WO2019004300A1 - Outil de cryoconservation de cellules biologiques et outil pour cryoconservation de cellules biologiques - Google Patents

Outil de cryoconservation de cellules biologiques et outil pour cryoconservation de cellules biologiques Download PDF

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Publication number
WO2019004300A1
WO2019004300A1 PCT/JP2018/024427 JP2018024427W WO2019004300A1 WO 2019004300 A1 WO2019004300 A1 WO 2019004300A1 JP 2018024427 W JP2018024427 W JP 2018024427W WO 2019004300 A1 WO2019004300 A1 WO 2019004300A1
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Prior art keywords
cell cryopreservation
living cell
water absorbing
base portion
biological cell
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PCT/JP2018/024427
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English (en)
Japanese (ja)
Inventor
井上　太
Original Assignee
株式会社北里コーポレーション
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Priority to JP2019526992A priority Critical patent/JP7108318B2/ja
Publication of WO2019004300A1 publication Critical patent/WO2019004300A1/fr

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    • 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

Definitions

  • the present invention relates to a biological cell cryopreservation device and a biological cell cryopreservation device for use in cryopreserving biological cells such as mammalian eggs, embryos such as eggs, spermatozoa, hematopoietic stem cells, pluripotent stem cells etc. .
  • Cryopreservation of mammalian embryos allows the preservation of the genetic resources of a particular strain or breed. It is also useful for maintaining animal species that are endangered. Furthermore, it is also useful in human infertility treatment.
  • a method for cryopreservation of mammalian embryo in Japanese Patent Application Laid-Open No. 2000-189155 (patent document 1), the inner surface of a cryopreservation container such as a frozen straw, a frozen vial or a frozen tube obtained by sterilizing mammalian embryo or egg is used. It has been proposed to stick with a minimal amount of vitrification solution sufficient to encapsulate these embryos or eggs, seal the cryopreservation container, and bring the container into contact with liquid nitrogen for rapid cooling. .
  • the cryopreservation container stored by the above method is removed from liquid nitrogen, one end of the container is opened, a dilution liquid of 33 to 39 ° C. is directly injected into this container, and embryo or egg Thaw and dilute the freeze.
  • a cryopreservation container such as a freezing straw, a freezing vial or a freezing tube with a sufficient amount and a small amount of a vitrification solution to cover an egg such as an embryo or an egg.
  • Patent document 1 JP-A-2000-189155 JP 2002-315573 (WO publication 02-085110) Japanese Patent Application Publication No. 2004-329202 (US Publication 2004-0259072) Unexamined-Japanese-Patent 2016-10359 (US publication 2017-0135335)
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2002-315573, WO Publication 02-085110.
  • the egg cryopreservation tool 1 of Patent Document 2 is attached to a main body 2 formed of a cold resistant material and one end of the main body 2 and is formed of a flexible, transparent and liquid nitrogen resistant material. It comprises a strip 3 and a cylindrical member 4 sealed at one end detachably attached to the main body 2 so as to be capable of enclosing the egg attachment holding strip 3 and made of a cold resistant material.
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2004-329202, US Publication No. 2004-0259072.
  • the egg cryopreservation tool 1 of Patent Document 3 includes an egg cryopreservation tube 2 formed of a liquid nitrogen resistant material, and a metallic cylindrical protective member 3 for protecting the tube 2.
  • the tube 2 includes a main body portion 21 and an egg storage narrow diameter portion 22 having an inner diameter of 0.1 mm to 0.5 mm, and can be heat-sealed on the tip end side of the narrow diameter portion. It can be heat sealed.
  • the cylindrical protective member 3 accommodates a cylindrical portion 31 for housing the distal end of the small diameter portion 22 of the tube 2, a portion not accommodated in the cylindrical portion 31 for the small diameter portion 22, and the distal end portion 21 a of the main body 21.
  • a semi-cylindrical portion 32 is provided.
  • a vitrification liquid absorber having an adhesive layer and a vitrification liquid absorption layer at least in this order is provided on a support.
  • a jig for vitrifying cryopreservation of cells or tissue having a portion where the adhesive layer is not present between the portion where the cell or tissue of the liquid absorbing layer is to be placed and the support.
  • Patent Documents 2, 3 and 4 are effective. Further, in the case of Patent Document 4, since the vitrified liquid absorption layer is not transparent, when mounting cells using a transmission type microscope, the tip of the pipette is disposed on the upper surface of the vitrified liquid absorption layer When it was difficult to visually recognize the tip, the workability was bad. For this reason, there is a need for an egg cryopreservation tool that can perform egg freezing operation more easily.
  • the object of the present invention is to facilitate the operation of placing a living cell on a living cell cryopreservation tool, and even if the excess vitrification solution is dropped, the removing operation is unnecessary, and freezing of the living cell is quick
  • the present invention provides a living cell cryopreservation tool that can be carried out.
  • a living cell cryopreservation tool comprising: a main body portion formed of a cold resistant material; and a biological cell holding portion formed of the cold resistant material,
  • the biological cell holding unit includes a light transmitting base unit, and a water absorbing unit fixed to the surface of the base unit, and the water absorbing unit includes a defective portion surrounded by the water absorbing unit,
  • the living cell cryopreservation tool in which the surface of the base part is exposed and has light permeability.
