WO2019004300A1 - Biological cell cryopreservation tool and tool for biological cell cryopreservation - Google Patents

Biological cell cryopreservation tool and tool for biological cell cryopreservation 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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WO
WIPO (PCT)
Prior art keywords
cell cryopreservation
living cell
water absorbing
base portion
biological cell
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PCT/JP2018/024427
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French (fr)
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/en
Publication of WO2019004300A1 publication Critical patent/WO2019004300A1/en

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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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Abstract

This biological cell cryopreservation tool 1 is equipped with a main body portion 2 formed from a cold-resistant material and a biological cell holding portion 3 formed from a cold-resistant material. The biological cell holding portion 3 is equipped with an optically transparent base portion 31 and a water-absorbing portion 32 secured to the surface of the base portion 31. The water-absorbing portion 32 is equipped with a missing portion 33 surrounded by the water-absorbing portion 32. The surface of the base portion 31 is exposed in the missing portion 33, and the missing portion is optically transparent.

Description

生体細胞凍結保存具および生体細胞凍結保存用具Living cell cryopreservation device and living cell cryopreservation device
　本発明は、哺乳動物の卵子、胚などの卵、精子、造血幹細胞、多能性幹細胞等の幹細胞などの生体細胞を凍結保存する際に使用する生体細胞凍結保存用具および生体細胞凍結保存用具に関する。 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. .
　哺乳動物胚の凍結保存は、特定の系統や品種の遺伝資源の保存を可能とする。また、絶滅の危機に瀕している動物種の維持にも有効である。さらに、ヒトの不妊治療においても有用である。
　哺乳動物胚の凍結保存方法としては、特開２０００－１８９１５５公報（特許文献１）において、哺乳動物胚または卵子を滅菌処理した凍結ストロー、凍結バイアルまたは凍結チューブ等の凍結保存用容器の内面に、これらの胚または卵子を包被するに充分な最少量のガラス化液で貼り付け、この凍結保存用容器を密封し、この容器を液体窒素に接触させて急速に冷却することが提案されている。そして、融解方法では、前記の方法で保存した凍結保存用容器を液体窒素から取り出し、容器の一端部を開口し、この容器内に３３～３９℃の希釈液を直接注入し、胚または卵子の凍結を融解希釈するものである。この方法によれば、哺乳動物胚または卵子をウイルスや細菌による感染のおそれがなく高い生存率で保存および融解希釈することのできるという優れた効果を備えている。
　しかし、凍結ストロー、凍結バイアルまたは凍結チューブ等の凍結保存用容器の内面に、胚または卵子などの卵を包被するに充分かつ少量のガラス化液で貼り付ける作業が必ずしも容易ではなかった。 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.
As 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. . Then, in the thawing method, 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. According to this method, it has an excellent effect that mammalian embryos or eggs can be stored and thaw-diluted at high survival rates without the risk of viral or bacterial infection.
However, it has not always been easy to attach the inside of 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.
特開２０００－１８９１５５Patent document 1: JP-A-2000-189155 特開２００２－３１５５７３（ＷＯ公開０２－０８５１１０）JP 2002-315573 (WO publication 02-085110) 特開２００４－３２９２０２（ＵＳ公開２００４－０２５９０７２）Japanese Patent Application Publication No. 2004-329202 (US Publication 2004-0259072) 特開２０１６－１０３５９（ＵＳ公開２０１７－０１３５３３５）Unexamined-Japanese-Patent 2016-10359 (US publication 2017-0135335)
　そこで、本願発明者は、特許文献２（特開２００２－３１５５７３、ＷＯ公開０２－０８５１１０）を提案している。特許文献２の卵凍結保存具１は、耐寒性材料により形成された本体部２と、本体部２の一端に取り付けられ、可撓性かつ透明性かつ液体窒素耐性材料により形成された卵付着保持用ストリップ３と、卵付着保持用ストリップ３を被包可能に本体部２に着脱自在に取り付けられる一端が封鎖され、かつ耐寒性材料により形成された筒状部材４とからなるものである。 Therefore, the inventor of the present invention has proposed 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.
　また、本願発明者は、特許文献３（特開２００４－３２９２０２、ＵＳ公開２００４－０２５９０７２）も提案している。特許文献３の卵凍結保存具１は、耐液体窒素材料で形成された卵凍結保存用チューブ２と、チューブ２を保護するための金属製筒状保護部材３とを備える。チューブ２は、本体部２１と、内径が０．１ｍｍ～０．５ｍｍの卵保存用細径部２２とを備えるとともに、細径部の先端側にてヒートシール可能であり、本体部２１にてヒートシール可能である。筒状保護部材３は、チューブ２の細径部２２の先端部を収納する筒状部３１と、細径部２２の筒状部３１に収納されない部分および本体部２１の先端部２１ａを収納する半筒状部３２とを備えている。
　また、特開２０１６－１０３５９（特許文献４、ＵＳ公開２０１７－０１３５３３５）には、支持体上に、接着層とガラス化液吸収層を少なくともこの順に有するガラス化液吸収体を有し、ガラス化液吸収層の細胞又は組織を載置する部分と支持体の間に接着層が存在しない部分を有する細胞又は組織のガラス化凍結保存用治具が開示されている。 In addition, the inventor of the present application has also proposed 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.
Further, in JP-A-2016-10359 (Patent Document 4, US Publication 2017-0135335), a vitrification liquid absorber having an adhesive layer and a vitrification liquid absorption layer at least in this order is provided on a support. There is disclosed 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.
　特許文献２、３および４の卵凍結保存具は、有効である。また、特許文献４のものでは、ガラス化液吸収層が透明でないため、透過型顕微鏡を用いて、細胞の載置作業を行う際、ガラス化液吸収層の上面にピペットの先端部を配置したとき、その先端が視認しにくく、作業性が悪いものであった。このため、より容易に卵凍結操作が行える卵凍結保存具が望まれている。 The egg cryopreservation devices of 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.
　本発明の目的は、生体細胞凍結保存具への生体細胞載置操作が容易であり、かつ、過剰なガラス化液が滴下されても、その除去操作が不要であり、生体細胞の凍結を迅速に行うことができる生体細胞凍結保存具を提供するものである。 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.
 上記目的を達成するものは、以下のものである。
　耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
　前記生体細胞保持部は、光透過性を有するベース部と、前記ベース部の表面に固定された吸水部とを備え、前記吸水部は、前記吸水部により取り囲まれた欠損部を備え、前記欠損部において、前記ベース部の前記表面が露出し、光透過性を有している生体細胞凍結保存具。 The objects to achieve the above object are 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, In part, the living cell cryopreservation tool in which the surface of the base part is exposed and has light permeability.
 また、上記目的を達成するものは、以下のものである。
　耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
　前記生体細胞保持部は、光透過性を有し、細長いベース部と、前記ベース部の少なくとも一方の表面の両側部に固定された細長い２つの吸水部を有し、前記２つの吸水部間にて露出する前記ベース部が、光透過性を有する細胞載置部位となっている生体細胞凍結保存具。 Moreover, what achieves the above object are 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 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.
 また、上記目的を達成するものは、以下のものである。
　耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
　前記生体細胞保持部は、光透過性を有し、細長いベース部と、前記ベース部の少なくとも一方の表面に、前記ベース部を横切るように固定された向かい合う２つの吸水部を有し、前記２つの吸水部間にて露出する前記ベース部が、光透過性を有する細胞載置部位となっている生体細胞凍結保存具。 Moreover, what achieves the above object are 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 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.
 また、上記目的を達成するものは、以下のものである。
　上記のいずれかに記載の生体細胞凍結保存具と、前記生体細胞凍結保存具を収納可能かつ耐寒性材料により形成された一端が閉塞した筒状収納部材とを備える生体細胞凍結保存用具。 Moreover, what achieves the above object are as follows.
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.
