WO2015115313A1 - Jig for use in cell or tissue vitrification cryopreservation - Google Patents

Jig for use in cell or tissue vitrification cryopreservation Download PDF

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Publication number
WO2015115313A1
WO2015115313A1 PCT/JP2015/051770 JP2015051770W WO2015115313A1 WO 2015115313 A1 WO2015115313 A1 WO 2015115313A1 JP 2015051770 W JP2015051770 W JP 2015051770W WO 2015115313 A1 WO2015115313 A1 WO 2015115313A1
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WIPO (PCT)
Prior art keywords
vitrification
cells
jig
cryopreservation
solution
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PCT/JP2015/051770
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French (fr)
Japanese (ja)
Inventor
松澤篤史
須崎活光
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三菱製紙株式会社
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Priority claimed from JP2014017015A external-priority patent/JP2015142523A/en
Priority claimed from JP2014069295A external-priority patent/JP2015188404A/en
Application filed by 三菱製紙株式会社 filed Critical 三菱製紙株式会社
Publication of WO2015115313A1 publication Critical patent/WO2015115313A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
    • A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen

Definitions

  • the present invention relates to a vitrification cryopreservation jig used for cryopreserving biological cells or tissues.
  • embryos used in bovine embryo transfer technology are transplanted in accordance with the estrous cycle of the recipient cow, and the embryo is cryopreserved and matched to the estrous cycle in order to match the embryo transfer to the estrous cycle.
  • the embryo is thawed and transplanted.
  • human infertility treatment after collecting an egg or ovary from a mother, it is stored frozen in order to match the timing suitable for transplantation, and thawed at the time of transplantation.
  • a slow freezing method is known as a method for preserving cells or tissues.
  • a preservation solution obtained by adding a freezing agent to a physiological solution such as phosphate buffered saline (freezing agent content is generally about 10% by volume or less, for example, J. Org. Mamm.Ova Res.Vol.21,2004 P65-68) is immersed in cells or tissues.
  • a physiological solution such as phosphate buffered saline
  • antifreeze compounds such as glycerol and ethylene glycol are used.
  • the cells or tissues are cooled or cooled to ⁇ 30 to ⁇ 35 ° C.
  • the solution inside and outside is sufficiently cooled and the viscosity becomes high.
  • a relatively slow cooling rate for example, 0.3 to 0.5 ° C./min
  • the micro solutions inside and outside the cells and the tissues remain solid and remain solid. Vitrification occurs.
  • the inside or outside of a cell or tissue is solidified by vitrification, the molecular movement is substantially eliminated. Therefore, it is considered that the vitrified cell or tissue can be stored semipermanently by storing it in liquid nitrogen.
  • the slow freezing method since it is necessary to cool at a relatively slow cooling rate, the operation for cryopreservation takes time. In addition, there is a problem of requiring a device or jig for temperature control. In addition, in the slow freezing method, since ice crystals are formed in the preservation solution outside the cells or tissues, the cells or tissues may be physically damaged by the ice crystals.
  • a vitrification preservation method has been proposed as a method for solving the problems in the slow freezing method.
  • the vitrification preservation method means that a large amount of antifreezing agents such as glycerol, ethylene glycol, DMSO (dimethyl sulfoxide), etc. (generally 30 to 40% by volume, for example, Journal of Japanese Society for Regenerative Medicine VOL13.No.1, P48-51). This is based on the principle that ice crystals are difficult to form even below freezing point due to the freezing point depression of the aqueous solution. When this aqueous solution is rapidly cooled in liquid nitrogen, it can be solidified without generating ice crystals. Such solidification is called vitrification freezing. An aqueous solution containing a large amount of antifreeze is called vitrification solution.
  • vitrification method cells or tissues are immersed in a vitrification solution, and then cooled at a liquid nitrogen temperature ( ⁇ 196 ° C.). Since this is a simple and quick process, it does not require time for the operation for cryopreservation, and does not require a device or jig for temperature control.
  • vitrification preservation method When vitrification preservation method is used, ice crystals do not form inside or outside the cell, so physical damage (freezing damage) to cells during freezing and thawing can be avoided, but it is included in the vitrification solution.
  • a high concentration of antifreeze has chemical toxicity, and it is preferable that the vitrification solution is not more than necessary when cryopreserving cells or tissues.
  • the time during which the cells or tissues are exposed to the vitrification solution that is, the time until freezing is short. Furthermore, it is necessary to dilute the vitrification solution immediately after thawing.
  • Patent Document 1 shows that application of a vitrification method to animal or human germ cells or somatic cells is extremely useful in terms of cryopreservation and survival after thawing.
  • the vitrification preservation method is a technique that has been developed mainly using human germ cells, but recently, its application to iPS cells and ES cells has been widely studied.
  • Non-Patent Document 1 shows that the vitrification preservation method was effective in preserving Drosophila embryos.
  • Patent Document 2 shows that the vitrification preservation method is effective in preservation of plant cultured cells and tissues.
  • the vitrification method is known to be useful for a wide variety of cells and tissues.
  • Patent Document 3 As a jig and operation method for performing the vitrification preservation method more efficiently, in Patent Document 3 and the like, an egg or an embryo is vitrified and frozen in a straw filled with a vitrification solution, and quickly when thawed. Attempts have been made to improve the regeneration rate by contacting with a diluent.
  • Patent Document 4 an excellent viability is obtained by removing an excess vitrification solution adhering to the periphery of an ovum or an embryo by placing the ovum or embryo together with a vitrification solution on a removal material for vitrification preservation and sucking from the lower part.
  • a method of cryopreserving is proposed.
  • a removal material for vitrification preservation save, what has a through-hole by the film-form thing which consists of natural products and synthetic resins, such as a wire net and paper, is described.
  • Patent Document 5 proposes a method of cryopreserving with an excellent survival rate by absorbing excess vitrification solution adhering to the periphery of an egg or embryo with an absorbent such as filter paper.
  • Patent Document 6 and Patent Document 7 a strip-like flexible, colorless and transparent film is used as a strip for holding eggs attached by the so-called cryotop method used in the field of human infertility treatment.
  • cryotop method used in the field of human infertility treatment.
  • Patent Document 8 a metal plate-like member having a plurality of holes with a size of about 2 to 8 mm 2 into which a cooling medium can enter, and a tissue piece for vitrifying and storing a collected tissue piece A method of cryopreserving using a cryopreservation plate has been proposed.
  • Patent Document 9 a method for producing a frozen cell sheet by vitrification is proposed.
  • a conveyance auxiliary membrane manufactured by Cellseed Co., Ltd., which is a sheet mainly made of cellulose.
  • a method is described in which a cell sheet is placed together with a cell shifter or a PVDF membrane), and after the excess vitrification solution is sucked or dropped, the cell sheet is cryopreserved.
  • Patent Document 3 has a problem that it takes a long time to freeze because the vitrification liquid is filled in the straw.
  • the size of cells or tissues that can be cryopreserved is limited to the inner diameter of the straw, it is difficult to preserve a sheet-shaped tissue such as a cell sheet.
  • Patent Document 4 proposes a method for cryopreserving these germ cells with an excellent survival rate by removing excess vitrification solution adhering to the periphery of the ovum or embryo.
  • the method described in the above publication requires a suction operation from the bottom when removing the excess vitrification solution, which is a complicated operation and is not suitable for a vitrification cryopreservation operation in a short time. is there.
  • the suction from the lower part is insufficient, there is a problem that excess vitrification liquid remains.
  • Patent Document 5 proposes a method of cryopreserving these germ cells with an excellent survival rate by absorbing excess vitrification solution adhering to the periphery of the egg or embryo into an absorbent such as filter paper.
  • an absorbent such as filter paper.
  • Patent Document 6 and Patent Document 7 describe a method for cryopreserving an egg or embryo together with a small amount of vitrification solution by limiting the width of the film on which the egg or embryo is placed. .
  • an egg or embryo is placed on a film together with a very small amount of vitrification solution by the operator's operation, but there is a problem that the operation is difficult.
  • the cryotop method in order to cryopreserve the egg or embryo with a smaller amount of vitrification solution, once the egg or embryo is placed on the film together with the vitrification solution, excess vitrification is performed.
  • a complicated operation of sucking the liquid and removing it from the film may be performed. For example, it is not suitable for application to cryopreservation of a tissue having a large area in a sheet shape such as a cell sheet.
  • Patent Document 8 a metal plate having excellent thermal conductivity is used, and further, a plurality of holes having a size of about 2 to 8 mm 2 in which a cooling solvent can enter can be preserved to preserve ovarian tissue.
  • a device has been devised.
  • excess vitrification liquid remains around the tissue and the cells constituting the tissue, and the toxicity of the vitrification liquid, the temperature drop during freezing and the temperature during melting Since the increase is delayed, the survival rate of the tissue and the cells constituting the tissue may be reduced.
  • Patent Document 9 for example, a cell sheet is placed on a pedestal such as a glass plate, a metal plate, a mesh-like net, and a non-woven fabric together with a transportation auxiliary film (Cell Shifter or PVDF film manufactured by Cellseed), and an extra glass
  • a transportation auxiliary film Cell Shifter or PVDF film manufactured by Cellseed
  • the method of cryopreserving after sucking or dropping the vitrification solution is described, but when the amount of vitrification solution dripped with cells or tissues is large, the absorption rate of the vitrification solution is not sufficient. There is a problem that excess vitrification liquid remains.
  • the main object of the present invention is to provide a vitrification cryopreservation jig capable of easily and reliably performing cryopreservation of cells or tissues. More specifically, when a cell or tissue is immersed in a vitrification solution and placed on the vitrification cryopreservation jig together with the vitrification solution, it has excellent absorption performance for absorbing excess vitrification solution. A jig for vitrification freezing storage is provided.
  • a jig for vitrification cryopreservation of cells or tissues having the following configuration.
  • a cell or tissue vitrification cryopreservation jig having a porous sintered body or a porous metal body as a vitrification liquid absorber.
  • the porous sintered compact heats and sinters the resin solid powder or metal oxide particles of a thermoplastic resin with a three-dimensional structure, and fuses the powder surface or the particle surface.
  • the jig for vitrification cryopreservation according to the above (1) which is a porous structure obtained by this.
  • the excess vitrification solution attached to the outer periphery of the cell or tissue is removed.
  • Other operations to remove excess vitrification liquid from absorption for example, suction removal operation from the bottom of the vitrification liquid absorber, or direct suction removal from cells or tissues using a micropipette etc.
  • suction removal operation from the bottom of the vitrification liquid absorber or direct suction removal from cells or tissues using a micropipette etc.
  • tool for vitrification cryopreservation of this invention is used, the vitrification freezing operation
  • FIG. 1 is an overall view showing an example of a jig for vitrification cryopreservation of cells or tissues of the present invention.
  • FIG. 2 is an enlarged view of the vitrification liquid absorber in FIG. 1.
  • FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used when a support is provided.
  • FIG. 4 is a schematic view showing an example of a vitrification liquid absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig.
  • FIG. 5 is a schematic view showing another example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig.
  • the vitrification cryopreservation jig of the present invention has a porous sintered body or a porous metal body as a vitrification liquid absorber.
  • the vitrification cryopreservation jig of the present invention is used when cryopreserving biological cells or tissues.
  • the cell includes not only a single cell but also a cell population composed of a plurality of cells.
  • the cell population composed of a plurality of cells may be a cell population composed of a single type of cell or a cell population composed of a plurality of types of cells.
  • the tissue may be a tissue composed of a single type of cell or a tissue composed of a plurality of types of cells, and includes non-cellular substances such as an extracellular matrix in addition to cells. Things can be used.
  • the jig for cryopreservation of vitrification according to the present invention is preferably such that cells or tissues are attached to a vitrification liquid absorber together with vitrification liquid, and the jig to which the cells or tissues are attached is immersed in a cooling substance such as liquid nitrogen. For freezing.
  • a cooling substance such as liquid nitrogen. For freezing.
  • the vitrification cryopreservation jig of the present invention can be restated as a cell or tissue cryopreservation tool or a cell or tissue vitrification storage tool.
  • the vitrification solution absorber absorbs excess vitrification solution, so that the cells or tissues immersed in the vitrification solution are combined with the vitrification solution.
  • the solution is dropped onto the cell, a stable cell or tissue survival rate can be expected even if the amount of vitrification solution adhering to the cell or tissue is large.
  • the cells or tissues thus manipulated are covered with a very small amount of vitrification solution, and can be quickly frozen even when freezing.
  • the vitrification solution can be diluted immediately after thawing the cryopreserved cells or tissues.