  • a living cell cryopreservation tool comprising: a main body portion formed of a cold resistant material; and a biological cell holding portion formed of the cold resistant material,
  • the living cell holding portion has light permeability and has an elongated base portion and two elongated water absorbing portions fixed on both sides of at least one surface of the base portion, and between the two water absorbing portions
  • the living cell cryopreservation tool, wherein the exposed base portion is a light transmitting cell mounting site.
  • a living cell cryopreservation tool comprising: a main body portion formed of a cold resistant material; and a biological cell holding portion formed of the cold resistant material,
  • the living cell holding portion is light transmitting, and has an elongated base portion, and two opposing water absorbing portions fixed on at least one surface of the base portion so as to cross the base portion,
  • a living cell cryopreservation tool wherein the base portion exposed between two water absorbing portions is a light transmitting cell mounting site.
  • a biological cell cryopreservation tool comprising: the biological cell cryopreservation device according to any of the above; and a cylindrical storage member capable of storing the biological cell cryopreservation device and having a cold resistant material and having one end closed.
  • FIG. 1 is a front view of a living cell cryopreservation tool according to an embodiment of the present invention.
  • FIG. 2 is a left side view of the living cell cryopreservation tool shown in FIG.
  • FIG. 3 is a longitudinal sectional view of the living cell cryopreservation tool shown in FIG.
  • FIG. 4 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG.
  • FIG. 5 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG. 6 is a longitudinal sectional view of the living cell cryopreservation tool shown in FIG.
  • FIG. 7 is an enlarged front view of a living cell holding portion of a living cell cryopreservation tool according to another embodiment of the present invention.
  • FIG. 8 is an enlarged front view of a living cell holding portion of a living cell cryopreservation tool according to another embodiment of the present invention.
  • FIG. 9 is an enlarged sectional view of a living cell holding portion of a living cell cryopreservation tool of another embodiment of the present invention.
  • FIG. 10 is a front view of an example cylindrical storage member used for the biological cell cryopreservation tool of the present invention.
  • FIG. 11 is a front view of the biological cell cryopreservation tool of the embodiment of the present invention.
  • 12 is a cross-sectional view taken along line AA of FIG.
  • FIG. 13 is a front view of a living cell cryopreservation tool of another embodiment of the present invention.
  • FIG. 10 is a front view of an example cylindrical storage member used for the biological cell cryopreservation tool of the present invention.
  • FIG. 11 is a front view of the biological cell cryopreservation tool of the embodiment of the present invention.
  • 12 is a cross-sectional view taken
  • FIG. 14 is an enlarged front view of a living cell holding portion of the living cell cryopreservation tool shown in FIG.
  • FIG. 15 is an enlarged left side view of a biological cell holding portion of the biological cell cryopreservation tool shown in FIG.
  • FIG. 16 is a longitudinal sectional view of the living cell cryopreservation tool shown in FIG.
  • FIG. 17 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG.
  • FIG. 18 is an enlarged view of the biological cell cryopreservation tool shown in FIG. 13 viewed from the tip side.
  • FIG. 19 is an explanatory view of a living cell cryopreservation tool of another embodiment of the present invention.
  • FIG. 19 is an explanatory view of a living cell cryopreservation tool of another embodiment of the present invention.
  • FIG. 20 is an enlarged view of the biological cell cryopreservation tool according to another embodiment of the present invention as viewed from the tip side.
  • FIG. 21 is a front view of a living cell cryopreservation device of another embodiment of the present invention.
  • FIG. 22 is a left side view of the living cell cryopreservation tool shown in FIG.
  • FIG. 23 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG.
  • FIG. 24 is a front view of a biological cell cryopreservation device of another embodiment of the present invention.
  • 25 is an enlarged view of a biological cell holding portion of the biological cell cryopreservation tool shown in FIG.
  • FIG. 26 is an enlarged front view of a biological cell holding portion of a biological cell cryopreservation tool of another embodiment of the present invention.
  • the living cell cryopreservation tool 1 of the present invention includes a main body 2 made of a cold resistant material and a living cell holding part 3 made of a cold resistant material.
  • the living cell holding unit 3 includes a light transmitting base portion 31 and a water absorbing portion 32 fixed to the surface of the base portion 31.
  • the water absorption portion 32 includes a defect portion 33 surrounded by the water absorption portion 32 (in other words, a defect portion provided in the water absorption portion 32), and in the defect portion 33, the surface of the base portion 31 is exposed to transmit light. Have sex.
  • the cell cryopreservation tool 1 of this embodiment is an egg cryopreservation tool
  • the living cell holding unit 3 is an egg holding unit.
  • the cell cryopreservation device of the present invention may be used to cryopreserve cells such as eggs such as embryos, ova, sperm, hematopoietic stem cells, stem cells such as pluripotent stem cells, and particularly such living cells as described above. it can.
  • the living cell cryopreservation tool 10 of the present invention comprises the above-described living cell cryopreservation tool 1 and the cylindrical storage member 4 capable of storing the living cell cryopreservation tool 1 and formed of a cold resistant material and closed at one end. Equipped with
  • the biological cell cryopreservation tool 1 of the present invention can be used for egg cryopreservation also as a biological cell cryopreservation tool alone without using the cylindrical storage member 4.
  • the living cell cryopreservation tool 1 is a living body cell holding part formed of a main body 2 made of a cold resistant material and a cold resistant material attached to one end of the main body 2. And 3.
  • the living cell holding unit 3 includes a light transmitting base portion 31 and a water absorbing portion 32 fixed to the surface of the base portion 31.
  • the water absorbing portion 32 includes the defective portion 33 surrounded by the water absorbing portion 32.
