WO2023026747A1 - Outil de cryoconservation pour ovule ou ovule fertilisé - Google Patents

Outil de cryoconservation pour ovule ou ovule fertilisé Download PDF

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Publication number
WO2023026747A1
WO2023026747A1 PCT/JP2022/028801 JP2022028801W WO2023026747A1 WO 2023026747 A1 WO2023026747 A1 WO 2023026747A1 JP 2022028801 W JP2022028801 W JP 2022028801W WO 2023026747 A1 WO2023026747 A1 WO 2023026747A1
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Prior art keywords
ovum
mesh
cryopreservation
ova
fertilized
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PCT/JP2022/028801
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English (en)
Japanese (ja)
Inventor
義隆 谷口
雅英 塩谷
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シーエステック株式会社
医療法人社団英ウィメンズクリニック
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Priority to CN202280057097.2A priority Critical patent/CN117980460A/zh
Priority to JP2022577775A priority patent/JP7292637B1/ja
Publication of WO2023026747A1 publication Critical patent/WO2023026747A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a cryopreservation tool for cryopreserving eggs or fertilized eggs.
  • the vitrification method is known as a technique for cryopreserving eggs or fertilized eggs (embryos).
  • the vitrification freezing method eggs or fertilized eggs (hereinafter collectively referred to as “eggs, etc.”) are immersed in liquid nitrogen together with a small amount of cryoprotectant (vitrification liquid) to rapidly cool them.
  • This is a method of freezing in an amorphous glass state without crystallizing the water inside and outside the cells. According to this method, it is possible to suppress the damage to the eggs and the like due to ice crystal formation, and to increase the survival rate of the eggs and the like during subsequent melting.
  • cryopreservation tools are widely used that include a rod-shaped main body provided with a thin plate-shaped mounting portion for placing eggs and the like on the tip, and a cylindrical cover that accommodates the main body. used (see, for example, Patent Document 1).
  • a cryopreservation tool When freezing ova or the like using such a cryopreservation tool, first, the ova that have been immersed in a cryoprotectant in advance are collected with a pipette or the like, and placed on the mounting section together with a small amount of the cryoprotectant. place. Then, after the tip of the main body including the mounting portion is immersed in liquid nitrogen for rapid cooling, the main body is fitted with the cover and stored in a liquid nitrogen tank.
  • Patent Document 2 proposes a cryopreservation tool that can remove excess cryoprotectant with a simple operation.
  • the placement section for placing the ova and the like is formed in a mesh shape. Excess cryoprotectant can be removed by manipulation.
  • the present invention has been made in view of the above points, and its object is to make the amount of cryoprotective solution around eggs, etc. appropriate without performing complicated work, and to thaw them. To provide a cryopreservation tool from which an ovum or the like can be easily removed from a placing part.
  • the oocyte or fertilized egg cryopreservation tool according to the present invention which was made to solve the above problems, a main body having a bar-shaped handle, and a mesh-shaped ovum or the like holding section provided at the tip of the handle; a tubular cover portion in which the main body portion is accommodated; , and the opening size of the mesh in the ovum or the like holding portion is 170 ⁇ m or more.
  • cryopreservation tool for oocytes or fertilized eggs having the above configuration, it is possible to make the amount of cryoprotectant around the oocytes appropriate without performing complicated work, and furthermore, during thawing, Eggs and the like can be easily removed from the cryopreservation device.
  • FIG. 1 is a schematic diagram showing the overall configuration of a cryopreservation tool according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing a procedure for freezing ova or fertilized eggs using the cryopreservation tool.
  • FIG. 4 is a schematic diagram showing a procedure for thawing oocytes or fertilized eggs cryopreserved using the cryopreservation tool. Photographs taken from directly above showing the state in which blastocysts are held in various holding parts for ova and the like having different mesh hole sizes (opening dimensions).
  • the oocyte or fertilized egg cryopreservation tool (hereinafter simply referred to as "cryopreservation tool") according to the present embodiment includes a main body 10 and a cylindrical cover part that accommodates the main body 10. 20 and.