  • the vitrification cryopreservation jig of the present invention is a vitrification cryopreservation jig having at least a vitrification solution absorber that absorbs a vitrification solution, and the vitrification solution absorber is formed by porous sintering. Or a porous metal body.
  • a vitrification cryopreservation jig having a porous sintered body as a vitrification liquid absorber is a vitrification cryopreservation jig A
  • a vitrification cryopreservation having a porous metal body as a vitrification liquid absorber The jig is referred to as a vitrification cryopreservation jig B.
  • the porous sintered compact that the vitrification cryopreservation jig A has as a vitrification liquid absorber is obtained by heating and sintering a resin solid powder or metal oxide particles of a thermoplastic resin with a three-dimensional structure.
  • the porous structure in the present invention is a structure having pores (pores) on the surface, preferably a structure having continuous pores on the surface and inside.
  • the jig A for vitrification cryopreservation has the porous sintered formed body as a vitrified liquid absorber, and the above-mentioned porous sintered formed body itself is vitrified without having a support. You may have as an absorber.
  • the vitrification liquid absorber which has an above-mentioned porous sintered compact on a support body may be sufficient.
  • an adhesive layer can be provided between the support and the porous sintered compact.
  • an undercoat layer may be provided for the purpose of obtaining a uniform adhesive layer on the support.
  • the support not only supports the porous sintered compact but also can provide the support itself with absorption performance.
  • the vitrification cryopreservation jig A When the cell or tissue immersed in the vitrification solution is placed on the vitrification solution absorber together with the vitrification solution, the vitrification cryopreservation jig A is vitrified through the pores on the surface of the porous sintered compact.
  • the liquid is absorbed by the vitrification liquid absorber, and excess vitrification liquid can be removed from the periphery of the cell or tissue.
  • the porous sintered formed body include a porous sintered formed body made of a resin and a porous sintered formed body made of a metal oxide.
  • the thermoplastic resin used to obtain the resin is low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene, polypropylene, polymethyl methacrylate, polystyrene, fluororesin, Examples thereof include ethylene-vinyl acetate copolymer, polyamide, styrene-acrylonitrile copolymer, styrene-butadiene-acrylonitrile terpolymer, polycarbonate, and polyvinyl chloride.
  • the porous sintered formed body made of resin may be a porous sintered formed body containing two or more kinds of resins.
  • the porous sintered compact of this invention consists of a metal oxide
  • a metal oxide used in order to obtain this a silica, an alumina, a zirconium, quartz glass etc. are mentioned. Since processing is easy, Preferably, polyethylene and quartz glass are mentioned.
  • a generally known method can be used as a method for manufacturing the porous sintered compact included in the jig A for vitrification cryopreservation.
  • the porous sintered body is made of a resin
  • a method described in JP 2009-235417 A can be used. More specifically, a solid powder of a thermoplastic resin obtained by a method such as emulsion polymerization or pulverization is filled in a mold, heated and sintered to fuse the powder particle surface, and cooled, A porous sintered compact can be produced.
  • the temperature at the time of heating and sintering varies depending on the type of thermoplastic resin to be sintered, but in order to maintain a high porosity, the temperature at which the particles are sufficiently fused in the vicinity of the melting point of each resin, A temperature that does not fill the particle gap is selected.
  • the heating temperature is preferably 110 ° C. to 180 ° C., more preferably 120 ° C. to 150 ° C.
  • the porous sintered body is made of a metal oxide, for example, the methods described in JP2009-29692A and JP2002-160930A can be used.
  • the produced porous sintered compact may be processed into an arbitrary shape by secondary processing such as cutting, cutting, and punching.
  • the surface of the porous sintered compact can be subjected to a hydrophilic treatment in order to improve the vitrification performance.
  • Hydrophilic treatment methods include graft modification methods, coating methods using hydrophilic polymer compounds, corona discharge, plasma treatment, general surface modification methods using various electron beams such as excimer laser, Can use surface modification methods such as surface roughening.
  • the hydrophilic treatment may be performed on the resin powder particles before the sintering step, or may be performed on the porous sintered body after the sintering step.
  • the porous sintered compact of the vitrification cryopreservation jig A preferably has a pore diameter of 1 ⁇ m or more from the viewpoint of the vitrification liquid absorbability.
  • the pore diameter is less than 1 ⁇ m, the absorption performance when the vitrified droplet is dropped is not sufficient, and it may take time until frozen storage, or a large amount of excess vitrified solution may remain around the cells or tissues. is there.
  • the pore diameter exceeds 1 mm, a part of the cell or sheet-shaped tissue is trapped in the pore gap, and in the operation of thawing and thawing after cryopreservation and the subsequent operation, the vitrification solution absorber There may be a problem that cells and tissues do not peel off from the surface.
  • the pore diameter of the porous sintered compact used in the present invention is preferably 1 ⁇ m to 1 mm. Further, the porosity of the porous sintered compact used in the present invention is preferably 20 to 80%, more preferably 30 to 60%.
  • the pore diameter, thickness, and porosity of the porous sintered compact used in the present invention can be appropriately set according to the type of cells or tissues to be used, the dripping amount of the vitrification solution dripped with the cells or tissues, and the like. .
  • the above-mentioned pore diameter represents the average diameter of the pores measured from image observation of the surface and the cross section.
  • the above porosity is defined by the following formula.
  • the area of the porous sintered compact used as the vitrification liquid absorber is not particularly limited as long as it is appropriately set according to the size of the cell or tissue and the amount of the vitrification liquid adhering to the cell or tissue.
  • the area of the porous sintered formed body is preferably larger than the bottom area of the sheet-like tissue.
  • a more preferable area of the porous sintered compact is 1.5 times or more with respect to the bottom area of the sheet-like structure.
  • the vitrification liquid 1 ⁇ L to per 2 mm 2 or more to be dropped, and more preferably in the 10 ⁇ 400 mm 2.
  • the porous metal body in the present invention is a structure having pores (pores) on the surface, preferably a structure having continuous pores on the surface and inside.
  • the vitrification cryopreservation jig B has the porous metal body as a vitrification liquid absorber, and has the above-described porous metal body itself as a vitrification liquid absorber without a support. You may do it. Or the vitrification liquid absorber which has the above-mentioned porous metal body on a support body may be sufficient.
  • an adhesive layer can be provided between the support body and the porous metal body.
  • an undercoat layer may be provided for the purpose of obtaining a uniform adhesive layer on the support.
  • the support not only supports the porous metal body, but also allows the support itself to have absorption performance.
  • the vitrification cryopreservation jig B is such that when a vitrification solution containing cells or tissues is dropped on the vitrification solution absorber, the vitrification solution passes through the pores on the surface of the porous metal body. And can remove excess vitrification fluid from around the cell or tissue.
  • porous metal body used as the vitrification liquid absorber in the present invention examples include copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, tin, zinc, lead, titanium, nickel, and stainless steel.
  • a porous metal body made of the above material can be used. From the viewpoint of availability as a material and ease of handling, a porous metal body made of copper, copper alloy, aluminum, aluminum alloy, titanium, nickel, or stainless steel is preferable.
  • a method for producing a porous metal body possessed by the vitrification cryopreservation jig B a general method for producing a porous metal body such as a powder metallurgy method or a spacer method can be used. Further, a so-called powder space holder method in which resin injection molding and powder metallurgy are combined can be preferably used. For example, a method described in International Publication No. 2006/0401118 pamphlet or Japanese Patent No. 4578062 can be used. More specifically, after mixing the metal powder and the resin serving as a spacer, forming by applying pressure, firing the metal powder by baking in a high temperature environment, vaporizing the resin serving as the spacer, A porous metal body can be obtained.
  • a resin binder can be mixed in addition to the metal powder and the resin serving as the spacer.
  • the metal powder is baked and hardened by baking in a high temperature environment to become a spacer. It is also possible to produce a porous metal body by vaporizing the fibers.
  • the manufacturing method of porous metal bodies such as the foaming melting method and gas expansion method which inject
  • a production method such as a slurry foaming method for producing a porous metal body using a foaming agent can also be used.
  • the surface of the above-described porous metal body can be subjected to a hydrophilic treatment in order to improve the vitrification solution absorption performance.
  • Hydrophilic treatment methods include graft modification methods, coating methods using hydrophilic polymer compounds, etc., corona discharge, plasma treatment, and general surface modification methods using various electron beams such as excimer lasers. can do.
  • the methods described in JP-A-11-256355, JP-A-2000-281936, JP-A-2008-161806, and JP-A-2011-7365 can be used.
  • the pore diameter of the porous metal body possessed by the vitrification cryopreservation jig B is preferably 0.05 to 500 ⁇ m, more preferably 0.5 to 50 ⁇ m.
  • the pore diameter is less than 0.05 ⁇ m, the absorption performance of the vitrification liquid may not be sufficient when the vitrification liquid is dropped.
  • the pore diameter exceeds 500 ⁇ m, the cells or tissues are trapped in the pores, and the cells may not be easily released from the porous metal body during the melting operation.
  • the absorption performance of vitrification liquid may not be enough.
  • the pore diameter of the porous metal body is an average pore diameter measured from image observation of the porous surface and cross section.
  • the thickness of the porous metal body is preferably 50 ⁇ m to 50 mm, more preferably 500 ⁇ m to 10 mm.
  • the porosity of the porous metal body is preferably 20% by volume or more, more preferably 40% by volume or more, and still more preferably 50% by volume or more.
  • the pores inside the porous metal body are preferably of a so-called open cell type that has a continuous structure not only in the thickness direction but also in a direction perpendicular to the thickness direction. With such a structure, since the pores inside the porous metal body can be used effectively, high absorption performance of the vitrification liquid can be obtained.
  • the thickness and porosity of the porous metal body can be appropriately selected according to the type of cells or tissues to be used, the dripping amount of the vitrification solution dripped with the cells or tissues, and the like.
  • the area of the porous metal body used as the vitrification liquid absorber is not particularly limited as long as it is appropriately set according to the dripping amount of the vitrification liquid dripped together with the cells or tissues, but for example, 1 ⁇ L of the dripping vitrification liquid It is preferably 1 mm 2 or more, more preferably 2 to 400 mm 2 .
  • the vitrification solution absorber which the jig A for vitrification cryopreservation and the jig B for vitrification cryopreservation of the present invention have has a support
  • various generally known supports can be used.
  • a support include various resin films, glass, rubber, metal, fiber-shaped substance, sponge-shaped substance, and the like.
  • Specific examples of the resin film include polyester resins such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN), acrylic resins, epoxy resins, silicone resins, polycarbonate resins, diacetate resins, triacetate resins, polyarylate resins, polychlorinated resins.
  • PET polyethylene terephthalate
  • PEN polyethylene naphthalate
  • acrylic resins epoxy resins
  • silicone resins polycarbonate resins
  • diacetate resins diacetate resins
  • triacetate resins polyarylate resins
  • polychlorinated resins polychlorinated resins.
  • the resin film examples include a vinyl resin, a polysulfone resin, a polyether sulfone resin, a polyimide resin, a polyamide resin, a polyolefin resin, a cyclic polyolefin resin, and a fluorine resin.
  • a metal plate can be used suitably from a viewpoint of being excellent in temperature conductivity and enabling rapid freezing.
  • the thickness of the support is preferably 10 ⁇ m to 100 mm.
  • the surface of the support can be easily adhered by an electrical method such as corona discharge treatment or a chemical method, and further roughened. You can also.
  • vitrification cryopreservation jig A or the vitrification cryopreservation jig B of the present invention has an adhesive layer
  • an instantaneous adhesive substance typified by a moisture-curable adhesive substance, a hot melt adhesive substance,
  • a general bonding method using a photo-curable adhesive material can be used.
  • water-soluble adhesive materials such as polyvinyl alcohol, hydroxy cellulose, polyvinyl pyrrolidone, starch paste, vinyl acetate adhesive materials, acrylic adhesive materials, Epoxy adhesives, urethane adhesives, elastomer adhesives, cyanoacrylate adhesives, fluorine adhesives, silicon adhesives, nitrocellulose adhesives, nitrile rubber adhesives, styrene-butadiene adhesives, Urea resin adhesives, styrene resin adhesives, phenol resin adhesives, polyimide adhesives Materials, polyamide series adhesive materials, polyester-based adhesive agent, bismaleimide adhesive material, an olefin-based adhesive material, a non-water soluble adhesive material, such as EVA-based adhesive materials can be preferably used.
  • water-soluble adhesive materials such as polyvinyl alcohol, hydroxy cellulose, polyvinyl pyrrolidone, starch paste, vinyl acetate adhesive materials, acrylic adhesive materials, Epoxy adhesives, urethane adhesives, elast
  • the adhesive layer may contain one kind of adhesive substance or may contain a plurality of kinds of adhesive substances.