  • the main body 2 is an elongated rod-like main body.
  • the light transmitting base portion 31 of the living cell holding portion 3 is a flexible, transparent and cold resistant (specifically, liquid nitrogen resistant) material In this embodiment, it is in the form of an elongated flat sheet.
  • the main body 2 holds one end of the living cell holding portion 3 (base portion 31).
  • the length of the main body is preferably about 50 to 200 mm.
  • the width of the main body is preferably about 2 to 7 mm, and the thickness is preferably about 1 to 7 mm.
  • the main body 2 includes a main spindle 21, a holder 22 for holding a biological cell holder 3 (base 31) provided at the front end of the main spindle 21, and a grip provided at the rear end of the main body 2. It has a portion 23 and a tapered portion 24 provided between the main shaft portion 21 and the grip portion 23.
  • the grip portion 23 In the biological cell cryopreservation tool 1 of this embodiment, except for the grip portion 23, it can be stored in the cylindrical storage member 4 described later, and the tapered portion 24 can be engaged with the opening of the cylindrical storage member 4 It has become.
  • the main body 2 is made of polyester (for example, polyethylene terephthalate, polybutylene terephthalate), polyolefin (for example, polyethylene, ultrahigh molecular weight polyethylene, polypropylene, ethylene-propylene copolymer, ethylene-vinyl acetate copolymer), styrene resin (for example, cold resistant resins such as polystyrene, methacrylate-styrene copolymer, methacrylate-butylene-styrene copolymer), polyamide (eg, 6 nylon, 66 nylon), polysulfone, fluoro resin, polyimide and the like are used, and in particular, liquid Those which can withstand the temperature of nitrogen are preferred.
  • polyester for example, polyethylene terephthalate, polybutylene terephthalate
  • polyolefin for example, polyethylene, ultrahigh molecular weight polyethylene, polypropylene, ethylene-propylene copolymer, ethylene-
  • the base portion 31 is in the form of an elongated flat sheet.
  • the base portion 31 is preferably in the form of a flat sheet having light transparency, preferably transparency, in order to facilitate attachment of living cells (eg, eggs).
  • a colorless and transparent flat sheet is preferred.
  • it may have flexibility.
  • a marker 34 is provided at the tip of the living cell holding unit 3.
  • the marker 34 is formed by coloring (coloring with an oil-based ink) on one surface (specifically, the surface of the water-absorbent sheet or the base portion) of the tip portion of the biological cell holding portion 3.
  • the base 31 has a length of about 10 to 70 mm, a width of about 0.5 to 4.0 mm, and a thickness of about 0.01 to 0.5 mm.
  • the base portion 31 has a base end portion fixed to the holding portion 22 of the main body portion 2 described above.
  • the sheet-forming material used for the base portion 31 may be a light-transmitting resin sheet, for example, 3-fluorinated polyethylene, low density polyethylene, medium density polyethylene, high density polyethylene, polycarbonate, nylon, polysulfone, polyester (for example, , PET), polystyrene, polyimide, ultra-high molecular weight polyethylene, ethylene-vinyl acetate copolymer, etc., films made of synthetic resins or laminates thereof.
  • cold resistance specifically, a liquid nitrogen resistant material, in other words, a material which does not become brittle when contacted with liquid nitrogen is preferable.
  • the living cell cryopreservation tool 1 of the present invention is provided with a water absorbing portion 32 fixed on the surface of the base portion 31 having light transmittance. Furthermore, the water absorbing portion 32 includes the defective portion 33 surrounded by the water absorbing portion 32. Then, the surface of the base portion 31 is exposed in the defect portion 33 and has light transparency. The surface of the base portion 31 in the defect portion 33 can be effectively used as a living cell mounting site.
  • the width of the water absorbing portion 32 is preferably about 2/3 the same as the width of the base portion.
  • the length of the water absorbing portion 32 is preferably 5 to 70 mm, and particularly preferably 10 to 50 mm.
  • the living cell holding unit 3 includes a plurality of defective parts (cell mounting sites) 33.
  • a plurality of members are provided in the longitudinal direction of the biological cell holding unit 3.
  • five defects (cell mounting sites) 33 are provided.
  • the number of defective parts (cell mounting sites) 33 is preferably about 1 to 7, particularly preferably 2 to 5.
  • the diameter is. About 0.5 to 1.5 mm is preferable.
  • deletion part 33 is circular shape or polygonal shape, and it is preferable that it is especially circular and an ellipse.
  • the water absorbing portion 32 is preferably a light impermeable sheet. By setting it like this, when the mounting operation of a living cell using a transmission type microscope is performed, the water absorbing portion 32 is impervious and light is transmitted to the defect portion 33, so the cells are mounted. It looks as if the target to be placed, that is, the defect (cell mounting site) 33, appears to float up, and recognition of the target is extremely easy.
  • the water absorbing portion 32 covers the entire one surface of the tip portion of the base portion 31 except for the defective portion 33. There is. By adopting such a configuration, recognition of the defective portion (cell mounting portion) 33 becomes easier.
  • the water absorbing portion 32 is fixed to the surface of the base portion 31.
  • the water absorbing portion 32 is formed of a cold resistant material.
  • the fixing of the water absorbing portion 32 to the base portion 31 is preferably performed using an adhesive, an adhesive substance such as double-sided tape, or the like. In addition, you may fix by a partial heat seal.
  • various sheets such as a sheet which consists of textiles, and a porous resin sheet, can be used.