図１は、本発明の実施例の生体細胞凍結保存具の正面図である。FIG. 1 is a front view of a living cell cryopreservation tool according to an embodiment of the present invention. 図２は、図１に示した生体細胞凍結保存具の左側面図である。FIG. 2 is a left side view of the living cell cryopreservation tool shown in FIG. 図３は、図１に示した生体細胞凍結保存具の縦断面図である。FIG. 3 is a longitudinal sectional view of the living cell cryopreservation tool shown in FIG. 図４は、図１に示した生体細胞凍結保存具の生体細胞保持部の拡大図である。FIG. 4 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG. 図５は、図２に示した生体細胞凍結保存具の生体細胞保持部の拡大図である。FIG. 5 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG. 図６は、図５に示した生体細胞凍結保存具の縦断面図である。6 is a longitudinal sectional view of the living cell cryopreservation tool shown in FIG. 図７は、本発明の他の実施例の生体細胞凍結保存具の生体細胞保持部の拡大正面図である。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. 図８は、本発明の他の実施例の生体細胞凍結保存具の生体細胞保持部の拡大正面図である。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. 図９は、本発明の他の実施例の生体細胞凍結保存具の生体細胞保持部の拡大断面図である。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. 図１０は、本発明の生体細胞凍結保存用具に使用される一例の筒状収納部材の正面図である。FIG. 10 is a front view of an example cylindrical storage member used for the biological cell cryopreservation tool of the present invention. 図１１は、本発明の実施例の生体細胞凍結保存用具の正面図である。FIG. 11 is a front view of the biological cell cryopreservation tool of the embodiment of the present invention. 図１２は、図１１のＡ－Ａ線断面図である。12 is a cross-sectional view taken along line AA of FIG. 図１３は、本発明の他の実施例の生体細胞凍結保存具の正面図である。FIG. 13 is a front view of a living cell cryopreservation tool of another embodiment of the present invention. 図１４は、図１３に示した生体細胞凍結保存具の生体細胞保持部の拡大正面図である。FIG. 14 is an enlarged front view of a living cell holding portion of the living cell cryopreservation tool shown in FIG. 図１５は、図１３に示した生体細胞凍結保存具の生体細胞保持部の拡大左側面図である。FIG. 15 is an enlarged left side view of a biological cell holding portion of the biological cell cryopreservation tool shown in FIG. 図１６は、図１５に示した生体細胞凍結保存具の縦断面図である。FIG. 16 is a longitudinal sectional view of the living cell cryopreservation tool shown in FIG. 図１７は、図１３に示した生体細胞凍結保存具の生体細胞保持部の拡大図である。FIG. 17 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG. 図１８は、図１３に示した生体細胞凍結保存具を先端側から見た拡大作用説明図である。FIG. 18 is an enlarged view of the biological cell cryopreservation tool shown in FIG. 13 viewed from the tip side. 図１９は、本発明の他の実施例の生体細胞凍結保存用具の説明図である。FIG. 19 is an explanatory view of a living cell cryopreservation tool of another embodiment of the present invention. 図２０は、本発明の他の実施例の生体細胞凍結保存具を先端側から見た拡大作用説明図である。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. 図２１は、本発明の他の実施例の生体細胞凍結保存用具の正面図である。FIG. 21 is a front view of a living cell cryopreservation device of another embodiment of the present invention. 図２２は、図２１に示した生体細胞凍結保存具の左側面図である。FIG. 22 is a left side view of the living cell cryopreservation tool shown in FIG. 図２３は、図２１に示した生体細胞凍結保存具の生体細胞保持部の拡大図である。FIG. 23 is an enlarged view of a living cell holding portion of the living cell cryopreservation tool shown in FIG. 図２４は、本発明の他の実施例の生体細胞凍結保存用具の正面図である。FIG. 24 is a front view of a biological cell cryopreservation device of another embodiment of the present invention. 図２５は、図２４に示した生体細胞凍結保存具の生体細胞保持部の拡大図である。25 is an enlarged view of a biological cell holding portion of the biological cell cryopreservation tool shown in FIG. 図２６は、本発明の他の実施例の生体細胞凍結保存用具の生体細胞保持部の拡大正面図である。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.
　本発明の生体細胞凍結保存具を図面に示す実施例を用いて説明する。
　本発明の生体細胞凍結保存具１は、耐寒性材料により形成された本体部２と、耐寒性材料により形成された生体細胞保持部３とを備える。生体細胞保持部３は、光透過性を有するベース部３１と、ベース部３１の表面に固定された吸水部３２とを備える。吸水部３２は、吸水部３２により取り囲まれた欠損部（言い換えれば、吸水部３２の中に設けられた欠損部）３３を備え、欠損部３３において、ベース部３１の表面が露出し、光透過性を有している。
　特に、この実施例の細胞凍結保存具１は、卵凍結保存具であり、生体細胞保持部３は、卵保持部となっている。なお、本発明の細胞凍結保存具は、胚などの卵、卵子、精子、造血幹細胞、多能性幹細胞等の幹細胞などの細胞、特に上記のような生体細胞を凍結保存するために用いることができる。
　また、本発明の生体細胞凍結保存用具１０は、上記の生体細胞凍結保存具１と、生体細胞凍結保存具１を収納可能かつ耐寒性材料により形成された一端が閉塞した筒状収納部材４とを備える。
　なお、本発明の生体細胞凍結保存具１は、筒状収納部材４を使用せず、生体細胞凍結保存用具単独としても卵凍結保存に使用することができる。 The living cell cryopreservation tool of the present invention will be described using an example shown in the drawings.
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.
In particular, the cell cryopreservation tool 1 of this embodiment is an egg cryopreservation tool, and 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.
Further, 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
In addition, 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.
　生体細胞凍結保存具１は、図１ないし図６に示すように、耐寒性材料により形成された本体部２と、本体部２の一端に取り付けられた耐寒性材料により形成された生体細胞保持部３とを備える。生体細胞保持部３は、光透過性を有するベース部３１と、ベース部３１の表面に固定された吸水部３２とを備える。吸水部３２は、吸水部３２により取り囲まれた欠損部３３を備える。 As shown in FIGS. 1 to 6, 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.
　この実施例の生体細胞凍結保存具１では、図１ないし図３に示すように、本体部２は、細長い棒状の本体部となっている。また、この実施例の生体細胞凍結保存具１では、生体細胞保持部３の光透過性を有するベース部３１は、可撓性かつ透明性かつ耐寒性（具体的には、液体窒素耐性）材料により形成されており、この実施例では、細長い平坦シート状のものとなっている。 In the living cell cryopreservation tool 1 of this embodiment, as shown in FIG. 1 to FIG. 3, the main body 2 is an elongated rod-like main body. Further, in the living cell cryopreservation tool 1 of this embodiment, 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.
　本体部２は、図１ないし図３に示すように、生体細胞保持部３（ベース部３１）の一端を保持している。本体部の長さとしては、５０～２００ｍｍ程度が好適である。また、本体部の幅としては、２～７ｍｍ程度が好適であり、厚さは、１～７ｍｍ程度が好適である。本体部２は、主軸部２１と、主軸部２１の先端部に設けられた生体細胞保持部３（ベース部３１）を保持する保持部２２と、本体部２の後端部に設けられた把持部２３と、主軸部２１と把持部２３間に設けられたテーパー部２４とを備えている。この実施例の生体細胞凍結保存具１では、把持部２３を除き、後述する筒状収納部材４内に、収納可能であり、テーパー部２４は、筒状収納部材４の開口部と係合可能なものとなっている。 As shown in FIGS. 1 to 3, 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. 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.
　本体部２は、ポリエステル（例えば、ポリエチレンテレフタレート、ポリブチレンテレフタレート）、ポリオレフィン（例えば、ポリエチレン、超高分子量ポリエチレン、ポリプロピレン、エチレン－プロピレン共重合体、エチレン－酢酸ビニル共重合体）、スチレン系樹脂（例えば、ポリスチレン、メタクリレート－スチレン共重合体、メタクリレート－ブチレン－スチレン共重合体）、ポリアミド（例えば、６ナイロン，６６ナイロン）、ポリサルホン、フッ素樹脂、ポリイミド等の耐寒性樹脂が使用され、特に、液体窒素の温度に耐え得るものが好ましい。  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.