  • the solid content of the adhesive layer is preferably in the range of 0.01 to 100 g / m 2 , more preferably in the range of 0.1 to 50 g / m 2 .
  • the vitrification liquid absorber in the present invention has been described above.
  • the structure of the vitrification cryopreservation jig using these will be described below.
  • the vitrification cryopreservation jig of the present invention may be anything as long as it has a vitrification solution absorber as described above, but the vitrification solution absorber is connected to the gripping part. May be. Having a gripping portion is preferable because workability during cryopreservation work and melting work is good.
  • FIG. 1 is an overall view showing an example of a vitrification cryopreservation jig according to the present invention.
  • a vitrification cryopreservation jig 5 includes a gripping portion 1 and a vitrification liquid absorber 2.
  • the grip 1 is preferably made of a liquid nitrogen resistant material.
  • various metals such as aluminum, iron, copper, and stainless alloy, ABS resin, polypropylene resin, polyethylene resin, fluorine resin, various engineer plastics, and glass can be preferably used.
  • the vitrification liquid absorber 2 is preferably strip-shaped or sheet-shaped for handling.
  • FIG. 2 is an enlarged view of the vitrification liquid absorber 2 of FIG.
  • the vitrification liquid absorber 2a of FIG. 2 is an example of a form in which a porous sintered body or the porous metal body 3 itself is used as a vitrification liquid absorber without having a support.
  • FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used when a support is provided, and the vitrification liquid absorber 2b is formed on the support 4 with a porous sintered body or a porous metal body. 3.
  • the vitrification liquid absorber 2b shown in FIG. 3 is an example of a form having a porous sintered body or a porous metal body on the entire surface of the vitrification liquid absorber.
  • the vitrification liquid absorber 2 can be connected to the holding part 1 by insert molding at the time of molding. Furthermore, the vitrification liquid absorber insertion part which is not shown in figure in the holding part 1 can be produced, and the vitrification liquid absorber 2 can be connected with an adhesive agent.
  • an adhesive agent e.g., a silicon-based or fluorine-based adhesive that is resistant to low temperatures can be suitably used.
  • the jig body shown in FIG. 1 can be covered with a cap to shut off from the outside for safety. Furthermore, liquid nitrogen is usually not sterilized, and when frozen by direct contact with liquid nitrogen, sterility may not be guaranteed even if the vitrification solution storage jig is sterilized. Therefore, a vitrified liquid absorber to which cells or tissues are attached before freezing may be capped and frozen without being in direct contact with liquid nitrogen. In the advanced countries such as the US and EU, the freezing method that does not directly contact liquid nitrogen as described above has become mainstream.
  • the cap is preferably made of various metals, various resins, glass, ceramic, etc., which are liquid nitrogen resistant materials.
  • the shape may be any shape, such as a pencil cap or a cylindrical straw cap, as long as the shape can be cut off from the outside without being in contact with the vitrification liquid absorber.
  • FIG. 4 is a schematic view showing an example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig.
  • FIG. 5 is a schematic view showing another example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. 4 and 5, the porous sintered body or porous metal body 3 is discontinuous and arranged on the support 4.
  • the porous sintered body or the porous metal body 3 has a continuous shape as shown in FIG. 3 described above, an attempt is made to attach a plurality of cells or tissues to the porous sintered body or the porous metal body 3. Then, since the vitrification liquid is a porous metal body and spreads in the lateral direction and the thickness direction, for example, when the second or subsequent cells or tissues are attached to the porous sintered body or the porous metal body 3, vitrification is performed. The liquid absorbency may decrease. However, if a plurality of porous sintered bodies or porous metal bodies 3 are provided on the support 4 in a discontinuous manner as shown in FIGS. Alternatively, one structure can be reliably attached to each porous sintered body or porous metal body 3.
  • the vitrification liquid absorber 2c and the vitrification liquid absorber 2d shown in FIGS. 4 and 5 may be an example of a vitrification liquid absorber having a porous sintered body or a porous metal body on a part of the support. is there.
  • the vitrification cryopreservation jig of the present invention is suitably used, for example, in the cryotop method.
  • the conventional cryotop method is usually used for storage of a single cell or a small number of cells of less than 10, but the vitrification cryopreservation jig of the present invention can store more cells (for example, (Preservation of 10 to 1000000 cells) can also be suitably used.
  • it can be suitably used for the preservation of sheet-like cells (so-called cell sheets) composed of a plurality of cells.
  • the jig for vitrification cryopreservation according to the present invention When the jig for vitrification cryopreservation according to the present invention is used, it is difficult to be damaged by the vitrification solution outside the cell or tissue during freezing and thawing, and the cell or tissue can be cryopreserved with an excellent survival rate. .
  • the method for cryopreserving cells or tissues using the vitrification cryopreservation jig of the present invention is not particularly limited.
  • a vitrification solution absorber comprising cells or tissues immersed in a vitrification solution together with the vitrification solution
  • the vitrification solution adhering to the periphery of the cell or the tissue is dropped onto the vitrification solution absorber.
  • the cells or the tissues can be frozen by immersing them in liquid nitrogen or the like while being held on the vitrification liquid absorber.
  • the vitrification solution those usually used for freezing cells such as eggs and embryos can be used.
  • the above-mentioned cryoprotectant such as glycerol, ethylene glycol, DMSO (dimethyl sulfoxide) or more is 30% by volume or more.
  • an aqueous solution containing 30 to 40% by volume of an antifreezing agent can be preferably used.
  • the vitrification cryopreservation jig is taken out from a cooling solvent such as liquid nitrogen, and the vitrified liquid absorber on which the frozen cells or tissues are placed is immersed in the melt. The melting operation can be performed quickly.
  • Examples of cells that can be cryopreserved using the vitrification cryopreservation jig of the present invention include, for example, mammals (eg, humans, cows, pigs, horses, rabbits, rats, mice, etc.) eggs or embryos. And germ cells such as sperm, iPS cells, and ES cells. Moreover, cultured cells, such as a primary cultured cell, a subcultured cell, and a cell line cell, are mentioned.
  • the cells are fibroblasts, cancer-derived cells such as pancreatic cancer / hepatoma cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, Examples include embryonic stem cells and adherent cells such as immune cells.
  • tissues that can be cryopreserved include tissues composed of allogeneic or heterogeneous cells, such as tissues such as ovary, skin, corneal epithelium, periodontal ligament, myocardium, and cartilage.
  • the vitrification cryopreservation jig of the present invention is not limited to cells or tissues collected directly from a living body, but also, for example, cultured skin grown and grown in vitro, a so-called cell sheet constructed in vitro, It can also be suitably used in vitrified cryopreservation of an artificial tissue such as a tissue model having a three-dimensional structure described in JP-A-205516.
  • the pore diameters of Examples 1 to 4 indicate average pore diameters measured from image observation of the surface and cross section. Moreover, the pore diameter of Comparative Example 1 and Comparative Example 4 indicates the diameter of the largest pore measured by the bubble point test described in JIS K3832.
  • Example 1 As the porous sintered compact, Sunfine AQ800 (pore diameter 35 ⁇ m, porosity 43%, thickness 500 ⁇ m) manufactured by Asahi Kasei Co., Ltd., which is a porous sintered compact made of polyethylene resin, is used as a vitrified liquid absorber.
  • the vitrification solution absorber 500 mm 2 (10 mm ⁇ 50 mm) was joined to a gripping part made of ABS resin, and the vitrification cryopreservation jig of Example 1 was produced in the form shown in FIG.
  • Example 2 As a porous sintered body, a quartz glass porous body (pore diameter 3.5 ⁇ m, porosity 33%, thickness 2 mm) manufactured by Covalent Materials, which is a ceramic porous sintered body, is used as a vitrified liquid absorber.
  • This vitrified liquid absorber 500 mm 2 (10 mm ⁇ 50 mm) was joined to a gripping part made of ABS resin, and a vitrification cryopreservation jig of Example 2 was produced in the form shown in FIG.
  • Comparative Example 1 a cellulose acetate membrane made of cellulose acetate (pore size 0.5 ⁇ m, porosity 68%, film thickness 125 ⁇ m) was used as a vitrification liquid absorber. Since the membrane is thin and the self-supporting property is not sufficient, the membrane is pasted onto a PET film having a thickness of 250 ⁇ m using a urethane-based adhesive substance, and the vitrification cryopreservation jig of Comparative Example 1 is used in the same manner as in Example 1. Produced.
  • Comparative Example 2 Filter paper No. 1 manufactured by Advantech, which is a filter paper.
  • a jig for vitrification cryopreservation of Comparative Example 2 was produced in the same manner as Comparative Example 1 except that 5C (basis weight 120 g / m 2 , density 0.57 g / cm 3 ) was used as the vitrification liquid absorber.
  • Comparative Example 3 The treatment for vitrification cryopreservation of Comparative Example 3 was performed in the same manner as Comparative Example 1 except that Cell Shifter made of Cellulose, which is a cell transport auxiliary membrane described in Patent Document 8, was used as a vitrification solution absorber. A tool was prepared.
  • Comparative Example 4 A hydrophilic durapore membrane (pore diameter 0.1 ⁇ m, porosity 70%, film thickness 125 ⁇ m) made of Merck Millipore, made of PVDF, which is a cell transport auxiliary membrane described in Patent Document 8, is used as a vitrification solution absorber. A jig for vitrification cryopreservation of Comparative Example 4 was produced in the same manner as Comparative Example 1 except that.
  • Comparative Example 5 The vitrification cryopreservation treatment of Comparative Example 5 was carried out in the same manner as in Example 1 except that a transparent PET film (porosity 0%, thickness 250 ⁇ m), which was a colorless transparent film, was used as it was to obtain a vitrification liquid absorber. A tool was prepared.
  • ⁇ Evaluation of absorbability of vitrification solution > 200 ⁇ L of a vitrification solution containing a plurality of glass beads (diameter: 100 ⁇ m) as pseudo cells is dropped on the vitrification solution absorber of each vitrification cryopreservation jig of Example 1, Example 2 and Comparative Examples 1 to 5. Attached.
  • the vitrification solution used was a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich. After dropping and adhering, the state where the vitrification solution around the pseudo cell placed on the vitrification solution absorber was absorbed with an epi-illuminated optical microscope (OM4, VC4500-S1) was observed. The evaluation was based on the following criteria. These results are shown in the item “Evaluation of absorbability of vitrification solution” in Table 1.
  • ⁇ The vitrification solution was absorbed within 10 seconds after adhering under the vitrification droplet.
  • X Absorption of the vitrification solution was not sufficient within 10 seconds after adhering under the vitrification droplet, and the vitrification solution remained around the pseudo cell.
  • the vitrification solution used was a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich.
  • the vitrification liquid absorber was completely immersed in liquid nitrogen.
  • the vitrification cryopreservation jig was taken out of liquid nitrogen and placed in a room temperature environment. Compared to before immersion in liquid nitrogen, the vitrification solution absorber was visually observed for deformation and breakage, and practical durability was evaluated according to the following criteria.
  • ⁇ No deformation or breakage of the vitrification liquid absorber was observed.
  • X Deformation or breakage was observed in the vitrified liquid absorber. Or, in a series of evaluation processes, work defects were recognized due to the occurrence of deformation or breakage.
  • the vitrification cryopreservation jigs of Examples 1 and 2 of the present invention both showed excellent vitrification solution absorption performance.
  • the vitrification cryopreservation jigs of Comparative Examples 1 to 5 had insufficient vitrification solution absorption performance.
  • the vitrification cryopreservation jigs of Comparative Examples 1 to 4 except Comparative Example 5 showed some absorption of the vitrification solution, but the vitrification solution around the pseudo cell was absorbed within 10 seconds. It was not seen, and it was seen that it remained.
  • vitrification cryopreservation jigs of Examples 1, 2 and Comparative Examples 2 to 5 of the present invention were not deformed or damaged when immersed in liquid nitrogen, and failed in a series of cryopreservation operations. Was not recognized.
  • the vitrification cryopreservation jig of Comparative Example 1 damage to the vitrification liquid absorber was observed after immersion in liquid nitrogen.
  • Example 3 As the porous metal body, a stainless porous body (pore diameter 1.5 ⁇ m, porosity 65 volume%, thickness 1 mm) manufactured by Taisei Kogyo Co., Ltd. was used as the vitrification liquid absorber, and this vitrification liquid absorber 500 mm 2 (10 mm ⁇ 50 mm) was joined to a gripping part made of ABS resin, and the jig for vitrification cryopreservation of Example 3 was produced in the form shown in FIG.