  • the thickness of the water absorbing portion is preferably 10 ⁇ m to 5 mm, more preferably 20 ⁇ m to 2.5 mm.
  • a paper or a nonwoven fabric can be used as a sheet which consists of textiles.
  • the paper When paper is used as the water absorbing portion, the paper preferably has a density of 0.1 to 0.6 g / cm 3 and a basis weight of 10 to 130 g / m 2 .
  • a paper having a density of 0.12 to 0.3 g / cm 3 and a basis weight of 10 to 100 g / m 2 is excellent in the absorptivity of a storage solution, and further, a mounting portion using a transmission type microscope It is preferable because it becomes possible to provide a cryopreservation jig excellent in the visibility of cells or tissues, which enables observation of the cells or tissues placed thereon.
  • cellulose fibers rayon fibers and cupra fibers which are regenerated fibers consisting of cellulose fibers, acetate fibers which are semi-synthetic fibers from cellulose fibers, polyester fibers, A nylon fiber, an acrylic fiber, a polypropylene fiber, a polyethylene fiber, a polyvinyl chloride fiber, a vinylidene fiber, a polyurethane fiber, a vinylon fiber, a glass fiber, a silk fiber etc. are mentioned, The nonwoven fabric which variously mixed these fibers can also be used.
  • the non-woven fabric is preferably a non-woven fabric having a density of 0.1 to 0.4 g / cm 3 and a basis weight of 10 to 130 g / m 2 .
  • those having a density of 0.12 to 0.3 g / cm 3 and a basis weight of 10 to 100 g / m 2 are preferable.
  • porous resin sheet which can be used as the water absorbing portion, for example, it is stretched in at least uniaxial direction described in JP-B-42-13560 or JP-A-08-283447, and heated to a temperature above the melting point of the resin.
  • the solid powder of the thermoplastic resin obtained by the method is filled in a mold, heated and sintered to fuse the surface of the powder particles and cooled, and a resin sheet or the like having a porous structure is mentioned.
  • various polyethylenes such as low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene, polypropylene, polymethyl methacrylate, polystyrene, polytetrafluoroethylene, polyvinylidene difluoride and the like
  • fluorine resin ethylene-vinyl acetate copolymer, polyamide, styrene-acrylonitrile copolymer, styrene-butadiene-acrylonitrile terpolymer, polycarbonate, polyvinyl chloride and the like.
  • the base exposed portion 35 may be provided on the side of the living cell holding unit 3a.
  • the living cell holding portion 3a has two exposed base portions 35 extending in the longitudinal direction of the living cell holding portion 3a on both sides of the water absorbing portion 32a.
  • a plurality of individual water absorbing units 32b each having a deficient portion 33 in the central portion may be provided.
  • the individual water absorbing portion 32 b is circular, and has a circular defect portion 33 inside.
  • a plurality of individual water absorbing portions 32 b are fixed to the surface of the base portion 31. Further, the surface of the base portion 31 is exposed at the outside of the individual water absorption portion 32 b and at the defect portion 33.
  • the shape of the individual water absorbing portion 32b is not limited to a circular shape, and may be an elliptical shape, a rectangular shape, or a polygonal shape.
  • the water absorbing part 32c may have a predetermined thickness (height).
  • the thickness (height) of the water absorbing portion 32c is preferably about 2 to 7 mm, and more preferably 2 to 5 mm.
  • a cell storage portion is formed by the inner surface of the water absorbing portion 32c in the defective portion 33a and the upper surface of the base portion 31, and cell detachment is suppressed.
  • the cylindrical storage member 4 is detachably attached to the main body portion 2 so as to encapsulate the living cell holding portion 3 and has a closed end and a cold resistant material. It is formed.
  • the cylindrical storage member 4 of this embodiment comprises a cylindrical main body 41 and a sealing member 42 stored in one end thereof.
  • One end of the cylindrical main body 41 is air-sealably sealed by a sealing member (specifically, a breathable sealing member) 42 to be stored.
  • a weight 43 is stored at the tip of the cylindrical main body 41.
  • the weight 43 is accommodated closer to the tip than the sealing member 42.
  • the cylindrical storage member 4 has an inner diameter and a length that can be encapsulated without contacting the biological cell holding unit 3.
  • the cylindrical main body 41 is equipped with the coloring part 44 for making the recognition of an opening part easy in the outer surface of a rear end (opening part).
  • the cylindrical storage member 4 for example, 3-fluorinated polyethylene, low density polyethylene, medium density polyethylene, high density polyethylene, polycarbonate, nylon, polysulfone, polyester (for example, PET), polystyrene, polyimide, super high Examples thereof include films made of synthetic resins such as molecular weight polyethylene and ethylene-vinyl acetate copolymer, and laminates thereof.
  • a liquid nitrogen resistant material in other words, a material which does not become embrittled even in contact with liquid nitrogen is preferable.
  • the living cell holding portion of the living cell cryopreservation tool 1 to which an ovum has been attached is immersed in liquid nitrogen prepared beforehand and frozen (vitrified). Then, the biological cell cryopreservation tool 1 to which the ova is attached is inserted into the tubular member 4, and the cylindrical storage member 4 housing the biological cell cryopreservation tool 1 is stored in the storage container (not shown). Then, put the storage container in a liquid nitrogen tank and store it.
  • the living cell cryopreservation tool and the living cell cryopreservation tool of the present invention may be a living cell cryopreservation tool 1a as shown in FIGS.