　この実施例では、ベース部３１は、細長い平坦シート状のものとなっている。ベース部３１は、生体細胞（例えば、卵）の付着作業を容易にするために、光透過性、好ましくは、透明性を有する平坦シート状のものが好ましい。特に、無色透明の平坦シートが好ましい。さらには、可撓性を有するものであってもよい。 In this embodiment, 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). In particular, a colorless and transparent flat sheet is preferred. Furthermore, it may have flexibility.
　生体細胞保持部３の先端部には、マーカー３４が設けられている。マーカー３４は、生体細胞保持部３の先端部の一方の表面（具体的には、吸水シートまたはベース部の表面）に、有色を付する（油性インクで着色する）ことにより形成される。ベース部３１の長さは、１０～７０ｍｍ程度、幅０．５～４．０ｍｍ程度、厚さが０．０１～０．５ｍｍ程度のものが用いられる。そして、ベース部３１は、基端部が、上述した本体部２の保持部２２に固着されている。 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.
　ベース部３１に使用されるシート形成材料としては、光透過性を有する樹脂シート、例えば、３－フッ化ポリエチレン、低密度ポリエチレン、中密度ポリエチレン、高密度ポリエチレン、ポリカーボネート、ナイロン、ポリスルホン、ポリエステル（例えば、ＰＥＴ）、ポリスチレン、ポリイミド、超高分子量ポリエチレン、エチレン－酢酸ビニル共重合体などの合成樹脂からなるフィルムあるいはそれらの積層体が挙げられる。特に、耐寒性、具体的には、液体窒素耐性材料、言い換えれば、液体窒素に接触しても脆化しないものが好ましい。 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. In particular, 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.
　そして、本発明の生体細胞凍結保存具１では、図１、図４ないし図６に示すように、光透過性を有するベース部３１表面に固定された吸水部３２を備える。さらに、吸水部３２は、吸水部３２により取り囲まれた欠損部３３を備える。そして、欠損部３３において、ベース部３１の表面が露出し、光透過性を有している。欠損部３３におけるベース部３１の表面が、生体細胞載置部位として有効に使用できる。吸水部３２の幅は、ベース部の幅と同じ～２／３程度であることが好ましい。また、吸水部３２の長さは、５～７０ｍｍが好ましく、特に、１０～５０ｍｍが好ましい。 And, as shown in FIG. 1 and FIG. 4 to FIG. 6, 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.
　また、この実施例の生体細胞凍結保存具１では、図５に示すように、生体細胞保持部３は、複数の欠損部（細胞載置部位）３３を備えている。具体的には、生体細胞保持部３の長手方向に、複数設けられている。具体的には、５つの欠損部（細胞載置部位）３３が設けられている。欠損部（細胞載置部位）３３の数としては、１～７程度が好ましく、特に、２～５が好適である。欠損部の大きさとしては、円形の場合、直径が．０．５～１．５ｍｍ程度が好適である。 Further, in the living cell cryopreservation tool 1 of this embodiment, as shown in FIG. 5, the living cell holding unit 3 includes a plurality of defective parts (cell mounting sites) 33. Specifically, a plurality of members are provided in the longitudinal direction of the biological cell holding unit 3. Specifically, 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. As the size of the defect, in the case of a circle, the diameter is. About 0.5 to 1.5 mm is preferable.
　また、欠損部３３は、円状または多角形状であることが好ましく、特に、円形、楕円形であることが好ましい。さらに、吸水部３２は、光難透過性性シートであることが好ましい。このようなものとすることにより、透過型顕微鏡を用いた生体細胞の載置作業を行う際に、吸水部３２が難透過性であり、欠損部３３には光が透過するため、細胞を載置するターゲットである欠損部（細胞載置部位）３３が浮かび上がるように見え、ターゲットの認識が極めて容易である。　 Moreover, it is preferable that the defect | deletion part 33 is circular shape or polygonal shape, and it is preferable that it is especially circular and an ellipse. Furthermore, 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.
　また、この実施例の生体細胞凍結保存具１では、図５に示すように、吸水部３２は、欠損部３３を除き、ベース部３１の先端部の一方の表面の全体を覆うものとなっている。このようなものとすることにより、欠損部（細胞載置部位）３３の認識がより容易となる。 Further, in the biological cell cryopreservation tool 1 of this embodiment, as shown in FIG. 5, 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.
　吸水部３２は、その全体もしくは一部が、ベース部３１の表面に固定されている。吸水部３２は、耐寒性材料により形成されている。吸水部３２のベース部３１への固定は、接着剤、両面テープなどの接着性物質により行うことが好ましい。なお、部分的なヒートシールによって、固定してもよい。 All or part of 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.
　吸水部としては、繊維からなるシート、多孔性樹脂シートなどの各種シートが使用できる。吸水部の厚みは１０μｍ～５ｍｍであることが好ましく、より好ましくは２０μｍ～２．５ｍｍである。そして、繊維からなるシートとしては、紙又は不織布が使用できる。 As a water absorption part, 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 μm to 5 mm, more preferably 20 μm to 2.5 mm. And a paper or a nonwoven fabric can be used as a sheet which consists of textiles.
　吸水部として紙を用いる場合、紙としては、密度が０．１～０．６ｇ／ｃｍ３であり、坪量が１０～１３０ｇ／ｍ２の紙であることが好ましい。特に密度が０．１２～０．３ｇ／ｃｍ３であり、坪量が１０～１００ｇ／ｍ２である紙は、保存液の吸収性に優れ、さらには、透過型顕微鏡を用いて載置部上に載置した細胞又は組織を観察することができるような、細胞又は組織の視認性に優れた凍結保存用治具を提供することが可能となるため好ましい。 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 . In particular, 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.
　吸水部として不織布を用いる場合、不織布を形成する繊維としては、セルロース繊維、セルロース繊維からなる再生繊維であるレーヨン繊維やキュプラ繊維、さらにはセルロース繊維からの半合成繊維であるアセテート繊維、ポリエステル繊維、ナイロン繊維、アクリル繊維、ポリプロピレン繊維、ポリエチレン繊維、ポリ塩化ビニル繊維、ビニリデン繊維、ポリウレタン繊維、ビニロン繊維、ガラス繊維、絹繊維などが挙げられ、これら繊維を各種混合した不織布も用いることができる。中でもセルロース繊維、セルロース繊維由来の繊維でセルロース再生繊維であるレーヨン繊維やキュプラ繊維、更にはセルロース繊維からの半合成繊維であるアセテート繊維が好ましい。また、不織布としては、密度が０．１～０．４ｇ／ｃｍ３であり、坪量が１０～１３０ｇ／ｍ２の不織布であることが好ましい。特に密度が０．１２～０．３ｇ／ｃｍ３であり、坪量が１０～１００ｇ／ｍ２であるものが好ましい。 When a non-woven fabric is used as the water absorbing portion, as fibers forming the non-woven fabric, 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. Among them, preferred are cellulose fibers, rayon fibers and cupra fibers which are cellulose regenerated fibers and fibers derived from cellulose fibers, and acetate fibers which are semi-synthetic fibers derived from cellulose fibers. 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 . In particular, 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.
　吸水部として、使用可能な多孔性樹脂シートとしては、例えば特公昭４２－１３５６０号公報や、特開平０８－２８３４４７号公報に記載される、少なくとも一軸方向に延伸し、樹脂の融点以上に加熱し焼結することで得た微細繊維状構造により多孔質構造を形成した樹脂シート、特開２００９－２３５４１７号公報（ＷＯ公開０３－０１４２０５、ＵＳＰ７７５８９５３、ＵＳＰ８１１０２８９）に記載される、乳化重合又は粉砕等の方法によって得られた熱可塑性樹脂の固体粉末を金型に充填し、加熱、焼結して粉末粒子表面を融着させて冷却することにより、多孔質構造を形成した樹脂シート等が挙げられる。 As a 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. A resin sheet having a porous structure formed by a fine fibrous structure obtained by sintering, such as emulsion polymerization or pulverization described in JP-A-2009-235417 (WO 03-014205, USP 7758953, USP 8110289) 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.