  • a stainless porous body pore diameter 1.5 ⁇ m, porosity 65 volume%, thickness 1 mm
  • this vitrification liquid absorber 500 mm 2 (10 mm ⁇ 50 mm) was joined to a gripping part made of ABS resin, and the jig for vitrification cryopreservation of Example 3 was produced in the form shown in FIG.
  • Example 4 As a porous metal body, a stainless porous body (pore diameter: 8 ⁇ m, porosity: 65 volume%, thickness: 1 mm) made by Taisei Kogyo Co., Ltd. was used as a vitrification liquid absorber, and this vitrification liquid absorber 500 mm 2 (10 mm ⁇ 50 mm). ) was bonded to a gripping part made of ABS resin, and a jig for vitrification cryopreservation of Example 4 was produced in the form shown in FIG.
  • ⁇ The vitrification solution was completely absorbed within 5 seconds after adhering under the vitrification droplet.
  • X Absorption of the vitrification solution was insufficient within 5 seconds after adhering under the vitrification droplet, and the vitrification solution remained around the pseudo cell.
  • Example 4 ⁇ Evaluation of immersion in cooling solvent>
  • glass beads on the vitrification absorber of each vitrification cryopreservation jig were used in the same manner as in the previous immersion evaluation in the cooling solvent.
  • the vitrification liquid absorber was completely immersed in liquid nitrogen.
  • the vitrification cryopreservation jig was taken out of liquid nitrogen and placed in a room temperature environment.
  • the vitrification cryopreservation jigs of Examples 3 and 4 of the present invention both showed excellent vitrification solution absorption performance.
  • the vitrification cryopreservation jigs of Comparative Examples 1 to 5 were insufficient in vitrification solution absorption performance because most of the dripped vitrification solution remained.
  • vitrification cryopreservation jigs of Examples 3 and 4 and Comparative Examples 2 to 5 of the present invention were not deformed or damaged when immersed in liquid nitrogen, and failed in a series of cryopreservation operations. Was not recognized.
  • the vitrification cryopreservation jig of Comparative Example 1 damage to the vitrification liquid absorber was observed after immersion in liquid nitrogen.
  • the present invention can be used for testing or transplanting from iPS cells, ES cells, commonly used cultured cells, living organisms, etc. in addition to embryo transfer or artificial insemination of humans such as cattle and animals, artificial insemination to humans, etc. It can be used for cryopreservation of cells or tissues, cells or tissues cultured in vitro, and the like.

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Abstract

Provided is a jig for use in vitrification cryopreservation, which enables a cell or tissue vitrification cryopreservation operation to be performed easily and reliably. A jig for use in cell or tissue vitrification cryopreservation, which contains a porous sintered molded body or a porous metallic body as a vitrification solution absorber. According to the present invention, an additional operation for removing an excess portion of a vitrification solution is not particularly required when cells or tissues immersed in the vitrification solution are placed on a vitrification solution absorber together with the vitrification solution, and therefore a cell or tissue cryopreservation operation can be performed easily and simply.

Description

細胞または組織のガラス化凍結保存用治具Jig for cryopreserving vitrified cells or tissues
　本発明は、生物の細胞または組織などを凍結保存する際に使用するガラス化凍結保存用治具に関する。 The present invention relates to a vitrification cryopreservation jig used for cryopreserving biological cells or tissues.
　生物の細胞または組織の優れた保存技術は、様々な産業分野で求められている。例えば、牛の胚移植技術に用いられる胚は、受胚牛の発情周期に合わせて移植が行われており、発情周期に胚の移植を合わせるために、胚を凍結保存し、発情周期に合わせて胚を融解して移植することが行われている。また、ヒトの不妊治療においては、母体から卵子または卵巣を採取後、移植に適したタイミングに合わせるために凍結保存しておき、移植時に融解して用いることがなされている。 Excellent preservation technology for biological cells or tissues is required in various industrial fields. For example, embryos used in bovine embryo transfer technology are transplanted in accordance with the estrous cycle of the recipient cow, and the embryo is cryopreserved and matched to the estrous cycle in order to match the embryo transfer to the estrous cycle. The embryo is thawed and transplanted. Moreover, in human infertility treatment, after collecting an egg or ovary from a mother, it is stored frozen in order to match the timing suitable for transplantation, and thawed at the time of transplantation.
　一般に、生体内から採取された細胞または組織は、たとえ培養液の中であっても、次第に活性が失われていくことから、生体外での細胞または組織の長期間の培養は好ましくない。そのため、生体活性を損なわずに長期間保存する技術が重要である。優れた保存技術によって、採取された細胞または組織をより正確に分析することが可能になる。また優れた保存技術によって、生体内から採取された細胞または組織は、より高い生体活性を保ったまま移植に用いることが可能となり、移植後の生着率が向上することが望める。さらには、生体外で培養した培養皮膚や生体外で構築したいわゆる細胞シートのような移植のための人工の組織を、順次生産して保存しておき、必要な時に使用することも可能となる。このため、生物の細胞または組織の保存技術は、医療の面だけではなく、産業面においても広く求められている技術である。 Generally, since cells or tissues collected from a living body gradually lose their activity even in a culture solution, long-term culture of cells or tissues in vitro is not preferable. Therefore, a technique for storing for a long time without impairing biological activity is important. Superior storage techniques allow for more accurate analysis of harvested cells or tissues. In addition, cells or tissues collected from the living body can be used for transplantation while maintaining higher biological activity by an excellent preservation technique, and it can be expected that the survival rate after transplantation is improved. Furthermore, artificial tissues for transplantation, such as cultured skin cultured in vitro or so-called cell sheets constructed in vitro, can be produced and stored in sequence and used when needed. . For this reason, the technique for preserving biological cells or tissues is a technique that is widely demanded not only in the medical field but also in the industrial field.
　細胞または組織の保存方法として、例えば緩慢凍結法が知られている。この方法では、まず、リン酸緩衝生理食塩水等の生理的溶液に耐凍剤を含有させることで得られた保存液（耐凍剤の含有量は一般的には大凡１０容量％以下、例えばＪ．Ｍａｍｍ．Ｏｖａ　Ｒｅｓ．Ｖｏｌ．２１，２００４　Ｐ６５－６８）に、細胞または組織を浸漬する。該耐凍剤としては、グリセロール、エチレングリコール等の化合物が用いられる。該保存液に、細胞または組織を浸漬後、比較的遅い冷却速度（例えば０．３～０．５℃／分の速度）で、－３０～－３５℃まで冷却することにより、細胞内外または組織内外の溶液が十分に冷却され、粘性が高くなる。このような状態で、該保存液中の細胞または組織をさらに液体窒素の温度（－１９６℃）まで冷却すると、細胞内または組織内とその外の周囲の微少溶液がいずれも非結晶のまま固体となるガラス化が起こる。ガラス化により、細胞内外または組織内外が固化すると、実質的に分子の動きがなくなるので、ガラス化された細胞または組織を液体窒素中に保存することで、半永久的に保存できると考えられる。 For example, a slow freezing method is known as a method for preserving cells or tissues. In this method, first, a preservation solution obtained by adding a freezing agent to a physiological solution such as phosphate buffered saline (freezing agent content is generally about 10% by volume or less, for example, J. Org. Mamm.Ova Res.Vol.21,2004 P65-68) is immersed in cells or tissues. As the antifreeze, compounds such as glycerol and ethylene glycol are used. After immersing the cells or tissues in the preservation solution, the cells or tissues are cooled or cooled to −30 to −35 ° C. at a relatively slow cooling rate (for example, 0.3 to 0.5 ° C./min), thereby allowing intracellular or extracellular or tissue The solution inside and outside is sufficiently cooled and the viscosity becomes high. In such a state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (−196 ° C.), the micro solutions inside and outside the cells and the tissues remain solid and remain solid. Vitrification occurs. When the inside or outside of a cell or tissue is solidified by vitrification, the molecular movement is substantially eliminated. Therefore, it is considered that the vitrified cell or tissue can be stored semipermanently by storing it in liquid nitrogen.
　しかしながら、前記緩慢凍結法では、比較的遅い冷却速度で冷却する必要があるために、凍結保存のための操作に時間を要する。また、温度制御をするための装置または治具を必要とする問題がある。加えて、前記緩慢凍結法では、細胞外または組織外の保存液中に氷晶が形成されるので、細胞または組織が該氷晶により物理的に損害を受けるおそれがある。 However, in the slow freezing method, since it is necessary to cool at a relatively slow cooling rate, the operation for cryopreservation takes time. In addition, there is a problem of requiring a device or jig for temperature control. In addition, in the slow freezing method, since ice crystals are formed in the preservation solution outside the cells or tissues, the cells or tissues may be physically damaged by the ice crystals.
　前記緩慢凍結法での問題点を解決するための方法として、ガラス化保存法が提案されている。ガラス化保存法とは、グリセロールやエチレングリコール、ＤＭＳＯ（ジメチルスルホキシド）などの耐凍剤を多量（一般的には３０～４０容量％、例えば日本再生医療学会誌　ＶＯＬ１３．Ｎｏ．１　Ｐ４８－５１）に含む水溶液の凝固点降下により、氷点下でも氷晶ができにくくなる原理を用いたものである。この水溶液を急速に液体窒素中で冷却させると氷晶を生じさせないまま固体化させることができる。このように固体化することをガラス化凍結という。また、耐凍剤を多量に含む水溶液をガラス化液という。 A vitrification preservation method has been proposed as a method for solving the problems in the slow freezing method. The vitrification preservation method means that a large amount of antifreezing agents such as glycerol, ethylene glycol, DMSO (dimethyl sulfoxide), etc. (generally 30 to 40% by volume, for example, Journal of Japanese Society for Regenerative Medicine VOL13.No.1, P48-51). This is based on the principle that ice crystals are difficult to form even below freezing point due to the freezing point depression of the aqueous solution. When this aqueous solution is rapidly cooled in liquid nitrogen, it can be solidified without generating ice crystals. Such solidification is called vitrification freezing. An aqueous solution containing a large amount of antifreeze is called vitrification solution.
　前記ガラス化法の具体的な操作としては、ガラス化液に細胞または組織を浸漬させ、その後、液体窒素の温度（－１９６℃）で冷却する。このような簡便かつ迅速な工程であるために、凍結保存のための操作に時間を必要としない他、温度制御をするための装置または治具を必要としない。 As a specific operation of the vitrification method, cells or tissues are immersed in a vitrification solution, and then cooled at a liquid nitrogen temperature (−196 ° C.). Since this is a simple and quick process, it does not require time for the operation for cryopreservation, and does not require a device or jig for temperature control.
　ガラス化保存法を用いると、細胞内外のいずれにも氷晶が生じないために凍結時及び融解時の細胞への物理的障害（凍害）を回避することができるが、ガラス化液に含まれる高濃度の耐凍剤には化学的毒性があり、細胞または組織の凍結保存時にはガラス化液が必要以上に多くないことが好ましい。また、細胞または組織がガラス化液に暴露される時間つまりは凍結までの時間が短時間であることが好ましい。さらには、解凍後ただちにガラス化液を希釈する必要がある。 When vitrification preservation method is used, ice crystals do not form inside or outside the cell, so physical damage (freezing damage) to cells during freezing and thawing can be avoided, but it is included in the vitrification solution. A high concentration of antifreeze has chemical toxicity, and it is preferable that the vitrification solution is not more than necessary when cryopreserving cells or tissues. Moreover, it is preferable that the time during which the cells or tissues are exposed to the vitrification solution, that is, the time until freezing is short. Furthermore, it is necessary to dilute the vitrification solution immediately after thawing.
　これらガラス化保存法を用いた細胞または組織の凍結保存については、様々な方法で、様々な種類の細胞または組織を用いた例が示されている。例えば、特許文献１では、動物またはヒトの生殖細胞または体細胞へのガラス化保存法の適用が凍結保存、融解後の生存率の点で、極めて有用であることが示されている。 For the cryopreservation of cells or tissues using these vitrification preservation methods, examples using various types of cells or tissues are shown by various methods. For example, Patent Document 1 shows that application of a vitrification method to animal or human germ cells or somatic cells is extremely useful in terms of cryopreservation and survival after thawing.