  • the living cell cryopreservation tool 1a of this embodiment includes a main body 2 made of a cold resistant material and a living cell holding part 3d made of a cold resistant material.
  • the biological cell holding unit 3d is light transmissive and includes an elongated base portion 31 and two elongated water absorbing portions 32a and 32b fixed on both sides of at least one surface of the base portion. And the base part 31 exposed between two water absorption part 32a, 32b is the cell mounting site
  • the basic configuration of the living cell cryopreservation tool 1a of this embodiment is the same as that of the above-described living cell cryopreservation tool 1, and the only difference is in the form of the water absorbing portion.
  • the light transmitting base portion 31 is the same as described above.
  • the water absorbent portions 32 a and 32 b are fixed to one surface of the base portion 31.
  • the water absorbing portions 32 a and 32 b may be provided on the front and back surfaces of the base portion 31.
  • the water absorbing portions 32 a and 32 b are elongated in the longitudinal direction so as to cover the side portions of the surface of the base portion 31.
  • the two water absorbing portions 32a and 32b are provided to face each other.
  • the width of the water absorbing portions 32a and 32b is preferably 0.2 to 1.5 mm, and particularly preferably 0.3 to 1.0 mm. And between the water absorption parts 32a and 32b which face each other, the surface of the base part 31 is exposed and it becomes the biological cell mounting site 33.
  • the length of the water absorbing portions 32a and 32b is preferably 5 to 70 mm, and more preferably 10 to 50 mm.
  • the distance between the water absorbing portions 32a and 32b, in other words, the width of the living cell mounting portion 33 is preferably 0.5 to 1.5 mm.
  • the constituent material of the water absorbing portions 32a and 32b those described for the water absorbing portion 32 described above can be used.
  • a marker 34 is provided at the tip of the living cell holding portion 3d.
  • the marker 34 is formed by coloring (coloring with an oil-based ink) on one surface (specifically, the surface of the water-absorbent sheet or the base) of the tip of the biological cell holding portion 3 d.
  • the cylindrical storage member 4 described above can be used also in the biological cell cryopreservation tool 1a of this embodiment.
  • the living cell cryopreservation tool 1 a and the cylindrical storage member 4 constitute a living cell cryopreservation tool.
  • a living cell cryopreservation tool 1b as shown in FIGS. 24 and 25 may be used as the living cell cryopreservation tool and the living cell cryopreservation tool of the present invention.
  • the living cell cryopreservation tool 1b of this embodiment includes a main body 2 formed of a cold resistant material and a living cell holding part 3e formed of a cold resistant material.
  • the living cell holding portion 3e has light permeability, and the two opposing water absorbing portions 37a and 37b fixed to the elongated base portion 31 and at least one surface of the base portion 31 so as to cross the base portion 31. , 37c, and 37d, and the base portion 31 exposed between the water absorbing portions 37a, 37b, 37c, and 37d is a cell mounting portion 33 having light transparency.
  • the basic configuration of the biological cell cryopreservation tool 1b of this embodiment is the same as that of the above-described biological cell cryopreservation tool 1, and the difference is only in the form of the water absorbing portion.
  • the light transmitting base portion 31 is the same as described above.
  • the water absorbent portions 37 a, 37 b, 37 c and 37 d are fixed to one surface of the base portion 31.
  • the water absorbing portions 37 a, 37 b, 37 c, 37 d may be provided on the front and back surfaces of the base portion 31.
  • a plurality of water absorbing portions 37 a, 37 b, 37 c and 37 d are provided so as to cross the surface of the base portion 31.
  • the water absorbing portions 37a, 37b, 37c, and 37d are provided such that the water absorbing portions adjacent in the longitudinal direction of the base portion 31 face each other.
  • An exposed base portion between adjacent water absorbent portions is a living cell mounting portion 33.
  • the water absorbing portions 37 a, 37 b, 37 c and 37 d extend over the entire width of the base portion 31.
  • the water absorbent portions 37a, 37b, 37c and 37d may extend to a certain length instead of the entire width of the base portion 31 as in the biological cell holding portion 3f of the embodiment shown in FIG. In this case, it is preferable that the water absorbing portions 37a, 37b, 37c and 37d extend by 1/2 or more of the width of the figure base portion 31.
  • the longitudinal length of the base portion 31 of the water absorbing portions 37a, 37b, 37c, 37d is preferably 0.2 to 5 mm, and particularly preferably 0.5 to 3 mm.
  • the number of water absorbing parts is preferably about 2 to 10, and particularly preferably 2 to 5.
  • the distance between the water absorbing portions 37a, 37b, 37c, 37d, in other words, the width of the living cell mounting portion 33 is preferably 0.5 to 1.5 mm.
  • the constituent material of the water absorbing portions 32a and 32b those described for the water absorbing portion 32 described above can be used.
  • the marker 34 is provided at the tip of the living cell holding parts 3d, 3e, 3f.
  • the marker 34 is formed by coloring (coloring with an oil-based ink) on one surface (specifically, the surface of the water-absorbent sheet or the base portion) of the tip portion of the biological cell holding portion.
  • the biological cell holding device of the biological cell cryopreservation device of the embodiment shown in FIG. may have a predetermined thickness (height).
  • the thickness (height) of the water absorbing portion is preferably about 2 to 7 mm, and more preferably 2 to 5 mm.
  • the living cell cryopreservation tool can be configured by using the cylindrical storage member 4 described above.
  • the living cell cryopreservation tool 5 of this embodiment includes a main body 51 made of a cold resistant material and a living cell holding part 52 made of a cold resistant material.