　上記した多孔性樹脂シートを形成する樹脂としては、低密度ポリエチレン、高密度ポリエチレン、超高分子ポリエチレン等の各種ポリエチレンやポリプロピレン、ポリメチルメタクリレート、ポリスチレン、ポリテトラフルオロエチレンやポリビニリデンジフロライド等のフッ素樹脂、エチレン－酢酸ビニル共重合体、ポリアミド、スチレン－アクリロニトリル共重合体、スチレン－ブタジエン－アクリロニトリル三元共重合体、ポリカーボネート、ポリ塩化ビニル等が挙げられる。 As a resin for forming the porous resin sheet described above, 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 Examples thereof include fluorine resin, ethylene-vinyl acetate copolymer, polyamide, styrene-acrylonitrile copolymer, styrene-butadiene-acrylonitrile terpolymer, polycarbonate, polyvinyl chloride and the like.
　なお、吸水部３２の形態としては、上記のものに限定されるものではない。例えば、図７に示す生体細胞保持部３ａのように、生体細胞保持部３ａの側部に、ベース部露出部３５を有するものであってもよい。この実施例のものでは、生体細胞保持部３ａは、吸水部３２ａの両側部に生体細胞保持部３ａの長手方向に延びる向かい合う２つのベース部露出部３５を有している。 In addition, as a form of the water absorption part 32, it is not limited to said thing. For example, as in the living cell holding unit 3a shown in FIG. 7, the base exposed portion 35 may be provided on the side of the living cell holding unit 3a. In this embodiment, 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.
　また、図８に示す生体細胞保持部３ｂのように、中央部に欠損部３３を有する個別吸水部３２ｂが複数設けられたものであってもよい。この実施例では、個別吸水部３２ｂは、円形状のものとなっており、内部に円形状の欠損部３３を備えている。そして、複数の個別吸水部３２ｂが、ベース部３１の表面に固定されている。また、個別吸水部３２ｂの外側および欠損部３３において、ベース部３１の表面は、露出している。なお、個別吸水部３２ｂの形状は、円形に限定されるものではなく、楕円状のもの、矩形状のもの、多角形状のものであってもよい。 Further, as in the living cell holding unit 3b shown in FIG. 8, a plurality of individual water absorbing units 32b each having a deficient portion 33 in the central portion may be provided. In this embodiment, 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.
　また、図９に示す実施例の生体細胞凍結保存具の生体細胞保持部３ｃのように、吸水部３２ｃは、所定の厚さ（高さ）を有するものであってもよい。この場合、吸水部３２ｃの厚さ（高さ）としては、２～７ｍｍ程度が好適であり、特に、２～５ｍｍが好ましい。このように、吸水部３２ｃがある程度の厚さを有する場合、欠損部３３ａにおける吸水部３２ｃの内面とベース部３１の上面により、細胞収納部が形成され、細胞の離出が抑制される。 In addition, as in the case of the biological cell holding part 3c of the biological cell cryopreservation tool of the embodiment shown in FIG. 9, the water absorbing part 32c may have a predetermined thickness (height). In this case, the thickness (height) of the water absorbing portion 32c is preferably about 2 to 7 mm, and more preferably 2 to 5 mm. As described above, when the water absorbing portion 32c has a certain thickness, 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.
　筒状収納部材４は、図１０ないし図１２に示すように、生体細胞保持部３を被包可能に本体部２に着脱自在に取り付けられるものであり、一端が封鎖され、かつ耐寒性材料により形成されたものである。特に、この実施例の筒状収納部材４は、筒状本体４１と、その一端部内に収納された封止部材４２とを備える。筒状本体４１の一端部は、収納する封止部材(具体的には、通気性封止部材）４２により通気可能に封止されている。 As shown in FIGS. 10 to 12, 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. In particular, 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.
　さらに、この実施例の筒状収納部材４では、筒状本体４１の先端部には、錘４３が収納されている。錘４３は、封止部材４２よりも先端側に収納されている。錘４３を設けることにより、液体窒素タンクに投入された筒状収納部材４は、その先端側が下方を確実に向くものとなる。また、筒状収納部材４は、生体細胞保持部３に接触することなく被包できる内径および長さを備えている。また、筒状本体４１は、後端（開口部）の外面には、開口部の認識を容易とするための着色部４４を備えている。 Furthermore, in the cylindrical storage member 4 of this embodiment, 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. By providing the weight 43, the tip end side of the cylindrical storage member 4 charged into the liquid nitrogen tank is surely directed downward. In addition, the cylindrical storage member 4 has an inner diameter and a length that can be encapsulated without contacting the biological cell holding unit 3. Moreover, 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).
　筒状収納部材４の形成材料としては、例えば、３－フッ化ポリエチレン、低密度ポリエチレン、中密度ポリエチレン、高密度ポリエチレン、ポリカーボネート、ナイロン、ポリスルホン、ポリエステル（例えば、ＰＥＴ）、ポリスチレン、ポリイミド、超高分子量ポリエチレン、エチレン－酢酸ビニル共重合体などの合成樹脂からなるフィルムあるいはそれらの積層体が挙げられる。特に、液体窒素耐性材料、言い換えれば、液体窒素に接触しても脆化しないものが好ましい。 As a forming material of 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. In particular, a liquid nitrogen resistant material, in other words, a material which does not become embrittled even in contact with liquid nitrogen is preferable.
　次に、本発明の生体細胞凍結保存具１および生体細胞凍結保存用具１０の使用方法について説明する。
　この説明では、生体細胞である卵子を凍結保存する場合を例にとり説明する。
　まず、ピペットの先端に卵子を採取し、卵子の細胞内液を平衡液に置換する作業を行い、さらに、細胞外液をガラス化液に置換する作業を行う。そして、顕微鏡下において、本発明の生体細胞凍結保存具１の生体細胞保持部３に設けられた吸水部３２の欠損部３３に卵子を少量のガラス化液とともに配置させ、ベース部材３１の表面に付着させる。卵子が付着した生体細胞凍結保存具１の生体細胞保持部をあらかじめ準備しておいた液体窒素に浸漬し凍結（ガラス化）させる。そして、筒状部材４内に、卵子が付着した生体細胞凍結保存具１を挿入し、収納容器（図示せず）内に、生体細胞凍結保存具１を収納した筒状収納部材４を収納させ、続いて、収納容器を液体窒素タンク内に入れ保存する。 Next, a method of using the living cell cryopreservation tool 1 and the living cell cryopreservation tool 10 of the present invention will be described.
In this explanation, the case of cryopreserving an ovum, which is a living cell, will be described as an example.
First, an egg is collected at the tip of the pipette, the intracellular fluid of the egg is replaced with the equilibration solution, and the extracellular fluid is replaced with the vitrification solution. Then, under a microscope, an egg is placed along with a small amount of vitrification liquid in the defective portion 33 of the water absorbing portion 32 provided in the biological cell holding portion 3 of the biological cell cryopreservation tool 1 of the present invention. Adhere. 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.
　また、本発明の生体細胞凍結保存具および生体細胞凍結保存用具としては、図２１ないし図２３に示すような生体細胞凍結保存具１ａであってもよい。
　この実施例の生体細胞凍結保存具１ａは、耐寒性材料により形成された本体部２と、耐寒性材料により形成された生体細胞保持部３ｄとを備える。そして、生体細胞保持部３ｄは、光透過性を有し、細長いベース部３１と、ベース部の少なくとも一方の表面の両側部に固定された細長い２つの吸水部３２ａ，３２ｂを備える。そして、２つの吸水部３２ａ，３２ｂ間にて露出するベース部３１が、光透過性を有する細胞載置部位３３となっている。 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 | part 33 which has light transmittance.