　ガラス化保存法は、主にヒトの生殖細胞を用いて発展してきた技術であるが、最近では、ｉＰＳ細胞やＥＳ細胞への応用も広く検討されている。また、非特許文献１では、ショウジョウバエの胚の保存にガラス化保存法が有効であったことが示されている。さらに、特許文献２では、植物培養細胞や組織の保存において、ガラス化保存法が有効であることが示されている。このように、ガラス化法は広く様々な種の細胞や組織で有用であることが知られている。 The vitrification preservation method is a technique that has been developed mainly using human germ cells, but recently, its application to iPS cells and ES cells has been widely studied. Non-Patent Document 1 shows that the vitrification preservation method was effective in preserving Drosophila embryos. Furthermore, Patent Document 2 shows that the vitrification preservation method is effective in preservation of plant cultured cells and tissues. Thus, the vitrification method is known to be useful for a wide variety of cells and tissues.
　ガラス化保存法をより効率的に行うための治具や操作方法としては、特許文献３などでは、ストローにガラス化液を充満させた中で卵子または胚をガラス化凍結保存させ、解凍時に素早く希釈液と接触させて再生率を向上させる試みがなされている。 As a jig and operation method for performing the vitrification preservation method more efficiently, in Patent Document 3 and the like, an egg or an embryo is vitrified and frozen in a straw filled with a vitrification solution, and quickly when thawed. Attempts have been made to improve the regeneration rate by contacting with a diluent.
　特許文献４では、卵子または胚をガラス化液と共にガラス化保存用除去材の上に載せ、下部から吸引することで卵子または胚の周囲に付着した余分なガラス化液を除き、優れた生存率で凍結保存させる方法が提案されている。なお、ガラス化保存用除去材としては、金網、紙などの天然物や合成樹脂からなるフィルム状物で貫通孔を有したものが記載されている。 In Patent Document 4, an excellent viability is obtained by removing an excess vitrification solution adhering to the periphery of an ovum or an embryo by placing the ovum or embryo together with a vitrification solution on a removal material for vitrification preservation and sucking from the lower part. A method of cryopreserving is proposed. In addition, as a removal material for vitrification preservation | save, what has a through-hole by the film-form thing which consists of natural products and synthetic resins, such as a wire net and paper, is described.
　特許文献５では卵子または胚の周囲に付着した余分なガラス化液を濾紙などの吸収体により吸収させることにより、優れた生存率で凍結保存させる方法が提案されている。 Patent Document 5 proposes a method of cryopreserving with an excellent survival rate by absorbing excess vitrification solution adhering to the periphery of an egg or embryo with an absorbent such as filter paper.
　特許文献６、特許文献７では、人の不妊治療分野で使用されているいわゆるクライオトップ法という方法で、卵付着保持用ストリップとして短冊状の可撓性かつ無色透明なフィルムを使用し、該フィルム上に極少量のガラス化液と共に卵子または胚を顕微鏡下で付着させ、凍結保存する方法が提案されている。 In Patent Document 6 and Patent Document 7, a strip-like flexible, colorless and transparent film is used as a strip for holding eggs attached by the so-called cryotop method used in the field of human infertility treatment. There has been proposed a method in which an egg or embryo is attached under a microscope together with a very small amount of vitrification solution and cryopreserved.
　特許文献８では、金属製の板状部材であって、冷却媒体が侵入可能な２～８ｍｍ２程度の大きさの穴を複数有した、採取した組織片をガラス化凍結保存するための組織片凍結保存用プレートを用いて凍結保存する方法が提案されている。 In Patent Document 8, a metal plate-like member having a plurality of holes with a size of about 2 to 8 mm 2 into which a cooling medium can enter, and a tissue piece for vitrifying and storing a collected tissue piece A method of cryopreserving using a cryopreservation plate has been proposed.
　特許文献９では、ガラス化法による凍結細胞シートの製造方法が提案されており、メッシュ状の網等からなる台座の上に、運搬補助膜（材質が主にセルロースからなるシートであるセルシード社製Ｃｅｌｌ　Ｓｈｉｆｔｅｒ又はＰＶＤＦ膜）と共に細胞シートをのせ、余分なガラス化液を吸い取る若しくは落とした後で、細胞シートを凍結保存する方法が記載されている。 In Patent Document 9, a method for producing a frozen cell sheet by vitrification is proposed. On a pedestal made of a mesh-like net or the like, a conveyance auxiliary membrane (manufactured by Cellseed Co., Ltd., which is a sheet mainly made of cellulose). A method is described in which a cell sheet is placed together with a cell shifter or a PVDF membrane), and after the excess vitrification solution is sucked or dropped, the cell sheet is cryopreserved.
特許第３０４４３２３号公報Japanese Patent No. 3443323 特開２００８－５８４６号公報JP 2008-5846 A 特開平１０－２４８８６０号公報JP-A-10-248860 国際公開第２０１１／０７０９７３号パンフレットInternational Publication No. 2011/077093 Pamphlet 特開２００５－４００７３号公報Japanese Patent Laid-Open No. 2005-40073 特開２００２－３１５５７３号公報JP 2002-315573 A 特開２００６－２７１３９５号公報JP 2006-271395 A 特開２００８－２２２６４０号公報JP 2008-222640 A 特開２０１３－１１１０１７号公報JP 2013-111017 A
　特許文献３で提案されている方法は、ストロー中にガラス化液を充満させることから、凍結までの時間が長い問題がある。また、凍結保存できる細胞または組織の大きさはストローの内径に制限されるため、細胞シートのようなシート形状の組織を保存することは困難である。 The method proposed in Patent Document 3 has a problem that it takes a long time to freeze because the vitrification liquid is filled in the straw. In addition, since the size of cells or tissues that can be cryopreserved is limited to the inner diameter of the straw, it is difficult to preserve a sheet-shaped tissue such as a cell sheet.
　特許文献４で提案されている方法は、卵子又は胚の周囲に付着した余分なガラス化液を除くことにより、優れた生存率でこれらの生殖細胞を凍結保存させる方法を提案している。しかしながら、上記公報記載の方法では、余分なガラス化液を除く際に、下部からの吸引操作を必要とするため、煩雑な操作となり、短時間でガラス化凍結保存操作を行う場合には不向きである。さらには、下部からの吸引が不十分であると余分なガラス化液が残存する問題がある。 The method proposed in Patent Document 4 proposes a method for cryopreserving these germ cells with an excellent survival rate by removing excess vitrification solution adhering to the periphery of the ovum or embryo. However, the method described in the above publication requires a suction operation from the bottom when removing the excess vitrification solution, which is a complicated operation and is not suitable for a vitrification cryopreservation operation in a short time. is there. Furthermore, if the suction from the lower part is insufficient, there is a problem that excess vitrification liquid remains.
　特許文献５では、卵子又は胚の周囲に付着した余分なガラス化液を濾紙などの吸収体に吸収させることにより、優れた生存率でこれらの生殖細胞を凍結保存させる方法を提案している。しかしながら、細胞シートのようなより大きな組織に用いる場合には、余分なガラス化液も多くなり、吸水性が不十分となる問題がある。 Patent Document 5 proposes a method of cryopreserving these germ cells with an excellent survival rate by absorbing excess vitrification solution adhering to the periphery of the egg or embryo into an absorbent such as filter paper. However, when it is used for a larger tissue such as a cell sheet, there is a problem that excessive vitrification liquid is increased and water absorption is insufficient.
　特許文献６、特許文献７で提案されている方法では、卵子又は胚をのせるフィルムの幅を制限することにより、少ない量のガラス化液とともに卵子又は胚を凍結保存する方法が記載されている。この方法では、作業者の操作によって、極少量のガラス化液とともに、卵子又は胚をフィルム上にのせるが、操作の難度が高いといった問題があった。この方法をもとにしたクライオトップ法では、より少ない量のガラス化液と共に卵子又は胚を凍結保存するために、一度ガラス化液と共に卵子又は胚をフィルム上に載せた後に、余分なガラス化液を吸引してフィルム上から除去するといった煩雑な操作がされることもある。また、例えば、細胞シートのようなシート形状で大面積を有する組織の凍結保存への適用には不向きである。 The methods proposed in Patent Document 6 and Patent Document 7 describe a method for cryopreserving an egg or embryo together with a small amount of vitrification solution by limiting the width of the film on which the egg or embryo is placed. . In this method, an egg or embryo is placed on a film together with a very small amount of vitrification solution by the operator's operation, but there is a problem that the operation is difficult. In the cryotop method based on this method, in order to cryopreserve the egg or embryo with a smaller amount of vitrification solution, once the egg or embryo is placed on the film together with the vitrification solution, excess vitrification is performed. A complicated operation of sucking the liquid and removing it from the film may be performed. For example, it is not suitable for application to cryopreservation of a tissue having a large area in a sheet shape such as a cell sheet.
　特許文献８に提案されている方法では、熱伝導性に優れた金属板を用い、さらに冷却溶媒が侵入可能な、大きさが２～８ｍｍ２程度の穴を複数有することにより、卵巣組織の保存において迅速な冷却速度を得るための工夫がなされている。しかしながら、特許文献６や特許文献７における問題と同様に、余分なガラス化液が組織や組織を構成する細胞の周囲に残り、ガラス化液の毒性や、凍結時の温度降下と融解時の温度上昇が遅延するために、組織や組織を構成する細胞の生存率が低下するおそれがある。 In the method proposed in Patent Document 8, a metal plate having excellent thermal conductivity is used, and further, a plurality of holes having a size of about 2 to 8 mm 2 in which a cooling solvent can enter can be preserved to preserve ovarian tissue. In order to obtain a rapid cooling rate, a device has been devised. However, like the problems in Patent Document 6 and Patent Document 7, excess vitrification liquid remains around the tissue and the cells constituting the tissue, and the toxicity of the vitrification liquid, the temperature drop during freezing and the temperature during melting Since the increase is delayed, the survival rate of the tissue and the cells constituting the tissue may be reduced.
　特許文献９には、例えば、ガラス板、金属板、メッシュ状の網、不織布等の台座の上に、運搬補助膜（セルシード社製Ｃｅｌｌ　Ｓｈｉｆｔｅｒ又はＰＶＤＦ膜）と共に細胞シートを載せて、余分なガラス化液を吸い取る若しくは落とした後で、凍結保存する方法が記載されているが、細胞または組織と共に滴下されるガラス化液の量が多い場合には、ガラス化液の吸収速度が十分でないために、余分なガラス化液が残存する問題がある。 In Patent Document 9, for example, a cell sheet is placed on a pedestal such as a glass plate, a metal plate, a mesh-like net, and a non-woven fabric together with a transportation auxiliary film (Cell Shifter or PVDF film manufactured by Cellseed), and an extra glass The method of cryopreserving after sucking or dropping the vitrification solution is described, but when the amount of vitrification solution dripped with cells or tissues is large, the absorption rate of the vitrification solution is not sufficient. There is a problem that excess vitrification liquid remains.
　本発明は、細胞または組織の凍結保存作業を容易にかつ確実に行うことが可能な、ガラス化凍結保存用治具を提供することを主な課題とする。より具体的には、細胞または組織をガラス化液に浸漬し、ガラス化液と共に該ガラス化凍結保存用治具に載せる際に、余分なガラス化液を吸収するための優れた吸収性能を備えたガラス化凍結保存用治具を提供する。 The main object of the present invention is to provide a vitrification cryopreservation jig capable of easily and reliably performing cryopreservation of cells or tissues. More specifically, when a cell or tissue is immersed in a vitrification solution and placed on the vitrification cryopreservation jig together with the vitrification solution, it has excellent absorption performance for absorbing excess vitrification solution. A jig for vitrification freezing storage is provided.
　本発明者らは、上記課題を解決するために鋭意検討した結果、上記した各種技術的な課題は、以下の構成を有する、細胞または組織のガラス化凍結保存用治具で解決できることを見出した。
（１）多孔質焼結形成体または多孔質金属体をガラス化液吸収体として有する、細胞または組織のガラス化凍結保存用治具。
（２）前記多孔質焼結形成体が、熱可塑性樹脂の樹脂固体粉末または金属酸化物粒子を三次元構造を持たせたまま加熱、焼結し、該粉末表面または該粒子表面を融着させることによって得られる多孔質構造体である、上記（１）に記載のガラス化凍結保存用治具。
（３）前記多孔質焼結形成体の細孔径が１μｍ以上である、上記（１）または（２）に記載のガラス化凍結保存用治具。
（４）前記多孔質焼結形成体の空隙率が２０～８０％である、上記（１）～（３）のいずれか一に記載のガラス化凍結保存用治具。
（５）前記多孔質金属体の細孔径が０．０５～５００μｍである、上記（１）に記載のガラス化凍結保存用治具。
（６）前記多孔質金属体の空隙率が２０％以上である、上記（１）または（５）に記載のガラス化凍結保存用治具 As a result of intensive studies to solve the above problems, the present inventors have found that the various technical problems described above can be solved by a jig for vitrification cryopreservation of cells or tissues having the following configuration. .