  • the living cell holding unit 52 includes a light transmitting base 53 and a water absorbing unit 54 fixed on the surface of the base 53.
  • the water absorption portion 54 includes a defect portion 61 surrounded by the water absorption portion 54, and in the defect portion 61, the surface of the base portion 53 is exposed and has optical transparency.
  • the base portion 53 of the living cell holding portion 52 has a substantially rectangular cross section, and includes a base end portion 59 connected to the main body portion 51. And in this embodiment, the base portion 53 is a narrow strip (thin plate) having a narrow width. And the water absorption part 54 is being fixed to the surface.
  • the width of the base portion 52 is preferably 0.4 to 1.0 mm, the length is preferably 5 to 30 mm, and the total thickness is preferably 0.08 to 1.0 mm.
  • the length of the thick base end of the base portion 53 is preferably 5 to 30 mm, and the length of the main portion is preferably 20 to 100 mm.
  • the main body 51 includes a protrusion 64 protruding in the distal direction, and the base 53 includes a recess 65 for receiving the protrusion 64.
  • the biological cell holding part 52 is provided with a plurality of defective parts (cell mounting parts) 61. ing. Specifically, a plurality of members are provided in the longitudinal direction of the biological cell holding unit 52. Specifically, five defective parts (cell mounting sites) 61 are provided. The number of defective parts (cell mounting sites) 61 is preferably about 1 to 7, and particularly preferably 2 to 5. As the size of the defect, in the case of a circle, the diameter is. About 0.5 to 1.5 mm is preferable.
  • the defect part 61 is a circular form.
  • the water absorbing portion 54 is preferably a light impermeable sheet. By setting it like this, when the mounting operation of a living cell using a transmission type microscope is performed, the water absorbing portion 54 is impervious and light is transmitted to the defect portion 61, so the cells are mounted. It looks as if the target to be placed, that is, the defect (cell mounting site) 61, appears to be floating, and recognition of the target is extremely easy.
  • the living cell holding portion 52 is opposed to each other extending in the longitudinal direction of the living cell holding portion 52 on both sides of the water absorbing portion 54.
  • the water-absorbent sheet 54 may cover the entire one surface of the tip portion of the base portion 53 except for the defective portion 61.
  • a plurality of individual water absorption parts having a defect part in the central part may be provided.
  • All or part of the water absorbing portion 54 is fixed to the surface of the base portion 53. Fixing of the water absorbing portion 54 to the base portion 53 is preferably performed using an adhesive, an adhesive substance such as double-sided tape, or the like. In addition, you may fix by a partial heat seal.
  • various sheets such as a sheet which consists of textiles, and a porous resin sheet, can be used.
  • the thickness of the water absorbing portion is preferably 10 ⁇ m to 5 mm, more preferably 20 ⁇ m to 2.5 mm.
  • a paper or a nonwoven fabric can be used as a sheet which consists of textiles.
  • those described above can be suitably used.
  • the living cell cryopreservation tool 5 of this embodiment as shown in FIG. 13, FIG. 14, and FIG. 16 to FIG. It has two side bulges 57 and 58 extending in the longitudinal direction of the part 52. For this reason, since the living cell cryopreservation tool 5 has the bulging parts on both sides of the water absorption part fixing part, movement of the living cell in the side direction at the time of mounting the living cell can be reliably suppressed.
  • the living cell holding portion 52 is a living cell holding portion 52 than the water absorption portion fixing portion.
  • the projection 63 is provided on the tip side of the The protruding portion 63 protrudes from the tip of the living cell holding portion 52 to the upper surface side (water absorbing portion side).
  • the tip end surface of the projecting portion 63 is an inclined surface which is slightly inclined to the base end side.
  • the main body portion 51, the base portion 53, and the water absorbing portion 54 are formed of a cold resistant material.
  • the main body portion 51, the base portion 53 and the water absorption portion 54 are preferably liquid nitrogen resistant materials, in other words, those which do not become brittle even when in contact with liquid nitrogen.
  • the base portion 53 preferably has transparency, and preferably has some flexibility.
  • Examples of materials for forming the main body 51 and the base 53 include 3-fluorinated polyethylene, low density polyethylene, medium density polyethylene, high density polyethylene, polycarbonate, nylon, polysulfone, polyester, polystyrene, polyimide, ultrahigh molecular weight polyethylene, A synthetic resin such as ethylene-vinyl acetate copolymer, or a laminate of a film formed of the synthetic resin is preferably used.
  • the living cell holding portion 52 extends in the longitudinal direction of the living cell holding portion 52 and the living cell holding portion 52 It has the thermally conductive bodies 55 and 56 which project from the tip.
  • the thermally conductive bodies 55, 56 are linear thermal conducting bodies, and are provided on both sides of the living cell holding portion 52.
  • the heat conductive members 55 and 56 extend beyond the water absorption portion fixing portion to the base end side of the base portion 53 and reach the base end of the living cell holding portion 52 or in the vicinity thereof. It has become a thing.
  • the heat conductive members 55, 56 are embedded in the side bulges 57, 58 except for the tip portions 55a, 56a. In other words, the tip portions 55a and 56a protrude from the tip of the living cell holding portion 52, and the outer surface is exposed.