　この実施例の生体細胞凍結保存具１ａの基本構成は、上述した生体細胞凍結保存具１と同じであり、相違点は、吸水部の形態のみである。
　光透過性を有するベース部３１は、上述したものと同じである。図２１および図２３に示すように、ベース部３１の一方の表面に吸水部３２ａ，３２ｂが固定されている。なお、吸水部３２ａ，３２ｂは、ベース部３１の表面および裏面に設けてもよい。吸水部３２ａ，３２ｂは、ベース部３１の表面の側部を被覆するように、長手方向に細長く延びるものとなっている。２つの吸水部３２ａ，３２ｂは、向かい合うように設けられている。 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. As shown in FIGS. 21 and 23, 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.
　吸水部３２ａ，３２ｂの幅としては、０．２～１．５ｍｍが好ましく、特に、０．３～１．０ｍｍが好ましい。そして、向かい合う吸水部３２ａ，３２ｂ間において、ベース部３１の表面が露出し、生体細胞載置部位３３となっている。また、吸水部３２ａ，３２ｂの長さとしては、５～７０ｍｍが好ましく、特に、１０～５０ｍｍが好ましい。また、吸水部３２ａ，３２ｂ間の離間距離、言い換えれば、生体細胞載置部位３３の幅は、０．５～１．５ｍｍが好ましい。吸水部３２ａ，３２ｂの構成材料としては、上述した吸水部３２にて説明したものが使用可能である。 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. In addition, 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. As the constituent material of the water absorbing portions 32a and 32b, those described for the water absorbing portion 32 described above can be used.
　この実施例の生体細胞凍結保存具１ａにおいても、図２３に示すように、生体細胞保持部３ｄの先端部には、マーカー３４が設けられている。マーカー３４は、生体細胞保持部３ｄの先端部の一方の表面（具体的には、吸水シートまたはベース部の表面）に、有色を付する（油性インクで着色する）ことにより形成される。
　また、この実施例の生体細胞凍結保存具１ａにおいても、上述した筒状収納部材４を用いることができる。生体細胞凍結保存具１ａと筒状収納部材４により、生体細胞凍結保存用具が、構成される。 Also in the living cell cryopreservation tool 1a of this embodiment, as shown in FIG. 23, 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.
Further, 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.
　また、本発明の生体細胞凍結保存具および生体細胞凍結保存用具としては、図２４および図２５に示すような生体細胞凍結保存具１ｂであってもよい。
　この実施例の生体細胞凍結保存具１ｂは、耐寒性材料により形成された本体部２と、耐寒性材料により形成された生体細胞保持部３ｅとを備える。そして、生体細胞保持部３ｅは、光透過性を有し、細長いベース部３１と、ベース部３１の少なくとも一方の表面に、ベース部３１を横切るように固定された向かい合う２つの吸水部３７ａ，３７ｂ，３７ｃ，３７ｄを有し、吸水部３７ａ，３７ｂ，３７ｃ，３７ｄ間にて露出するベース部３１が、光透過性を有する細胞載置部位３３となっている。 In addition, as the living cell cryopreservation tool and the living cell cryopreservation tool of the present invention, a living cell cryopreservation tool 1b as shown in FIGS. 24 and 25 may be used.
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.
　この実施例の生体細胞凍結保存具１ｂの基本構成は、上述した生体細胞凍結保存具１と同じであり、相違点は、吸水部の形態のみである。
　光透過性を有するベース部３１は、上述したものと同じである。図２４および図２５に示すように、ベース部３１の一方の表面に吸水部３７ａ，３７ｂ，３７ｃ，３７ｄが固定されている。なお、吸水部３７ａ，３７ｂ，３７ｃ，３７ｄは、ベース部３１の表面および裏面に設けてもよい。
　吸水部３７ａ，３７ｂ，３７ｃ，３７ｄは、ベース部３１の表面を横切るように、複数設けられている。各吸水部３７ａ，３７ｂ，３７ｃ，３７ｄは、ベース部３１の長手方向に隣り合う吸水部が向かい合うように設けられている。隣り合う吸水部間における露出するベース部部分が、生体細胞載置部位３３となっている。 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. As shown in FIG. 24 and FIG. 25, 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.
　吸水部３７ａ，３７ｂ，３７ｃ，３７ｄは、ベース部３１の幅全体に延びるものとなっている。なお、吸水部３７ａ，３７ｂ，３７ｃ，３７ｄは、図２６に示す実施例の生体細胞保持部３ｆのように、ベース部３１の幅全体ではなく、ある程度の長さ延びるものであってもよい。この場合、吸水部３７ａ，３７ｂ，３７ｃ，３７ｄは、図ベース部３１の幅の１／２以上延びるものであることが好ましい。 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.
　吸水部３７ａ，３７ｂ，３７ｃ，３７ｄのベース部３１の長手方向長は、０．２～５ｍｍが好ましく、特に、０．５～３ｍｍが好ましい。吸水部の数としては、２～１０程度が好ましく、特に、２～５が好ましい。また、吸水部３７ａ，３７ｂ，３７ｃ，３７ｄの離間距離、言い換えれば、生体細胞載置部位３３の幅は、０．５～１．５ｍｍが好ましい。吸水部３２ａ，３２ｂの構成材料としては、上述した吸水部３２にて説明したものが使用可能である。 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. In addition, 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. As the constituent material of the water absorbing portions 32a and 32b, those described for the water absorbing portion 32 described above can be used.
　上述した実施例の生体細胞凍結保存具においても、図２３、図２５および図２６に示すように、生体細胞保持部３ｄ，３ｅ、３ｆの先端部には、マーカー３４が設けられている。マーカー３４は、生体細胞保持部の先端部の一方の表面（具体的には、吸水シートまたはベース部の表面）に、有色を付する（油性インクで着色する）ことにより形成される。 Also in the living cell cryopreservation tool of the embodiment described above, as shown in FIG. 23, FIG. 25 and FIG. 26, 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.
　また、上述した実施例の生体細胞凍結保存具１ａ，１ｂおよび図２６に示した生体細胞保持部３ｆを有する実施例のすべてにおいて、図９に示す実施例の生体細胞凍結保存具の生体細胞保持部３ｃのように、吸水部は、所定の厚さ（高さ）を有するものであってもよい。この場合、吸水部の厚さ（高さ）としては、２～７ｍｍ程度が好適であり、特に、２～５ｍｍが好ましい。
　また、上述したの実施例の生体細胞凍結保存具１ａ，１ｂにおいても、上述した筒状収納部材４を用いることにより、生体細胞凍結保存用具を構成することができる。 In all of the embodiments having the biological cell cryopreservation device 1a, 1b of the above-described embodiment and the biological cell holding portion 3f shown in FIG. 26, the biological cell holding device of the biological cell cryopreservation device of the embodiment shown in FIG. Like the part 3c, the water absorption part may have a predetermined thickness (height). In this case, the thickness (height) of the water absorbing portion is preferably about 2 to 7 mm, and more preferably 2 to 5 mm.
Also, in the living cell cryopreservation tools 1a and 1b of the above-described embodiment, the living cell cryopreservation tool can be configured by using the cylindrical storage member 4 described above.
　また、本発明の生体細胞凍結保存具および生体細胞凍結保存用具としては、図１３ないし図１９に示すような生体細胞凍結保存具５および生体細胞凍結保存用具２０であってもよい。
　この実施例の生体細胞凍結保存具５は、耐寒性材料により形成された本体部５１と、耐寒性材料により形成された生体細胞保持部５２とを備える。生体細胞保持部５２は、光透過性を有するベース部５３と、ベース部５３の表面に固定された吸水部５４とを備える。吸水部５４は、吸水部５４により取り囲まれた欠損部６１を備え、欠損部６１において、ベース部５３の表面が露出し、光透過性を有している。 Further, as the living cell cryopreservation tool and the living cell cryopreservation tool of the present invention, a living cell cryopreservation tool 5 and a living cell cryopreservation tool 20 as shown in FIGS. 13 to 19 may be used.