(1) A cell or tissue vitrification cryopreservation jig having a porous sintered body or a porous metal body as a vitrification liquid absorber.
(2) The porous sintered compact heats and sinters the resin solid powder or metal oxide particles of a thermoplastic resin with a three-dimensional structure, and fuses the powder surface or the particle surface. The jig for vitrification cryopreservation according to the above (1), which is a porous structure obtained by this.
(3) The jig for vitrification cryopreservation according to the above (1) or (2), wherein the porous sintered compact has a pore diameter of 1 μm or more.
(4) The vitrification cryopreservation jig according to any one of (1) to (3) above, wherein the porosity of the porous sintered compact is 20 to 80%.
(5) The vitrification cryopreservation jig according to (1), wherein the porous metal body has a pore diameter of 0.05 to 500 μm.
(6) The jig for vitrification cryopreservation according to (1) or (5) above, wherein the porosity of the porous metal body is 20% or more.
　本発明により、ガラス化液に浸漬させた細胞または組織をガラス化液と共にガラス化液吸収体上に載せる際に、ガラス化液吸収体が細胞または組織の外周に付着した余分なガラス化液を吸収することから、余分なガラス化液を除くためのその他の操作（例えば、ガラス化液吸収体下部からの吸引除去操作や、マイクロピペット等を用いた細胞または組織周囲からの直接的な吸引除去操作）を特に必要とせずに、細胞または組織の凍結保存作業が容易かつ簡便に行うことが可能な、細胞または組織のガラス化凍結保存用治具を提供することができる。また、本発明のガラス化凍結保存用治具を用いると、細胞または組織のガラス化凍結作業及び融解作業を効率よく行うことができる。 According to the present invention, when the cell or tissue immersed in the vitrification solution is placed on the vitrification solution absorber together with the vitrification solution, the excess vitrification solution attached to the outer periphery of the cell or tissue is removed. Other operations to remove excess vitrification liquid from absorption (for example, suction removal operation from the bottom of the vitrification liquid absorber, or direct suction removal from cells or tissues using a micropipette etc. It is possible to provide a jig for vitrification cryopreservation of cells or tissues, which can be easily and conveniently performed for cryopreservation of cells or tissues without particularly requiring operation. Moreover, if the jig | tool for vitrification cryopreservation of this invention is used, the vitrification freezing operation | work and melting | fusing operation | work of a cell or a structure | tissue can be performed efficiently.
図１は、本発明の細胞または組織のガラス化凍結保存用治具の一例を示す全体図である。FIG. 1 is an overall view showing an example of a jig for vitrification cryopreservation of cells or tissues of the present invention. 図２は、図１中のガラス化液吸収体の拡大図である。FIG. 2 is an enlarged view of the vitrification liquid absorber in FIG. 1. 図３は、支持体を有する場合に用いるガラス化液吸収体の一例を示す概略図である。FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used when a support is provided. 図４は、複数個の細胞または組織を１つの当該ガラス化凍結保存用治具で凍結保存させる場合に用いるガラス化液吸収体の一例を示す概略図である。FIG. 4 is a schematic view showing an example of a vitrification liquid absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. 図５は、複数個の細胞または組織を１つの当該ガラス化凍結保存用治具で凍結保存させる場合に用いるガラス化液吸収体の別の一例を示す概略図である。FIG. 5 is a schematic view showing another example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig.
　以下に本発明のガラス化凍結保存用治具を詳細に説明する。本発明のガラス化凍結保存用治具は、多孔質焼結形成体または多孔質金属体をガラス化液吸収体として有する。 Hereinafter, the vitrification cryopreservation jig of the present invention will be described in detail. The jig for vitrification cryopreservation of the present invention has a porous sintered body or a porous metal body as a vitrification liquid absorber.
　本発明のガラス化凍結保存用治具は、生物の細胞または組織を凍結保存する際に用いられるものである。本明細書中で細胞とは、単一の細胞のみならず、複数の細胞からなる細胞集団を含むものである。複数の細胞からなる細胞集団とは単一の種類の細胞から構成される細胞集団でも良いし、複数の種類の細胞から構成される細胞集団でも良い。また、組織とは、単一の種類の細胞から構成される組織でも良いし、複数の種類の細胞から構成される組織でも良く、細胞以外に細胞外マトリックスのような非細胞性の物質を含むものでも良い。本発明のガラス化凍結保存用治具は、好ましくは、ガラス化液吸収体に細胞または組織をガラス化液と共に付着させて、細胞または組織が付着した治具を液体窒素等の冷却物質に浸漬し凍結させるためのものである。上記ガラス化液吸収体を有することにより、細胞または組織をガラス化液と共に容易に保持することができ、さらには細胞または組織の液体窒素への浸漬作業も容易に行うことができる。本発明のガラス化凍結保存用治具は、細胞または組織凍結保存用具、細胞または組織ガラス化保存用具と言い換えることができる。 The vitrification cryopreservation jig of the present invention is used when cryopreserving biological cells or tissues. In the present specification, the cell includes not only a single cell but also a cell population composed of a plurality of cells. The cell population composed of a plurality of cells may be a cell population composed of a single type of cell or a cell population composed of a plurality of types of cells. The tissue may be a tissue composed of a single type of cell or a tissue composed of a plurality of types of cells, and includes non-cellular substances such as an extracellular matrix in addition to cells. Things can be used. The jig for cryopreservation of vitrification according to the present invention is preferably such that cells or tissues are attached to a vitrification liquid absorber together with vitrification liquid, and the jig to which the cells or tissues are attached is immersed in a cooling substance such as liquid nitrogen. For freezing. By having the vitrification liquid absorber, cells or tissues can be easily held together with the vitrification liquid, and further, the operation of immersing the cells or tissues in liquid nitrogen can be easily performed. The vitrification cryopreservation jig of the present invention can be restated as a cell or tissue cryopreservation tool or a cell or tissue vitrification storage tool.
　本発明のガラス化凍結保存用治具は、該ガラス化液吸収体が余分なガラス化液を吸収するので、ガラス化液に浸漬した細胞または組織を、ガラス化液と共に該ガラス化液吸収体上に滴下付着させる際に、細胞または組織に付着したガラス化液の量が多くても安定した細胞または組織の生存率が期待できる。さらに、そのように操作された細胞または組織はごく少量のガラス化液に覆われており、凍結操作する場合でも速やかに凍結状態にすることができる。さらには凍結保存した細胞または組織を解凍後ただちにガラス化液を希釈することができる。 In the vitrification cryopreservation jig of the present invention, the vitrification solution absorber absorbs excess vitrification solution, so that the cells or tissues immersed in the vitrification solution are combined with the vitrification solution. When the solution is dropped onto the cell, a stable cell or tissue survival rate can be expected even if the amount of vitrification solution adhering to the cell or tissue is large. Furthermore, the cells or tissues thus manipulated are covered with a very small amount of vitrification solution, and can be quickly frozen even when freezing. Furthermore, the vitrification solution can be diluted immediately after thawing the cryopreserved cells or tissues.
　本発明のガラス化凍結保存用治具は、ガラス化液を吸収するガラス化液吸収体を少なくとも有するガラス化凍結保存用治具であって、該ガラス化液吸収体が、多孔質焼結形成体または多孔質金属体であることを特徴とする。以下、多孔質焼結形成体をガラス化液吸収体として有するガラス化凍結保存用治具をガラス化凍結保存用治具Ａ、多孔質金属体をガラス化液吸収体として有するガラス化凍結保存用治具をガラス化凍結保存用治具Ｂと記載する。 The vitrification cryopreservation jig of the present invention is a vitrification cryopreservation jig having at least a vitrification solution absorber that absorbs a vitrification solution, and the vitrification solution absorber is formed by porous sintering. Or a porous metal body. Hereinafter, a vitrification cryopreservation jig having a porous sintered body as a vitrification liquid absorber is a vitrification cryopreservation jig A, and a vitrification cryopreservation having a porous metal body as a vitrification liquid absorber The jig is referred to as a vitrification cryopreservation jig B.
　ガラス化凍結保存用治具Ａがガラス化液吸収体として有する多孔質焼結形成体は、熱可塑性樹脂の樹脂固体粉末や金属酸化物粒子を三次元構造を持たせたまま加熱、焼結し、該粉末表面または該粒子表面を融着させることによって得られる多孔質構造体である。本発明における多孔質構造体は表面に気孔（細孔）を有する構造体であり、好ましくは表面及び内部に連続的な気孔を有する構造体である。ガラス化凍結保存用治具Ａは、該多孔質焼結形成体をガラス化液吸収体として有するものであり、支持体を有さずに、前記した多孔質焼結形成体そのものをガラス化液吸収体として有していても良い。あるいは支持体上に前記した多孔質焼結形成体を有するガラス化液吸収体であっても良い。支持体上に多孔質焼結形成体を積層する場合には、支持体と多孔質焼結形成体との間に、接着層を設けることができる。さらには、該多孔質焼結形成体と該接着層以外にも、例えば支持体上に均一な接着層を得ることを目的に、例えば下塗り層等を設けても良い。支持体は、多孔質焼結形成体を支持する目的だけでなく、支持体自体に吸収性能を持たせることもできる。 The porous sintered compact that the vitrification cryopreservation jig A has as a vitrification liquid absorber is obtained by heating and sintering a resin solid powder or metal oxide particles of a thermoplastic resin with a three-dimensional structure. A porous structure obtained by fusing the powder surface or the particle surface. The porous structure in the present invention is a structure having pores (pores) on the surface, preferably a structure having continuous pores on the surface and inside. The jig A for vitrification cryopreservation has the porous sintered formed body as a vitrified liquid absorber, and the above-mentioned porous sintered formed body itself is vitrified without having a support. You may have as an absorber. Or the vitrification liquid absorber which has an above-mentioned porous sintered compact on a support body may be sufficient. When laminating a porous sintered compact on a support, an adhesive layer can be provided between the support and the porous sintered compact. Furthermore, in addition to the porous sintered body and the adhesive layer, for example, an undercoat layer may be provided for the purpose of obtaining a uniform adhesive layer on the support. The support not only supports the porous sintered compact but also can provide the support itself with absorption performance.
　ガラス化凍結保存用治具Ａは、ガラス化液に浸漬された細胞または組織をガラス化液と共にガラス化液吸収体上に載せた時に、多孔質焼結形成体の表面の細孔を通してガラス化液がガラス化液吸収体に吸収され、細胞または組織の周囲から余分なガラス化液を除くことができる。多孔質焼結形成体としては、例えば、樹脂からなる多孔質焼結形成体、金属酸化物からなる多孔質焼結形成体が挙げられる。 When the cell or tissue immersed in the vitrification solution is placed on the vitrification solution absorber together with the vitrification solution, the vitrification cryopreservation jig A is vitrified through the pores on the surface of the porous sintered compact. The liquid is absorbed by the vitrification liquid absorber, and excess vitrification liquid can be removed from the periphery of the cell or tissue. Examples of the porous sintered formed body include a porous sintered formed body made of a resin and a porous sintered formed body made of a metal oxide.
　多孔質焼結形成体が樹脂からなる場合には、これを得るために用いられる熱可塑性樹脂として、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、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、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。樹脂からなる多孔質焼結形成体は、２種類以上の樹脂を含有する多孔質焼結形成体であっても良い。また、本発明の多孔質焼結形成体が金属酸化物からなる場合には、これを得るために用いられる金属酸化物としては、シリカ、アルミナ、ジルコニウム、石英ガラス等が挙げられる。加工が容易であることから、好ましくは、ポリエチレン、石英ガラスが挙げられる。 When the porous sintered body is made of a resin, the thermoplastic resin used to obtain the resin is low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene, polypropylene, polymethyl methacrylate, polystyrene, fluororesin, Examples thereof include ethylene-vinyl acetate copolymer, polyamide, styrene-acrylonitrile copolymer, styrene-butadiene-acrylonitrile terpolymer, polycarbonate, and polyvinyl chloride. The porous sintered formed body made of resin may be a porous sintered formed body containing two or more kinds of resins. Moreover, when the porous sintered compact of this invention consists of a metal oxide, as a metal oxide used in order to obtain this, a silica, an alumina, a zirconium, quartz glass etc. are mentioned. Since processing is easy, Preferably, polyethylene and quartz glass are mentioned.