  • the thermally conductive members 55, 56 are formed of linear members or thin rod-like members. Furthermore, the shape of the distal end 55a, 56a of the heat conductive members 55, 56 may be a curved distal end that curves toward the proximal end. In this case, as shown in FIG. 14, the curved tip portions 55 a and 56 a preferably protrude forward of the tip portion of the biological cell cryopreservation tool 5 and protect the protruding portion 63.
  • the thermally conductive bodies 55 and 56 are formed of a thermally conductive material. As the heat conductive material, metals such as silver, copper, aluminum and stainless steel, and heat conductive ceramics such as aluminum nitride, silicon nitride and alumina can be suitably used.
  • the biological cell cryopreservation tool 20 of the embodiment shown in FIG. 19 is a cylindrical storage member capable of storing the above-mentioned biological cell cryopreservation tool 5 and the biological cell cryopreservation tool 5 and being closed by one end formed of a cold resistant material.
  • the cylindrical storage member 7 is a cylindrical body which can store the living cell cryopreservation tool 5 and is formed of a cold-resistant material and is closed at one end, as shown in FIG.
  • the cylindrical storage member 7 includes a cylindrical body 70 having a living cell holding member storage portion 71 therein, and a heat conductive member 72 provided at the tip of the cylindrical body 70.
  • the cylindrical storage member 7 includes a distal end closing portion 73, a proximal end opening portion 74, a cylindrical body 70 having a living cell holding member storage portion 71 therein, and a distal end of the cylindrical body 70. It is comprised by the thermally-conductive member 72 accommodated in the part.
  • the heat conductive member 72 is housed immovably inside the cylindrical body 70 and is housed so as to abut on the inner surface of the tip end closing portion 73.
  • the heat conductive member 72 is in the form of a metal cylinder.
  • the upper surface of the heat conductive member 72, that is, the tip portions 55a and 56a of the heat conductive members 55 and 56 of the living cell cryopreservation tool 5 can be contacted.
  • the heat conductive member 72 may be such that the tip end surface, the tip end side surface, etc. are exposed from the cylindrical body 70.
  • the water absorbing portion 54a provided on the upper surface of the base portion 53 has a predetermined thickness (height). It may be one.
  • the thickness (height) of the water absorbing portion 54a is preferably about 2 to 7 mm, and more preferably 2 to 5 mm.
  • the cylindrical body 70 is formed of a cold resistant material.
  • the cylindrical body 70 be a liquid nitrogen resistant material, in other words, one that does not become embrittled when it comes in contact with liquid nitrogen.
  • the cylindrical body 70 is a transparent or semi-transparent body whose inside is visible.
  • a forming material of the cylindrical body 70 for example, 3-fluorinated polyethylene, low density polyethylene, medium density polyethylene, high density polyethylene, polycarbonate, nylon, polysulfone, polyester, polystyrene, polyimide, ultra high molecular weight polyethylene, ethylene-acetic acid Synthetic resins such as vinyl copolymers, and laminates of films formed of these synthetic resins are preferably used.
  • the length of the living cell holding member storage portion 71 of the cylindrical storage member 7 is preferably 10 to 50 mm longer than the total length of the living cell cryopreservation tool 5. Further, the total length of the cylindrical storage member 7 (the cylindrical body 70) is preferably 50 to 100 mm, and the inner diameter is preferably 2 to 5 mm.
  • cryopreserving an ovum which is a living cell
  • the case of cryopreserving an ovum which is a living cell
  • the intracellular fluid of the ova is replaced with the equilibration solution
  • the extracellular fluid is replaced with the vitrification solution.
  • the ovum 68 is placed along with a small amount of vitrification liquid in the defective portion 61 of the water absorbing portion 54 provided in the biological cell holding portion 52 of the biological cell cryopreservation tool 5 It adheres to the surface of the member 53.
  • the biological cell cryopreservation tool 5 to which an ovum is attached is inserted into the cylindrical storage member 7 from the biological cell holding unit 52 side, and as shown in FIG. 19, the tip of the biological cell holding unit 52 of the biological cell cryopreservation tool 5
  • the distal end portions 55 a and 56 a of the heat conductive members 55 and 56 further protruding are brought into contact with the heat conductive member 72 in the cylindrical storage member 7.
  • the cylindrical storage member 7 storing the biological cell cryopreservation tool 5 is immersed in liquid nitrogen prepared in advance and frozen (vitrified) from the tip end side (conductive member 72 side).
  • the heat conductive member 72 in the cylindrical storage member 7 is rapidly cooled by the contact of the cylindrical storage member 7 with liquid nitrogen, and the cooling is performed by the living body of the biological cell cryopreservation tool 5 in contact with the heat conductive member 72.
  • the temperature is rapidly taken away from the heat conductive members 55, 56 of the cell holding unit 52, and the ova held by the living cell holding unit 52 is also rapidly cooled.
  • the biological cell cryopreservation tool 5 for adhering and holding the vitrified ovum is stored together with the cylindrical storage member 7 in a storage container (cane), the storage container is put in a liquid nitrogen tank and stored.
  • the living cell cryopreservation device of the present invention is as follows.
  • a living cell cryopreservation tool comprising: a main body portion formed of a cold resistant material; and a biological cell holding portion formed of the cold resistant material,
  • the biological cell holding unit includes a light transmitting base unit, and a water absorbing unit fixed to the surface of the base unit, and the water absorbing unit includes a defective portion surrounded by the water absorbing unit,
  • the living cell cryopreservation tool in which the surface of the base part is exposed and has light permeability.
  • the tip of the pipette is disposed on the upper surface of the defect part when mounting the cells.