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.
　生体細胞保持部５２のベース部５３は、断面が略矩形状であり、本体部５１と接続された基端部５９を備えている。そして、この実施例のものでは、ベース部５３は、細長い幅の狭い帯状（薄板状）のものとなっている。そして、その表面に、吸水部５４が固定されている。ベース部５２の幅は、０．４～１．０ｍｍが好ましく、長さは、５～３０ｍｍが好ましく、全体の厚さは、０．０８～１．０ｍｍが好ましい。また、ベース部５３の肉厚な基端部の長さは、５～３０ｍｍが好ましく、本体部の長さは、２０～１００ｍｍが好ましい。本体部５１は、先端方向に突出する突出部６４を備え、ベース部５３は、突出部６４を収納する凹部６５を備えている。 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.
　そして、本発明の生体細胞凍結保存具５においても、図１３、図１４、図１６および図１７に示すように、生体細胞保持部５２は、複数の欠損部（細胞載置部位）６１を備えている。具体的には、生体細胞保持部５２の長手方向に、複数設けられている。具体的には、５つの欠損部（細胞載置部位）６１が設けられている。欠損部（細胞載置部位）６１の数としては、１～７程度が好ましく、特に、２～５が好適である。欠損部の大きさとしては、円形の場合、直径が．０．５～１．５ｍｍ程度が好適である。 Also in the biological cell cryopreservation tool 5 of the present invention, as shown in FIGS. 13, 14, 16 and 17, 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.
　また、欠損部６１は、円状形態であることが好ましい。さらに、吸水部５４は、光難透過性性シートであることが好ましい。このようなものとすることにより、透過型顕微鏡を用いた生体細胞の載置作業を行う際に、吸水部５４が難透過性であり、欠損部６１には光が透過するため、細胞を載置するターゲットである欠損部（細胞載置部位）６１が浮かび上がるように見え、ターゲットの認識が極めて容易である。　 Moreover, it is preferable that the defect part 61 is a circular form. Furthermore, 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.
　また、この実施例の生体細胞凍結保存具５では、図１４および図１６に示すように、生体細胞保持部５２は、吸水部５４の両側部に生体細胞保持部５２の長手方向に延びる向かい合う２つのベース部露出部（後述する膨出部５７，５８）を有している。なお、吸水シート５４は、欠損部６１を除き、ベース部５３の先端部の一方の表面の全体を覆うものであってもよい。さらには、図７に示した生体細胞保持部３ｂのように、中央部に欠損部を有する個別吸水部が複数設けられたものであってもよい。 Further, in the living cell cryopreservation tool 5 of this embodiment, as shown in FIGS. 14 and 16, 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. There are two exposed base portions ( expansion portions 57 and 58 described later). 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. Furthermore, as in the case of the biological cell holding part 3b shown in FIG. 7, a plurality of individual water absorption parts having a defect part in the central part may be provided.
　吸水部５４は、その全体もしくは一部が、ベース部５３の表面に固定されている。吸水部５４のベース部５３への固定は、接着剤、両面テープなどの接着性物質により行うことが好ましい。なお、部分的なヒートシールによって、固定してもよい。
　吸水部としては、繊維からなるシート、多孔性樹脂シートなどの各種シートが使用できる。吸水部の厚みは１０μｍ～５ｍｍであることが好ましく、より好ましくは２０μｍ～２．５ｍｍである。そして、繊維からなるシートとしては、紙又は不織布が使用できる。吸水部としては、上述したものが好適に使用できる。 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.
As a water absorption part, 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 μm to 5 mm, more preferably 20 μm to 2.5 mm. And a paper or a nonwoven fabric can be used as a sheet which consists of textiles. As the water absorbing portion, those described above can be suitably used.
　さらに、この実施例の生体細胞凍結保存具５では、図１３、図１４、図１６ないし図１８に示すように、ベース部５３の吸水部５４の固定部の両側部に設けられかつ生体細胞保持部５２の長手方向に延びる２つの側部膨出部５７，５８を有している。このため、生体細胞凍結保存具５は、吸水部固定部の両側部に膨出部を有するため、生体細胞載置時における生体細胞の側部方向への移動を確実に抑制できる。 Furthermore, in 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.
　さらに、この実施例の生体細胞凍結保存具５では、図１３ないし図１８（特に、図１５，図１６）に示すように、生体細胞保持部５２は、吸水部固定部より生体細胞保持部５２の先端側に設けられた突出部６３を備えている。突出部６３は、生体細胞保持部５２の先端部より、上面側（吸水部側）に突出するものとなっている。また、突出部６３は、図１７に示すように、先端面は、若干基端側に傾斜する傾斜面となっている。このような突出部６３を形成することにより、もし、生体細胞収納部より生体細胞が離脱して、先端側に移動したとき、生体細胞凍結保存具５、言い換えれば、生体細胞保持部５２からの落下を防止する。特に、この実施例の生体細胞凍結保存具５では、上述したように、生体細胞保持部５２の両側部に設けられた膨出部５７，５８と協同することにより、生体細胞の落下を防止する。 Furthermore, in the living cell cryopreservation tool 5 of this embodiment, as shown in FIGS. 13 to 18 (in particular, FIGS. 15 and 16), 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). Further, as shown in FIG. 17, the tip end surface of the projecting portion 63 is an inclined surface which is slightly inclined to the base end side. By forming such a projecting part 63, if the living cell is detached from the living cell storage part and moved to the tip side, the living cell cryopreservation tool 5, in other words, from the living cell holding part 52. Prevent falling. In particular, in the living cell cryopreservation tool 5 of this embodiment, as described above, the falling of living cells is prevented by cooperating with the bulging parts 57 and 58 provided on both sides of the living cell holding part 52. .
　本体部５１、ベース部５３および吸水部５４は、耐寒性材料により形成されている。特に、本体部５１、ベース部５３および吸水部５４は、液体窒素耐性材料、言い換えれば、液体窒素に接触しても脆化しないものが好ましい。また、ベース部５３は、透明性を有することが好ましく、また、多少の可撓性を有するものであることが好ましい。本体部５１およびベース部５３の形成材料としては、例えば、３－フッ化ポリエチレン、低密度ポリエチレン、中密度ポリエチレン、高密度ポリエチレン、ポリカーボネート、ナイロン、ポリスルホン、ポリエステル、ポリスチレン、ポリイミド、超高分子量ポリエチレン、エチレン－酢酸ビニル共重合体などの合成樹脂、また、それら合成樹脂により形成されたフィルムの積層体が好適に使用される。 The main body portion 51, the base portion 53, and the water absorbing portion 54 are formed of a cold resistant material. In particular, 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. In addition, 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.
　そして、この実施例の生体細胞凍結保存具５では、図１３ないし図１９に示すように、生体細胞保持部５２は、生体細胞保持部５２の長手方向に延び、かつ、生体細胞保持部５２の先端より突出する熱伝導性体５５，５６を有している。特に、この実施例の生体細胞凍結保存具５では、熱伝導性体５５，５６は、線状熱伝導性体となっており、生体細胞保持部５２の両方の側部に設けられている。さらに、この実施例のものでは、熱伝導性体５５，５６は、吸水部固定部を越えて、ベース部５３の基端側に延び、生体細胞保持部５２の基端もしくはその付近に到達するものとなっている。また、熱伝導性体５５，５６は、先端部５５ａ，５６ａを除き、側部膨出部５７，５８内に埋設されている。言い換えれば、先端部５５ａ，５６ａは、生体細胞保持部５２の先端より突出し、外面が露出した状態となっている。 In the living cell cryopreservation tool 5 of this embodiment, as shown in FIGS. 13 to 19, 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. In particular, in the living cell cryopreservation tool 5 of this embodiment, the thermally conductive bodies 55, 56 are linear thermal conducting bodies, and are provided on both sides of the living cell holding portion 52. Furthermore, in this embodiment, 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.