　ガラス化凍結保存用治具Ａが有する多孔質焼結形成体の製造方法としては、一般に知られている方法を使用することができる。多孔質焼結形成体が樹脂からなる場合には、例えば、特開２００９－２３５４１７号公報に記載された方法などを用いることができる。より具体的には、乳化重合また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、多孔質焼結形成体を製造することができる。加熱、焼結する際の温度は、焼結する熱可塑性樹脂の種類によって異なるが、高い空隙率を維持するために、それぞれの樹脂の融点付近で、粒子同士が十分に融着する温度で、かつ、粒子間隙を埋めることのない温度が選択される。例えば、ポリエチレンの場合には、加熱温度は、１１０℃～１８０℃が好ましく、１２０℃～１５０℃がより好ましい。多孔質焼結形成体が金属酸化物からなる場合には、例えば、特開２００９－２９６９２号公報や特開２００２－１６０９３０号公報に記載された方法などを用いることができる。製造された多孔質焼結形成体は、切断、切削、打ち抜き加工等の二次加工によって、任意の形状に加工してもよい。 A generally known method can be used as a method for manufacturing the porous sintered compact included in the jig A for vitrification cryopreservation. When the porous sintered body is made of a resin, for example, a method described in JP 2009-235417 A can be used. More specifically, a solid powder of a thermoplastic resin obtained by a method such as emulsion polymerization or pulverization is filled in a mold, heated and sintered to fuse the powder particle surface, and cooled, A porous sintered compact can be produced. The temperature at the time of heating and sintering varies depending on the type of thermoplastic resin to be sintered, but in order to maintain a high porosity, the temperature at which the particles are sufficiently fused in the vicinity of the melting point of each resin, A temperature that does not fill the particle gap is selected. For example, in the case of polyethylene, the heating temperature is preferably 110 ° C. to 180 ° C., more preferably 120 ° C. to 150 ° C. When the porous sintered body is made of a metal oxide, for example, the methods described in JP2009-29692A and JP2002-160930A can be used. The produced porous sintered compact may be processed into an arbitrary shape by secondary processing such as cutting, cutting, and punching.
　上記した多孔質焼結形成体の表面は、ガラス化液の吸収性能を高めるために、親水化処理することもできる。親水化処理の方法としては、グラフト改質法、親水性高分子化合物等を用いたコーティング法、コロナ放電、プラズマ処理、エキシマレーザー等の各種電子線を用いた一般的な表面改質方法、さらには粗面化処理等による表面改質方法等を使用することできる。なお、親水化処理は、焼結工程前の樹脂粉末粒子に対して行っても良いし、焼結工程後の多孔質焼結形成体に対して行っても良い。 The surface of the porous sintered compact can be subjected to a hydrophilic treatment in order to improve the vitrification performance. Hydrophilic treatment methods include graft modification methods, coating methods using hydrophilic polymer compounds, corona discharge, plasma treatment, general surface modification methods using various electron beams such as excimer laser, Can use surface modification methods such as surface roughening. The hydrophilic treatment may be performed on the resin powder particles before the sintering step, or may be performed on the porous sintered body after the sintering step.
　ガラス化凍結保存用治具Ａが有する多孔質焼結形成体は、ガラス化液の吸収性の観点から、細孔径が１μｍ以上であることが好ましい。細孔径が１μｍ未満の範囲では、ガラス化液滴下時の吸収性能が十分でなく、凍結保存までに時間を要したり、余分なガラス化液が細胞または組織の周辺に多量に残存するおそれがある。一方で、細孔径が１ｍｍを超える範囲では、細胞やシート形状の組織の一部等が細孔間隙にトラップされ、凍結保存後の解凍・融解する操作やその後の操作において、ガラス化液吸収体表面から、細胞や組織がはがれないといった問題が発生する場合がある。以上の観点から、本発明に用いる多孔質焼結形成体が有する細孔径は、好ましくは１μｍ～１ｍｍである。また、本発明に用いる多孔質焼結形成体の空隙率は２０～８０％であることが好ましく、より好ましくは３０～６０％である。本発明に用いる多孔質焼結形成体の細孔径、厚み、空隙率は、用いる細胞または組織の種類や細胞または組織と共に滴下されるガラス化液の滴下量等に応じて適宜設定することができる。 The porous sintered compact of the vitrification cryopreservation jig A preferably has a pore diameter of 1 μm or more from the viewpoint of the vitrification liquid absorbability. When the pore diameter is less than 1 μm, the absorption performance when the vitrified droplet is dropped is not sufficient, and it may take time until frozen storage, or a large amount of excess vitrified solution may remain around the cells or tissues. is there. On the other hand, in the range where the pore diameter exceeds 1 mm, a part of the cell or sheet-shaped tissue is trapped in the pore gap, and in the operation of thawing and thawing after cryopreservation and the subsequent operation, the vitrification solution absorber There may be a problem that cells and tissues do not peel off from the surface. From the above viewpoint, the pore diameter of the porous sintered compact used in the present invention is preferably 1 μm to 1 mm. Further, the porosity of the porous sintered compact used in the present invention is preferably 20 to 80%, more preferably 30 to 60%. The pore diameter, thickness, and porosity of the porous sintered compact used in the present invention can be appropriately set according to the type of cells or tissues to be used, the dripping amount of the vitrification solution dripped with the cells or tissues, and the like. .
　上記した細孔径とは、表面及び断面の画像観察から測定した細孔の平均直径を表す。 The above-mentioned pore diameter represents the average diameter of the pores measured from image observation of the surface and the cross section.
　上記した空隙率とは、以下の式で定義される。ここで空隙容量Ｖは水銀圧入法を用いて測定することができる。
Ｐ＝（Ｖ／Ｔ）×１００（％）
　Ｐ：空隙率（％）
　Ｖ：空隙容量（ｍＬ／ｍ２）
　Ｔ：厚み（μｍ） The above porosity is defined by the following formula. Here, the void volume V can be measured using a mercury intrusion method.
P = (V / T) × 100 (%)
P: Porosity (%)
V: void volume (mL / m 2 )
T: Thickness (μm)
　ガラス化液吸収体として用いる多孔質焼結形成体の面積は、細胞または組織の大きさと細胞または組織に付着したガラス化液の量等に応じて適宜設定すればよく特に限定されないが、例えば、凍結保存する細胞または組織がシート状の組織である場合には、シート状の組織の底面積よりも、多孔質焼結形成体の面積が大きいことが好ましい。多孔質焼結形成体のより好ましい面積は、シート状の組織の底面積に対して１．５倍以上である。また、凍結保存する細胞が粒状で懸濁された状態の場合には、滴下するガラス化液１μＬにつき２ｍｍ２以上とすることが好ましく、１０～４００ｍｍ２とすることがより好ましい。 The area of the porous sintered compact used as the vitrification liquid absorber is not particularly limited as long as it is appropriately set according to the size of the cell or tissue and the amount of the vitrification liquid adhering to the cell or tissue. When the cell or tissue to be cryopreserved is a sheet-like tissue, the area of the porous sintered formed body is preferably larger than the bottom area of the sheet-like tissue. A more preferable area of the porous sintered compact is 1.5 times or more with respect to the bottom area of the sheet-like structure. In the case of state cells cryopreserved are suspended in particulate is preferably in the vitrification liquid 1μL to per 2 mm 2 or more to be dropped, and more preferably in the 10 ~ 400 mm 2.
　次に、ガラス化凍結保存用治具Ｂがガラス化液吸収体として有する多孔質金属体について説明する。本発明における多孔質金属体は、表面に気孔（細孔）を有する構造体であり、好ましくは表面及び内部に連続的な気孔を有する構造体である。ガラス化凍結保存用治具Ｂは、該多孔質金属体をガラス化液吸収体として有するものであり、支持体を有さずに、前記した多孔質金属体そのものをガラス化液吸収体として有していても良い。あるいは支持体上に前記した多孔質金属体を有するガラス化液吸収体であっても良い。支持体を多孔質金属体とともに用いる場合には、支持体と多孔質金属体との間に、接着層を設けることができる。さらには、該多孔質金属体と該接着層以外にも、例えば支持体上に均一な接着層を得ることを目的に、例えば下塗り層等を設けても良い。支持体は、多孔質金属体を支持する目的だけでなく、支持体自体に吸収性能を持たせることもできる。 Next, the porous metal body that the vitrification cryopreservation jig B has as a vitrification liquid absorber will be described. The porous metal body in the present invention is a structure having pores (pores) on the surface, preferably a structure having continuous pores on the surface and inside. The vitrification cryopreservation jig B has the porous metal body as a vitrification liquid absorber, and has the above-described porous metal body itself as a vitrification liquid absorber without a support. You may do it. Or the vitrification liquid absorber which has the above-mentioned porous metal body on a support body may be sufficient. When using a support body with a porous metal body, an adhesive layer can be provided between the support body and the porous metal body. Furthermore, in addition to the porous metal body and the adhesive layer, for example, an undercoat layer may be provided for the purpose of obtaining a uniform adhesive layer on the support. The support not only supports the porous metal body, but also allows the support itself to have absorption performance.
　ガラス化凍結保存用治具Ｂは、細胞または組織を含むガラス化液がガラス化液吸収体上に滴下された時、多孔質金属体の表面の細孔を通してガラス化液がガラス化液吸収体に吸収され、細胞または組織の周囲から余分なガラス化液を除くことができる。 The vitrification cryopreservation jig B is such that when a vitrification solution containing cells or tissues is dropped on the vitrification solution absorber, the vitrification solution passes through the pores on the surface of the porous metal body. And can remove excess vitrification fluid from around the cell or tissue.
　本発明においてガラス化液吸収体として用いる多孔質金属体は、例えば、銅、銅合金、アルミニウム、アルミニウム合金、金、金合金、銀、銀合金、錫、亜鉛、鉛、チタン、ニッケル、ステンレスなどの素材からなる多孔質金属体を用いることができる。素材としての入手のしやすさと扱いやすさの観点から、銅、銅合金、アルミニウム、アルミニウム合金、チタン、ニッケル、ステンレスの素材からなる多孔質金属体であることが好ましい。 Examples of the porous metal body used as the vitrification liquid absorber in the present invention include copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, tin, zinc, lead, titanium, nickel, and stainless steel. A porous metal body made of the above material can be used. From the viewpoint of availability as a material and ease of handling, a porous metal body made of copper, copper alloy, aluminum, aluminum alloy, titanium, nickel, or stainless steel is preferable.
　ガラス化凍結保存用治具Ｂが有する多孔質金属体の製造方法としては、粉末冶金法、スペーサー法などの一般的な多孔質金属体の製造方法を使用することができる。また、樹脂射出成型と粉末冶金法を組み合わせた所謂パウダースペースホルダー法も好ましく使用できる。例えば、国際公開第２００６／０４０１１１８号パンフレットや特許第４５７８０６２号公報に記載された方法などを用いることができる。より具体的には、金属粉末とスペーサーとなる樹脂を混合後、圧力をかけて成型した後、高温環境下で焼成することで、金属粉末を焼き固め、スペーサーとなる樹脂を気化させて、多孔質金属体を得ることができる。パウダースペースホルダー法等を用いる場合には、金属粉末とスペーサーとなる樹脂に加えて、樹脂のバインダーを混合することができる。類似の方法として、一般に知られている所謂ファイバースペースホルダー法のように、金属粉末と繊維状のスペーサー素材を混合した後に、高温環境下で焼成することで、金属粉末を焼き固め、スペーサーとなる繊維を気化させて、多孔質金属体を製造することもできる。また、金属粉末を高温で加熱した後に、ガスを注入して空隙を作製する発泡溶融法、ガス膨張法などの多孔質金属体の製造方法も使用することができる。さらには、発泡剤を用いて多孔質金属体を製造するスラリー発泡法のような製造方法も使用することができる。 As a method for producing a porous metal body possessed by the vitrification cryopreservation jig B, a general method for producing a porous metal body such as a powder metallurgy method or a spacer method can be used. Further, a so-called powder space holder method in which resin injection molding and powder metallurgy are combined can be preferably used. For example, a method described in International Publication No. 2006/0401118 pamphlet or Japanese Patent No. 4578062 can be used. More specifically, after mixing the metal powder and the resin serving as a spacer, forming by applying pressure, firing the metal powder by baking in a high temperature environment, vaporizing the resin serving as the spacer, A porous metal body can be obtained. In the case of using the powder space holder method or the like, a resin binder can be mixed in addition to the metal powder and the resin serving as the spacer. As a similar method, as in the generally known so-called fiber space holder method, after mixing metal powder and a fibrous spacer material, the metal powder is baked and hardened by baking in a high temperature environment to become a spacer. It is also possible to produce a porous metal body by vaporizing the fibers. Moreover, after heating metal powder at high temperature, the manufacturing method of porous metal bodies, such as the foaming melting method and gas expansion method which inject | pour gas and produce a space | gap, can also be used. Furthermore, a production method such as a slurry foaming method for producing a porous metal body using a foaming agent can also be used.