  • the body portion 10 includes a handle portion 11 and an ovum or the like holding portion 12 provided at the tip of the handle portion 11 .
  • the handle portion 11 is a rod-shaped member made of a low temperature resistant material.
  • the length of the handle 11 is not particularly limited, but is preferably 9 cm to 11 cm.
  • a material forming the handle portion 11 for example, a metal such as stainless steel or a synthetic resin can be used.
  • the ovum holding part 12 is composed of a mesh made of fibers 13 and has a flat plate shape with a width of 3 mm to 10 mm and a length of 5 mm to 30 mm as a whole.
  • the fiber 13 synthetic resin such as polyester can be suitably used, but the fiber is not limited to this, and for example, metal such as stainless steel may be used. If the mesh holes of the ovum holding part 12 are too small, it becomes difficult for the ova to come out of the mesh holes during melting.
  • the size of the mesh holes (opening size) of the ovum holding portion 12 in the present embodiment is 170 ⁇ m. 190 ⁇ m or more (preferably 190 ⁇ m or more).
  • the upper limit of the opening size is not particularly limited as long as the cryoprotectant can be retained by surface tension. It is desirable that the thickness is 250 ⁇ m or less (more preferably 250 ⁇ m or less).
  • the wire diameter (dimension B in FIG. 2) of the fibers 13 forming the mesh is not particularly limited, but is, for example, 40 ⁇ m to 70 ⁇ m (more preferably 50 ⁇ m to 60 ⁇ m).
  • the cover portion 20 is a thin tubular member made of a low-temperature resistant material (for example, a synthetic resin such as polypropylene, or a metal such as stainless steel). It's becoming In addition, the cover portion 20 has a length that can accommodate the entire ovum holding portion 12 of the main body portion 10 and at least a portion of the handle portion 11 .
  • a low-temperature resistant material for example, a synthetic resin such as polypropylene, or a metal such as stainless steel.
  • the procedure for freezing eggs or fertilized eggs using the cryopreservation tool according to the present embodiment will be described with reference to FIG.
  • the cryopreservation tool according to the present embodiment can be suitably used particularly for cryopreservation of human ova or fertilized eggs, but is not limited to this, and is not limited to this, and can be used for ova or fertilized eggs of animals other than humans. It can also be used for cryopreservation.
  • the "egg" to be frozen using the cryopreservation tool according to the present embodiment can include mature eggs, and the "fertilized egg” includes pronuclear stage embryos, division stage embryos (early embryos ), morula, or blastocyst, but are not limited thereto.
  • ova or fertilized eggs to be cryopreserved (hereinafter referred to as "eggs 30") are immersed in the cryoprotectant 41 to permeate the ova 30 with the cryoprotectant 41.
  • the operator collects the ovum or the like 30 together with a small amount of the cryoprotectant 41 using the pipette 42, and discharges them onto the ovum or the like holding portion 12 of the cryopreservation tool according to the present embodiment under a microscope. do.
  • the ova and the like 30 and a small amount of the cryoprotectant 41 are retained in the meshes (mesh holes) of the ovum and the like holding section 12 due to the surface tension of the cryoprotectant 41 .
  • the surplus cryoprotectant 41 that cannot be retained by the surface tension falls through the mesh of the mesh and falls below the ovum holding section 12. 41 need not be removed.
  • the operator since the mesh size of the mesh forming the ovum holding unit 12 is relatively large, the operator can visually recognize the ova 30 held in the ovum holding unit 12. It also has the advantage of being easy. Subsequently, the operator grips the handle 11 and immerses the tip of the main body 10 in the liquid nitrogen 43, thereby rapidly cooling the ova 30 and the cryoprotectant 41 held in the ovum holding unit 12. .
  • the ovum or the like 30 is held in the mesh of the mesh by the surface tension of the cryoprotectant 41, and is cooled from all directions. It is possible to improve the freezing speed of the ovum or the like 30.
  • the body portion 10 is housed in the cover portion 20 and the body portion 10 and the cover portion 20 are stored in the liquid nitrogen tank 44 .
  • an operator takes out the cryopreservation tool according to the present embodiment from the liquid nitrogen tank 44 , removes the cover part 20 from the body part 10 , and then immerses the tip of the body part 10 in the melting liquid 45 .