　また、この実施例では、熱伝導性体５５，５６は、線状部材もしくは細い棒状部材により形成されている。さらに、熱伝導性体５５，５６の先端部５５ａ，５６ａの形状は、基端側に向かって湾曲する湾曲先端部となっているものであってもよい。この場合、湾曲先端部５５ａ，５６ａは、図１４に示すように、生体細胞凍結保存具５の先端部の前方に突出し、かつ、突出部６３を保護するものであることが好ましい。
　熱伝導性体５５，５６は、熱伝導性材料により形成されている。熱伝導性材料としては、銀、銅、アルミニウム、ステンレス鋼などの金属類、窒化アルミニウム、窒化ケイ素、アルミナなどの熱伝導性セラミックスなどが好適に使用できる。 Further, in this embodiment, 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.
　図１９に示す実施例の生体細胞凍結保存用具２０は、上述した生体細胞凍結保存具５と、生体細胞凍結保存具５を収納可能かつ耐寒性材料により形成された一端が閉塞した筒状収納部材７とを備える。
　筒状収納部材７は、図１９に示すように、生体細胞凍結保存具５を収納可能かつ耐寒性材料により形成された一端が閉塞した筒状体である。筒状収納部材７は、内部に生体細胞保持部材収納部７１を有する筒状体７０と、筒状体７０の先端に設けられた熱伝導性部材７２とを備える。また、この実施例では、筒状収納部材７は、先端閉塞部７３と、基端開口部７４と、内部に生体細胞保持部材収納部７１を有する筒状体７０と、筒状体７０の先端部内に収納された熱伝導性部材７２とにより構成されている。 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. And 7.
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. Further, in this embodiment, 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.
　熱伝導性部材７２は、筒状体７０の内部に移動不能に収納されており、また、先端閉塞部７３の内面に当接するように収納されている。また、熱伝導性部材７２は、金属製の円柱状のものが用いられている。熱伝導性部材７２の上面、すなわち、生体細胞凍結保存具５の熱伝導性体５５，５６の先端部５５ａ，５６ａと接触可能なものとなっている。なお、熱伝導性部材７２は、先端面、先端側側面などが筒状体７０より露出するものであってもよい。 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.
　また、このタイプの生体細胞凍結保存具においても、図２０に示す生体細胞凍結保存具５ａのように、ベース部５３の上面に設けられる吸水部５４ａは、所定の厚さ（高さ）を有するものであってもよい。この場合、吸水部５４ａの厚さ（高さ）としては、２～７ｍｍ程度が好適であり、特に、２～５ｍｍが好ましい。このように、吸水部５４ａがある程度の厚さを有する場合、欠損部６１における吸水部５４ａの内面とベース部５３の上面により、細胞収納部が形成され、載置される細胞６８の離出が抑制される。 Also in this type of biological cell cryopreservation tool, as in the biological cell cryopreservation tool 5a shown in FIG. 20, the water absorbing portion 54a provided on the upper surface of the base portion 53 has a predetermined thickness (height). It may be one. In this case, the thickness (height) of the water absorbing portion 54a is preferably about 2 to 7 mm, and more preferably 2 to 5 mm. Thus, when the water absorbing portion 54a has a certain thickness, a cell storage portion is formed by the inner surface of the water absorbing portion 54a in the defective portion 61 and the upper surface of the base portion 53, and the cells 68 to be mounted are detached. Be suppressed.
　筒状体７０は、耐寒性材料により形成されている。特に、筒状体７０は、液体窒素耐性材料、言い換えれば、液体窒素に接触しても脆化しないものが好ましい。また、筒状体７０は、内部が視認可能な透明もしくは半透明体であることが好ましい。筒状体７０の形成材料としては、例えば、３－フッ化ポリエチレン、低密度ポリエチレン、中密度ポリエチレン、高密度ポリエチレン、ポリカーボネート、ナイロン、ポリスルホン、ポリエステル、ポリスチレン、ポリイミド、超高分子量ポリエチレン、エチレン－酢酸ビニル共重合体などの合成樹脂、また、それら合成樹脂により形成されたフィルムの積層体が好適に使用される。 The cylindrical body 70 is formed of a cold resistant material. In particular, it is preferable that 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. Moreover, it is preferable that the cylindrical body 70 is a transparent or semi-transparent body whose inside is visible. As 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.
　また、筒状収納部材７の生体細胞保持部材収納部７１の長さは、生体細胞凍結保存具５の全長より、１０～５０ｍｍ長いものであることが好ましい。また、筒状収納部材７（筒状体７０）の全長は、５０～１００ｍｍが好ましく、内径は、２～５ｍｍが好ましい。 In addition, 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.
　次に、本発明の生体細胞凍結保存用具５の使用方法について説明する。
　この説明では、生体細胞である卵子を凍結保存する場合を例にとり説明する。
　まず、複数の卵子を採取し、卵子の細胞内液を平衡液に置換する作業を行い、さらに、細胞外液をガラス化液に置換する作業を行う。そして、顕微鏡下において、図１８に示すように、生体細胞凍結保存具５の生体細胞保持部５２に設けられた吸水部５４の欠損部６１に卵子６８を少量のガラス化液とともに配置させ、ベース部材５３の表面に付着させる。卵子が付着した生体細胞凍結保存具５を生体細胞保持部５２側より、筒状収納部材７内に挿入し、図１９に示すように、生体細胞凍結保存具５の生体細胞保持部５２の先端より突出する熱伝導性体５５，５６の先端部５５ａ，５６ａが、筒状収納部材７内の熱伝導性部材７２と接触する状態とする。 Next, a method of using the living cell cryopreservation tool 5 of the present invention will be described.
In this explanation, the case of cryopreserving an ovum, which is a living cell, will be described as an example.
First, a plurality of ova are collected, the intracellular fluid of the ova is replaced with the equilibration solution, and the extracellular fluid is replaced with the vitrification solution. Then, under a microscope, as shown in FIG. 18, 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.
　そして、生体細胞凍結保存具５を収納した筒状収納部材７を先端側（伝導性部材７２側）より、あらかじめ準備しておいた液体窒素に浸漬し凍結（ガラス化）させる。筒状収納部材７の液体窒素との接触により、筒状収納部材７内の熱伝導性部材７２は急激冷却され、その冷却は、熱伝導性部材７２と接触する生体細胞凍結保存具５の生体細胞保持部５２の熱伝導性体５５，５６より温度を急激に奪うものとなり、生体細胞保持部５２に保持されてる卵子も急激に冷却される。収納容器（ケーン）に、ガラス化した卵子を付着保持する生体細胞凍結保存具５を筒状収納部材７ごと収納した後、収納容器を液体窒素タンク内に入れ保存する。 Then, 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. After 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.
　本発明の生体細胞凍結保存具は、以下のものである。
　（１）　耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
　前記生体細胞保持部は、光透過性を有するベース部と、前記ベース部の表面に固定された吸水部とを備え、前記吸水部は、前記吸水部により取り囲まれた欠損部を備え、前記欠損部において、前記ベース部の前記表面が露出し、光透過性を有している生体細胞凍結保存具。 The living cell cryopreservation device of the present invention is as follows.
(1) 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, In part, the living cell cryopreservation tool in which the surface of the base part is exposed and has light permeability.
　この生体細胞凍結保存具では、吸水部により取り囲まれた欠損部を備え、欠損部は光透過性を有するため、細胞の載置作業を行う際、欠損部の上面にピペットの先端部を配置したとき、透過型顕微鏡によるピペット先端の確認が容易である。また、過剰なガラス化液が、細胞とともに欠損部に滴下されても、ガラス化液は、欠損部を取り囲む吸水部に吸収されるため、除去操作も不要である。このため、生体細胞の凍結を迅速に行うことができる。 In this living cell cryopreservation tool, since the defect part surrounded by the water absorption part is provided and the defect part has light permeability, the tip of the pipette is disposed on the upper surface of the defect part when mounting the cells. When, it is easy to confirm the pipette tip with a transmission microscope. In addition, even if excessive vitrification liquid is dropped onto the defect with 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.
