designed to receive the support (1) hermetically and the distal end (5) with pointed shape facilitates the introduction of the support in the sheath (7) when operating under liquid nitrogen and said sheath (7) also protecting the sample from a potential microbial and viral infection / contamination during its storage in the biobank.
    [0069]
    [0070] Figure 3 shows a detailed view of the deposition zone of the ovules or embryos at the tip of the support (1) in the through hole (6) of elliptical configuration, taking advantage of the surface tension generated by the vitrification solution containing the ovules or embryos to deposit them in the device, ensuring a minimum volume around them and avoiding the need to do more operations to eliminate the excess vitrification solution (for example, direct suction in a micropipette, or by contact with absorbent elements). In addition, the device allows easy, reliable and efficient thawing of ovules and embryos.
    [0071]
    [0072] The device allows a minimum layer of vitrification medium around the ovule and the embryo, taking advantage of the forces due to the surface tension of the liquid, to retain and wrap the cells by a minimum layer of vitrification medium, favoring even more reaching Ultra-fast cooling and heating speeds which means a remarkable technical improvement compared to the solutions in the state of the art.
    [0073]
    [0074] The device is easy to use and its geometry allows the ovules or embryos to be surrounded by the minimum volume (minimum essential volume), which contributes to generating high speeds to reach the viral state. This also entails a great ease of use by the manipulator, since, once the sample is deposited in the support, no further steps are required to reduce the volume of cryoprotective solution. Likewise, the device allows individualized storage as well as a high number of ovules or embryos. In Figure 2, the holding portion (2) of the support (1) allows a labeling with the relevant information of easy visualization. In addition, the geometry of the fastening portion (2) ensures a tight seal with the protective cover (7). In this way, the direct contact of the biological sample with the nitrogen of the storage bank is prevented thus preventing! a potential microbial and viral infection / contamination during storage in the biobank. The finishing of the device in the form of a pointed tip (5) facilitates its introduction into the cover when operating under immersion of liquid nitrogen used for vitrification.
    权利要求:
    Claims (1)
    [0001]
    1. - Device for the cryopreservation of a sample of ovules or embryos by vitrification comprising a support (1) that has a single piece and comprises a portion of slender hold (2) configured to hold and label the sample and that joins with a intermediate portion (3) that connects with a tip (4), characterized in that said tip (4) has a distal end (5) of pointed shape and comprises a through hole (6) with an elliptical configuration that has a maximum width of 2.5 mm and a maximum length of 20 mm intended to form a thin film of vitrification medium on which the ovules or embryos are deposited to vitrify by direct contact with liquid nitrogen.
    2. - Device for cryopreservation of ovules or embryos according to claim 1, characterized in that it comprises a protective sheath (7) designed to receive the support (1) hermetically where the distal end (5) with pointed shape facilitates the introduction of support in the sheath (7) when operating under liquid nitrogen and protecting said sheath (7) from a potential microbial and viral infection / contamination of the sample during storage.
    3. - Device for the cryopreservation of ovules or embryos according to claim 1, characterized in that the through hole (6) with elliptical configuration has a length of 8.8 mm.
    4. - Device for the cryopreservation of ovules or embryos according to re v indication 3, characterized in that the through hole (6) with elliptical configuration has a width of 0.6 mm.
    5. - Device for the cryopreservation of ovules or embryos according to re v indication 1, characterized in that the tip (4) has a covering of the through hole (6) and has an elliptical configuration with a maximum width of 2.5 mm and a length minimum of 20 mm.
    6. - Device for the cryopreservation of ovules or embryos according to re v indication 1, characterized in that the tip (4) has a coating of the through hole (6) and has an elliptical configuration with a width of 1.8 mm and a length of 10 mm.
    7. - Device for the cryopreservation of ovules or embryos according to claim 1, characterized in that the support (1) is made of plastic material suitable for injection and / or 3D printing.
    8. - Device for the cryopreservation of ovules or embryos according to claim 1, characterized in that the support (1) longitudinally measures between 60-140 mm.
    9. - Device for the cryopreservation of ovules or embryos according to re vindication 8, characterized in that the intermediate portion (3) is frustoconical and longitudinally measures 30 50% of the total length of the support.
    10. - Device for the cryopreservation of ovules or embryos according to re v indication 8, characterized in that the slender hold portion (2) measures substantially 30-40% of the length of the support.
    11. - Device for the cryopreservation of ovules or embryos according to re v indication 1, characterized in that the slender fastening portion (2) is rectangular with a length / width ratio of a magnitude 8-12.
    12. Device for the cryopreservation of ovules or embryos according to re v indication 1, characterized in that the rectangular slender holding portion (2) comprises a width of 3 mm and a length of 30 mm.
    13. Device for the cryopreservation of ovules or embryos according to re v indication 1, which also allows the cryopreservation of spermatozoa and somatic cells.
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    同族专利:
    公开号 | 公开日
    EP3957715A1|2022-02-23|
    WO2020212638A1|2020-10-22|
    ES2713800B2|2020-06-24|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2212641T3|1998-10-14|2004-07-16|Katrina T. Forest|VITRIFICATION PROCEDURE OF A BIOLOGICAL SPECIMEN.|
    CN205196832U|2015-11-09|2016-05-04|中国农业大学|Freezing carrier of oocyte embryo glass ization|
    法律状态:
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    优先权:
    申请号 | 申请日 | 专利标题
    ES201930351A|ES2713800B2|2019-04-16|2019-04-16|DEVICE FOR THE CRYOPRESERVATION OF A SAMPLE OF OVULES AND EMBRYOS BY VITRIFICATION|ES201930351A| ES2713800B2|2019-04-16|2019-04-16|DEVICE FOR THE CRYOPRESERVATION OF A SAMPLE OF OVULES AND EMBRYOS BY VITRIFICATION|
    PCT/ES2020/070244| WO2020212638A1|2019-04-16|2020-04-16|Device for the cryopreservation of a sample of ova and embryos by means of vitrification|
    EP20791398.9A| EP3957715A1|2019-04-16|2020-04-16|Device for the cryopreservation of a sample of ova and embryos by means of vitrification|
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