  • the mesh size of the mesh forming the ovum holding section 12 is relatively large, and the ova 30 can be retained by simply immersing the ovum holding section 12 in the lysing liquid 45. Since it is separated from the part 12, it is not necessary for the operator to manually remove the ovum or the like 30 from the ovum or the like holding part 12 using a pipette or the like.
  • each cryopreservation device was almost the same as that shown in FIG. 1, and the meshes constituting the ovum holding part were all made of polyester fibers with a wire diameter of 55 ⁇ m.
  • the opening size of the mesh was 100 ⁇ m (Comparative Example 1), 150 ⁇ m (Comparative Example 2), 200 ⁇ m (Example 1), 250 ⁇ m (Example 2), and 300 ⁇ m (Example 3).
  • the photograph in Fig. 5 is taken from directly above the state in which the blastocyst is held in the ovum, etc. holding part made of mesh with each of the above-mentioned opening sizes.
  • the photograph of FIG. 6 is a side view of the blastocyst and the cryoprotectant held in the ovum holding part with an opening size of 200 ⁇ m. As shown in these photographs, the blastocyst and the cryoprotectant could be retained in the meshes of the ovum, etc., regardless of the opening size of the ovum holder.
  • FIG. 7 shows the retention of early embryos 2 days after fertilization (D2)
  • FIG. 7(b) shows the retention of early embryos 3 days after fertilization (D3)
  • FIG. 7(c) shows grade 3 (G3) blastocysts (BL embryos) retained
  • FIG. 7(d) shows grade 4 (G4) blastocysts.
  • ) shows grade 5 (G5) blastocysts
  • FIG. 7(f) shows grade 6 (G6) blastocysts.
  • the size of each fertilized egg is indicated at the top of each figure.
  • fertilized eggs of various types and sizes can be stored in the mesh of the mesh. , was confirmed to be retained by the surface tension of the cryoprotectant.
  • the fertilized egg could be removed from the holding part for ova and the like simply by immersing the holding part for ova and the like after freezing in the thawing solution.
  • a cryopreservation tool having a mesh opening size of 167 ⁇ m and a cryopreservation tool having a mesh opening size of 183 ⁇ m are prepared, and human fertilized eggs are frozen using these cryopreservation tools. / Melting experiments were performed.
  • the structure of each cryopreservation device is almost the same as that shown in Fig. 1, but for the cryopreservation device with an opening size of 167 ⁇ m, the mesh that constitutes the ovum holding part is made of polyester fiber with a wire diameter of 45 ⁇ m.
  • a cryopreservation tool with an opening size of 183 ⁇ m was made of polyester fiber with a wire diameter of 71 ⁇ m.
  • the mesh opening size is smaller than that of the cryopreservation tools of Examples 1 to 3 (that is, the mesh opening size is 200 ⁇ m, 250 ⁇ m, or 300 ⁇ m), it is difficult to hold the fertilized egg. There was an impression that it was difficult to visually check the mesh holes when confirming whether or not the was held. In addition, when the opening size is 167 ⁇ m, it may not be possible to hold the fertilized egg and the cryoprotectant in the mesh holes at the same time. A two-stage operation was required, first holding the fertilized egg and then placing the fertilized egg in the mesh hole.
  • a cryopreservation tool with a mesh opening size of 200 ⁇ m ie, the cryopreservation tool of Example 1
  • a cryopreservation tool with a mesh opening size of 250 ⁇ m ie, the cryopreservation tool of Example 2.
  • a frozen mouse early embryo Day 3 embryo, 4 to 8 cells
  • the embryos were frozen using a tool and then thawed immediately. Freezing and thawing were performed in the same manner as described above.
  • Embryos were cultured at 37.0°C using SAGE 1 step medium (Origio).
  • Table 1 shows the culture results of embryos after freezing and thawing using the cryopreservation tool of Example 1
  • BL incidence indicates the ratio of embryos that have grown to blastocyst
  • good BL incidence means the ratio of embryos with a grade of 3AA or higher.
  • the culture performance of the embryos after thawing was evaluated. Specifically, first, a frozen mouse early embryo (same as above) was thawed and cultured for 1 or 2 days until the early embryo grew into a blastocyst. The culture was performed under the same conditions as above. The blastocysts were then frozen and thawed using the cryopreservation device of Example 1 or the cryopreservation device of Example 2. Freezing and thawing were performed in the same manner as described above.
  • the thawed embryos were cultured for 1 day under the same conditions as above, and the cultured embryos (that is, Day 5 embryos and Day 6 embryos) were observed.
  • Table 3 shows the culture results of embryos frozen and thawed using the cryopreservation tool of Example 1
  • Table 4 shows the culture results of embryos frozen and thawed using the cryopreservation tool of Example 2.