　また、上記の実施態様は、以下のものであってもよい。
　（２）　前記欠損部における前記ベース部の表面が、細胞載置部位である上記（１）に記載の生体細胞凍結保存具。
　（３）　前記生体細胞凍結保存具は、複数の前記欠損部を備えている上記（１）または（２）に記載の生体細胞凍結保存具。
　（４）　前記欠損部は、円状または多角形状である上記（１）ないし（３）のいずれかに記載の生体細胞凍結保存具。 Also, the above embodiment may be as follows.
(2) The biological cell cryopreservation tool according to the above (1), wherein the surface of the base portion in the defective portion is a cell mounting site.
(3) The living cell cryopreservation tool according to (1) or (2), wherein the living cell cryopreservation tool comprises a plurality of the defect parts.
(4) The living cell cryopreservation tool according to any one of the above (1) to (3), wherein the defect portion is circular or polygonal.
　また、本発明の生体細胞凍結保存具は、以下のものである。
　（５）　耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
　前記生体細胞保持部は、光透過性を有し、細長いベース部と、前記ベース部の少なくとも一方の表面の両側部に固定された細長い２つの吸水部を有し、前記２つの吸水部間にて露出する前記ベース部が、光透過性を有する細胞載置部位となっている生体細胞凍結保存具。 Moreover, 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.
　また、本発明の生体細胞凍結保存具は、以下のものである。
　（６）　耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
　前記生体細胞保持部は、光透過性を有し、細長いベース部と、前記ベース部の少なくとも一方の表面に、前記ベース部を横切るように固定された向かい合う２つの吸水部を有し、前記２つの吸水部間にて露出する前記ベース部が、光透過性を有する細胞載置部位となっている生体細胞凍結保存具。 Moreover, 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.
　これら生体細胞凍結保存具においても、吸水部に近接する細胞載置部位は、光透過性を有するため、細胞の載置作業を行う際、細胞載置部位の上面にピペットの先端部を配置したとき、透過型顕微鏡によるピペット先端の確認が容易である。また、過剰なガラス化液が、細胞とともに細胞載置部位に滴下されても、ガラス化液は、細胞載置部位に近接する吸水部に吸収されるため、除去操作も不要である。このため、生体細胞の凍結を迅速に行うことができる。 Also in these living cell cryopreservation tools, since the cell mounting site adjacent to the water absorbing portion has light permeability, the tip of the pipette is disposed on the upper surface of the cell mounting site when performing the cell mounting operation. When, it is easy to confirm the pipette tip with a transmission microscope. In addition, even if the excess vitrification liquid is dropped onto the cell mounting site along with the cells, 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.
　また、上記の実施態様は、以下のものであってもよい。
　（７）　前記ベース部は、無色透明である上記（１）ないし（６）のいずれかに記載の生体細胞凍結保存具。
　（８）　前記ベース部は、透明性シートである上記（１）ないし（６）のいずれかに記載の生体細胞凍結保存具。
　（９）　前記ベース部は、細長い平板状の可撓性シートである上記（１）ないし（８）のいずれかに記載の生体細胞凍結保存具。
　（１０）　前記ベース部は、細長い硬質平板部材である上記（１）ないし（７）のいずれかに記載の生体細胞凍結保存具。
　（１１）　前記吸水部は、光難透過性性シートである上記（１）ないし（１０）のいずれかに記載の生体細胞凍結保存具。 Also, 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.
　また、本発明の生体細胞凍結保存用具は、以下のものである。
　（１２）　上記（１）ないし（１１）のいずれかに記載の生体細胞凍結保存具と、前記生体細胞凍結保存具を収納可能かつ耐寒性材料により形成された一端が閉塞した筒状収納部材とを備える生体細胞凍結保存用具。 Moreover, 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:

Claims (12)

  1. 耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
    　前記生体細胞保持部は、光透過性を有するベース部と、前記ベース部の表面に固定された吸水部とを備え、前記吸水部は、前記吸水部により取り囲まれた欠損部を備え、前記欠損部において、前記ベース部の前記表面が露出し、光透過性を有していることを特徴とする生体細胞凍結保存具。     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, In part, the surface of the base part is exposed and has optical transparency.
  2. 前記欠損部における前記ベース部の表面が、細胞載置部位である請求項１に記載の生体細胞凍結保存具。 The biological cell cryopreservation tool according to claim 1, wherein the surface of the base portion in the defect portion is a cell mounting site.
  3. 前記生体細胞凍結保存具は、複数の前記欠損部を備えている請求項１または２に記載の生体細胞凍結保存具。 The living cell cryopreservation apparatus according to claim 1, wherein the living cell cryopreservation apparatus comprises a plurality of the defects.
  4. 前記欠損部は、円状または多角形状である請求項１ないし３のいずれかに記載の生体細胞凍結保存具。 The living cell cryopreservation tool according to any one of claims 1 to 3, wherein the defect portion is circular or polygonal.
  5. 耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
    　前記生体細胞保持部は、光透過性を有し、細長いベース部と、前記ベース部の少なくとも一方の表面の両側部に固定された細長い２つの吸水部を有し、前記２つの吸水部間にて露出する前記ベース部が、光透過性を有する細胞載置部位となっていることを特徴とする生体細胞凍結保存具。     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.
  6. 耐寒性材料により形成された本体部と、耐寒性材料により形成された生体細胞保持部とを備える生体細胞凍結保存具であって、
    　前記生体細胞保持部は、光透過性を有し、細長いベース部と、前記ベース部の少なくとも一方の表面に、前記ベース部を横切るように固定された向かい合う２つの吸水部を有し、前記２つの吸水部間にて露出する前記ベース部が、光透過性を有する細胞載置部位となっていることを特徴とする生体細胞凍結保存具。     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 characterized in that the base portion exposed between two water absorbing portions is a light transmitting cell mounting site.
  7. 前記ベース部は、無色透明である請求項１ないし６のいずれかに記載の生体細胞凍結保存具。 The biological cell cryopreservation device according to any one of claims 1 to 6, wherein the base portion is colorless and transparent.
  8. 前記ベース部は、透明性シートである請求項１ないし６のいずれかに記載の生体細胞凍結保存具。 The biological cell cryopreservation tool according to any one of claims 1 to 6, wherein the base portion is a transparent sheet.
  9. 前記ベース部は、細長い平板状の可撓性シートである請求項１ないし８のいずれかに記載の生体細胞凍結保存具。 The biological cell cryopreservation device according to any one of claims 1 to 8, wherein the base portion is an elongated flat flexible sheet.
  10. 前記ベース部は、細長い硬質平板部材である請求項１ないし７のいずれかに記載の生体細胞凍結保存具。 The biological cell cryopreservation device according to any one of claims 1 to 7, wherein the base portion is an elongated hard flat plate member.
  11. 前記吸水部は、光難透過性性シートである請求項１ないし１０のいずれかに記載の生体細胞凍結保存具。 The biological cell cryopreservation tool according to any one of claims 1 to 10, wherein the water absorbing portion is a light impermeable sheet.
  12. 請求項１ないし１１のいずれかに記載の生体細胞凍結保存具と、前記生体細胞凍結保存具を収納可能かつ耐寒性材料により形成された一端が閉塞した筒状収納部材とを備える生体細胞凍結保存用具。 A biological cell cryopreservation apparatus comprising: the biological cell cryopreservation device according to any one of claims 1 to 11; and a cylindrical storage member capable of storing the biological cell cryopreservation device and having one end blocked by a cold resistant material Tools.
PCT/JP2018/024427 2017-06-30 2018-06-27 Biological cell cryopreservation tool and tool for biological cell cryopreservation WO2019004300A1 (en)

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