  • the vitrification liquid is absorbed by the water absorbing portion surrounding the defect, and therefore the removal operation is also unnecessary. For this reason, living cells can be rapidly frozen.
  • the above embodiment may be as follows.
  • the living cell cryopreservation tool of the present invention is as follows. (5) A living cell cryopreservation tool comprising: a main body portion formed of a cold resistant material; and a biological cell holding portion formed of the cold resistant material, The living cell holding portion has light permeability and has an elongated base portion and two elongated water absorbing portions fixed on both sides of at least one surface of the base portion, and between the two water absorbing portions The living cell cryopreservation tool, wherein the exposed base portion is a light transmitting cell mounting site.
  • the living cell cryopreservation tool of the present invention is as follows. (6) A living cell cryopreservation tool comprising: a main body portion formed of a cold resistant material; and a biological cell holding portion formed of the cold resistant material, The living cell holding portion is light transmitting, and has an elongated base portion, and two opposing water absorbing portions fixed on at least one surface of the base portion so as to cross the base portion, A living cell cryopreservation tool, wherein the base portion exposed between two water absorbing portions is a light transmitting cell mounting site.
  • the tip of the pipette is disposed on the upper surface of the cell mounting site when performing the cell mounting operation.
  • the vitrification liquid is absorbed by the water absorbing portion in the vicinity of the cell mounting site, and the removing operation is also unnecessary. For this reason, living cells can be rapidly frozen.
  • the above embodiment may be as follows. (7) The living cell cryopreservation tool according to any one of the above (1) to (6), wherein the base part is colorless and transparent. (8) The living cell cryopreservation tool according to any one of the above (1) to (6), wherein the base part is a transparent sheet. (9) The living cell cryopreservation tool according to any one of the above (1) to (8), wherein the base portion is an elongated flat flexible sheet. (10) The living cell cryopreservation tool according to any one of the above (1) to (7), wherein the base portion is an elongated hard flat plate member. (11) The living cell cryopreservation tool according to any one of the above (1) to (10), wherein the water absorbing part is a light impermeable sheet.
  • the biological cell cryopreservation tool of the present invention is as follows. (12) The living cell cryopreservation tool according to any one of the above (1) to (11), and a tubular housing member capable of storing the living cell cryopreservation tool and having a closed end formed of a cold resistant material
  • a living cell cryopreservation tool comprising:

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  • Bioinformatics & Cheminformatics (AREA)
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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

L'outil de cryoconservation des cellules biologiques 1 est équipé d'une partie de corps principal 2 formée à partir d'un matériau résistant au froid, et d'une partie de support de cellules biologiques 3 formée à partir d'un matériau résisstant au froid. La partie de support de cellules biologiques 3 est équipée d'une partie de base optiquement transparente 31 et d'une partie d'absorption d'eau 32 fixée à la surface de la partie de base 31. La partie d'absorption d'eau 32 est équipée d'une partie manquante 33 entourée par la partie d'absorption d'eau 32. La surface de la partie de base 31 est exposée dans la partie manquante 33, et la partie manquante est optiquement transparente.
PCT/JP2018/024427 2017-06-30 2018-06-27 Outil de cryoconservation de cellules biologiques et outil pour cryoconservation de cellules biologiques WO2019004300A1 (fr)

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JP2020171223A (ja) * 2019-04-10 2020-10-22 三菱製紙株式会社 凍結保存用治具
WO2020250766A1 (fr) 2019-06-12 2020-12-17 三菱製紙株式会社 Dispositif de fixation de gabarit de cryoconservation et procédé de congélation et de décongélation utilisant ledit dispositif de fixation
JP2021029170A (ja) * 2019-08-23 2021-03-01 三菱製紙株式会社 細胞又は組織の凍結保存用治具
WO2023132279A1 (fr) * 2022-01-07 2023-07-13 株式会社北里コーポレーション Instrument de congélation/conservation de cellules biologiques

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JP2020120627A (ja) * 2019-01-31 2020-08-13 三菱製紙株式会社 細胞又は組織のガラス化凍結保存用治具
JP7163207B2 (ja) 2019-01-31 2022-10-31 三菱製紙株式会社 細胞又は組織のガラス化凍結保存用治具
JP2020171223A (ja) * 2019-04-10 2020-10-22 三菱製紙株式会社 凍結保存用治具
JP7232693B2 (ja) 2019-04-10 2023-03-03 三菱製紙株式会社 凍結保存用治具
WO2020250766A1 (fr) 2019-06-12 2020-12-17 三菱製紙株式会社 Dispositif de fixation de gabarit de cryoconservation et procédé de congélation et de décongélation utilisant ledit dispositif de fixation
KR20220020895A (ko) 2019-06-12 2022-02-21 미쓰비시 세이시 가부시키가이샤 동결 보존용 지그의 고정구 및 당해 고정구를 이용한 동결 융해 방법
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WO2021039330A1 (fr) 2019-08-23 2021-03-04 三菱製紙株式会社 Gabarit destiné à la cryoconservation de cellules ou de tissus
KR20220035239A (ko) 2019-08-23 2022-03-21 미쓰비시 세이시 가부시키가이샤 세포 또는 조직의 동결 보존용 지그
JP7144380B2 (ja) 2019-08-23 2022-09-29 三菱製紙株式会社 細胞又は組織の凍結保存用治具
WO2023132279A1 (fr) * 2022-01-07 2023-07-13 株式会社北里コーポレーション Instrument de congélation/conservation de cellules biologiques

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