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Abstract

Outil de cyopréservation pour un ovule ou ovule fécondé selon la présente invention, comprenant les éléments suivants : une partie de corps principal (10) munie d'une partie de poignée en forme de tige (11) et d'une partie de maintien d'ovule en forme de maille (12) prévue à l'extrémité de la partie de poignée ; et une partie de couvercle cylindrique (20) contenant la partie de corps principal, la taille d'ouverture de la maille de la partie de maintien d'ovule étant de 170 μm ou plus. Par conséquent, la quantité de solution cryoprotectrice autour de l'ovule peut être ajustée à une quantité appropriée sans effectuer une opération compliquée, et l'ovule peut être facilement retiré pendant la décongélation.
PCT/JP2022/028801 2021-08-23 2022-07-26 Outil de cryoconservation pour ovule ou ovule fertilisé WO2023026747A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202280057097.2A CN117980460A (zh) 2021-08-23 2022-07-26 卵子或受精卵的冷冻保存用具
JP2022577775A JP7292637B1 (ja) 2021-08-23 2022-07-26 卵子又は受精卵の凍結保存用具

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JP2021-135532 2021-08-23
JP2021135532 2021-08-23

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010213692A (ja) * 2009-02-19 2010-09-30 Hiroaki Inui 細胞をガラス化保存する方法および細胞のガラス化保存用容器
WO2011070973A1 (fr) * 2009-12-08 2011-06-16 学校法人北里研究所 Tube étroit pour la conservation par vitrification d'un embryon ou d'un ovule animal
JP2012140422A (ja) * 2010-12-17 2012-07-26 Nipro Corp 凍結保存器具
WO2017187543A1 (fr) * 2016-04-27 2017-11-02 有限会社 乾メディカル Récipient de vitrification-cryoconservation dans un liquide, trousse dotée d'un récipient et tube destiné à le recevoir, et procédé de vitrification-cryoconservation dans un liquide

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010213692A (ja) * 2009-02-19 2010-09-30 Hiroaki Inui 細胞をガラス化保存する方法および細胞のガラス化保存用容器
WO2011070973A1 (fr) * 2009-12-08 2011-06-16 学校法人北里研究所 Tube étroit pour la conservation par vitrification d'un embryon ou d'un ovule animal
JP2012140422A (ja) * 2010-12-17 2012-07-26 Nipro Corp 凍結保存器具
WO2017187543A1 (fr) * 2016-04-27 2017-11-02 有限会社 乾メディカル Récipient de vitrification-cryoconservation dans un liquide, trousse dotée d'un récipient et tube destiné à le recevoir, et procédé de vitrification-cryoconservation dans un liquide

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JP7292637B1 (ja) 2023-06-19
CN117980460A (zh) 2024-05-03
JPWO2023026747A1 (fr) 2023-03-02

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