　上記した多孔質金属体の表面は、ガラス化液吸収性能を高めるために、親水化処理することもできる。親水化処理の方法としては、グラフト改質法、親水性高分子化合物等を用いたコーティング法、コロナ放電、プラズマ処理、エキシマレーザー等の各種電子線を用いた一般的な表面改質方法を使用することができる。例えば、特開平１１－２５６３５５号公報、特開２０００－２８１９３６号公報、特開２００８－１６１８０６号公報、特開２０１１－７３６５号公報に記載される方法を用いることができる。 The surface of the above-described porous metal body can be subjected to a hydrophilic treatment in order to improve the vitrification solution absorption performance. Hydrophilic treatment methods include graft modification methods, coating methods using hydrophilic polymer compounds, etc., corona discharge, plasma treatment, and general surface modification methods using various electron beams such as excimer lasers. can do. For example, the methods described in JP-A-11-256355, JP-A-2000-281936, JP-A-2008-161806, and JP-A-2011-7365 can be used.
　ガラス化凍結保存用治具Ｂが有する多孔質金属体の細孔径は、０．０５～５００μｍであることが好ましく、より好ましくは０．５～５０μｍである。細孔径が０．０５μｍ未満の範囲では、ガラス化液の滴下時にガラス化液の吸収性能が十分でない場合がある。また、多孔質金属体の製造が難しいという問題がある。一方、細孔径が５００μｍを超える範囲では、細胞または組織が細孔中にトラップされ、融解作業時に、細胞が多孔質金属体上から遊離しにくくなる場合がある。また、ガラス化液の吸収性能が十分でない場合がある。なお、多孔質金属体の細孔径は、該多孔質の表面及び断面の画像観察から測定した平均細孔直径である。該多孔質金属体の厚みは、５０μｍ～５０ｍｍであることが好ましく、より好ましくは５００μｍ～１０ｍｍである。該多孔質金属体の空隙率は２０容量％以上であることが好ましく、より好ましくは４０容量％以上、さらに好ましくは５０容量％以上である。多孔質金属体の内部の気孔は、厚み方向のみならず、厚み方向に対して垂直な方向に連続的な構造である、所謂オープンセル型であることが好ましい。このような構造を有すると、多孔質金属体内部の気孔を有効に用いることができるために、ガラス化液の高い吸収性能が得られる。多孔質金属体の厚み、空隙率は、用いる細胞または組織の種類や細胞または組織と共に滴下されるガラス化液の滴下量等に応じて適宜選択することができる。 The pore diameter of the porous metal body possessed by the vitrification cryopreservation jig B is preferably 0.05 to 500 μm, more preferably 0.5 to 50 μm. When the pore diameter is less than 0.05 μm, the absorption performance of the vitrification liquid may not be sufficient when the vitrification liquid is dropped. In addition, there is a problem that it is difficult to produce a porous metal body. On the other hand, when the pore diameter exceeds 500 μm, the cells or tissues are trapped in the pores, and the cells may not be easily released from the porous metal body during the melting operation. Moreover, the absorption performance of vitrification liquid may not be enough. The pore diameter of the porous metal body is an average pore diameter measured from image observation of the porous surface and cross section. The thickness of the porous metal body is preferably 50 μm to 50 mm, more preferably 500 μm to 10 mm. The porosity of the porous metal body is preferably 20% by volume or more, more preferably 40% by volume or more, and still more preferably 50% by volume or more. The pores inside the porous metal body are preferably of a so-called open cell type that has a continuous structure not only in the thickness direction but also in a direction perpendicular to the thickness direction. With such a structure, since the pores inside the porous metal body can be used effectively, high absorption performance of the vitrification liquid can be obtained. The thickness and porosity of the porous metal body can be appropriately selected according to the type of cells or tissues to be used, the dripping amount of the vitrification solution dripped with the cells or tissues, and the like.
　ガラス化液吸収体として用いる多孔質金属体の面積は、細胞または組織と共に滴下されるガラス化液の滴下量等に応じて適宜設定すればよく特に限定されないが、例えば、滴下するガラス化液１μＬにつき１ｍｍ２以上とすることが好ましく、２～４００ｍｍ２とすることがより好ましい。 The area of the porous metal body used as the vitrification liquid absorber is not particularly limited as long as it is appropriately set according to the dripping amount of the vitrification liquid dripped together with the cells or tissues, but for example, 1 μL of the dripping vitrification liquid It is preferably 1 mm 2 or more, more preferably 2 to 400 mm 2 .
　本発明のガラス化凍結保存用治具Ａおよびガラス化凍結保存用治具Ｂが有するガラス化液吸収体が支持体を有する場合、一般に知られる様々な支持体を使用することができる。このような支持体としては例えば、各種樹脂フィルム、ガラス、ゴム、金属、繊維形状物質、スポンジ形状物質等が挙げられる。樹脂フィルムの具体例としては、ポリエチレンテレフタレート（ＰＥＴ）やポリエチレンナフタレート（ＰＥＮ）等のポリエステル樹脂、アクリル樹脂、エポキシ樹脂、シリコーン樹脂、ポリカーボネート樹脂、ジアセテート樹脂、トリアセテート樹脂、ポリアリレート樹脂、ポリ塩化ビニル樹脂、ポリスルフォン樹脂、ポリエーテルスルフォン樹脂、ポリイミド樹脂、ポリアミド樹脂、ポリオレフィン樹脂、環状ポリオレフィン樹脂、フッ素樹脂等からなる樹脂フィルムが挙げられる。また温度伝導性に優れ、急速な凍結を可能にするという観点で金属板を好適に用いることができる。該支持体の厚さは１０μｍ～１００ｍｍであることが好ましい。また、接着層との接着強度を高めるために、支持体の表面をコロナ放電処理のような電気的な方法や、化学的な方法により易接着処理することができ、さらには粗面化することもできる。 When the vitrification solution absorber which the jig A for vitrification cryopreservation and the jig B for vitrification cryopreservation of the present invention have has a support, various generally known supports can be used. Examples of such a support include various resin films, glass, rubber, metal, fiber-shaped substance, sponge-shaped substance, and the like. Specific examples of the resin film include polyester resins such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN), acrylic resins, epoxy resins, silicone resins, polycarbonate resins, diacetate resins, triacetate resins, polyarylate resins, polychlorinated resins. Examples of the resin film include a vinyl resin, a polysulfone resin, a polyether sulfone resin, a polyimide resin, a polyamide resin, a polyolefin resin, a cyclic polyolefin resin, and a fluorine resin. Moreover, a metal plate can be used suitably from a viewpoint of being excellent in temperature conductivity and enabling rapid freezing. The thickness of the support is preferably 10 μm to 100 mm. In addition, in order to increase the adhesive strength with the adhesive layer, the surface of the support can be easily adhered by an electrical method such as corona discharge treatment or a chemical method, and further roughened. You can also.
　本発明のガラス化凍結保存用治具Ａまたはガラス化凍結保存用治具Ｂが接着層を有する場合には、湿気硬化性の接着物質に代表されるような瞬間接着物質、ホットメルト接着物質、光硬化性接着物質などを用いた一般的な接着方法が利用でき、例えば、ポリビニルアルコール、ヒドロキシセルロース、ポリビニルピロリドン、澱粉糊のような水溶性接着物質、酢酸ビニル系接着物質、アクリル系接着物質、エポキシ系接着物質、ウレタン系接着物質、エラストマー系接着物質、シアノアクリレート系接着物質、フッ素系接着物質、シリコン系接着物質、ニトロセルロース系接着物質、ニトリルゴム系接着物質、スチレン－ブタジエン系接着物質、ユリア樹脂系接着物質、スチレン系樹脂接着物質、フェノール樹脂系接着物質、ポリイミド系接着物質、ポリアミド系接着物質、ポリエステル系接着物質、ビスマレイミド系接着物質、オレフィン系接着物質、ＥＶＡ系接着物質などの非水溶性接着物質が好ましく利用できる。該接着層は、一種類の接着物質を含有してもよいし、複数種類の接着物質を含有してもよい。接着層の固形分量は、０．０１～１００ｇ／ｍ２の範囲が好ましく、更に０．１～５０ｇ／ｍ２の範囲がより好ましい。 In the case where the vitrification cryopreservation jig A or the vitrification cryopreservation jig B of the present invention has an adhesive layer, an instantaneous adhesive substance typified by a moisture-curable adhesive substance, a hot melt adhesive substance, A general bonding method using a photo-curable adhesive material can be used. For example, water-soluble adhesive materials such as polyvinyl alcohol, hydroxy cellulose, polyvinyl pyrrolidone, starch paste, vinyl acetate adhesive materials, acrylic adhesive materials, Epoxy adhesives, urethane adhesives, elastomer adhesives, cyanoacrylate adhesives, fluorine adhesives, silicon adhesives, nitrocellulose adhesives, nitrile rubber adhesives, styrene-butadiene adhesives, Urea resin adhesives, styrene resin adhesives, phenol resin adhesives, polyimide adhesives Materials, polyamide series adhesive materials, polyester-based adhesive agent, bismaleimide adhesive material, an olefin-based adhesive material, a non-water soluble adhesive material, such as EVA-based adhesive materials can be preferably used. The adhesive layer may contain one kind of adhesive substance or may contain a plurality of kinds of adhesive substances. The solid content of the adhesive layer is preferably in the range of 0.01 to 100 g / m 2 , more preferably in the range of 0.1 to 50 g / m 2 .
　以上、本発明におけるガラス化液吸収体を説明してきた。以下にこれらを用いたガラス化凍結保存用治具の構成について説明する。本発明のガラス化凍結保存用治具は上述したようなガラス化液吸収体を有するものであればどのようなものであってもよいが、該ガラス化液吸収体が把持部に接続されていてもよい。把持部を有すると、凍結保存作業時及び融解作業時の作業性が良好であるため、好ましい。 The vitrification liquid absorber in the present invention has been described above. The structure of the vitrification cryopreservation jig using these will be described below. The vitrification cryopreservation jig of the present invention may be anything as long as it has a vitrification solution absorber as described above, but the vitrification solution absorber is connected to the gripping part. May be. Having a gripping portion is preferable because workability during cryopreservation work and melting work is good.
　図１は本発明のガラス化凍結保存用治具の一例を示す全体図である。図１においてガラス化凍結保存用治具５は、把持部１とガラス化液吸収体２から構成される。把持部１は耐液体窒素素材であることが好ましい。このような素材としては、例えばアルミ、鉄、銅、ステンレス合金などの各種金属、ＡＢＳ樹脂、ポリプロピレン樹脂、ポリエチレン樹脂、フッ素系樹脂や各種エンジニアプラスチック、更にはガラスなどを好適に用いることができる。また基本的にガラス化液吸収体２はハンドリング上、短冊状又はシート状であることが好ましい。 FIG. 1 is an overall view showing an example of a vitrification cryopreservation jig according to the present invention. In FIG. 1, a vitrification cryopreservation jig 5 includes a gripping portion 1 and a vitrification liquid absorber 2. The grip 1 is preferably made of a liquid nitrogen resistant material. As such a material, for example, various metals such as aluminum, iron, copper, and stainless alloy, ABS resin, polypropylene resin, polyethylene resin, fluorine resin, various engineer plastics, and glass can be preferably used. Basically, the vitrification liquid absorber 2 is preferably strip-shaped or sheet-shaped for handling.
　本発明におけるガラス化液吸収体の一例を、図２に示す。図２は、図１のガラス化液吸収体２の拡大図である。図２のガラス化液吸収体２ａは支持体を有さずに多孔質焼結形成体あるいは多孔質金属体３そのものをガラス化液吸収体とする形態の一例である。また、図３は支持体を有する場合に用いるガラス化液吸収体の一例を示す概略図であり、該ガラス化液吸収体２ｂは支持体４上に多孔質焼結形成体あるいは多孔質金属体３を有する。図３に示すガラス化液吸収体２ｂは、ガラス化液吸収体の全面に多孔質焼結形成体あるいは多孔質金属体を有する形態の一例である。 An example of the vitrification liquid absorber in the present invention is shown in FIG. FIG. 2 is an enlarged view of the vitrification liquid absorber 2 of FIG. The vitrification liquid absorber 2a of FIG. 2 is an example of a form in which a porous sintered body or the porous metal body 3 itself is used as a vitrification liquid absorber without having a support. FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used when a support is provided, and the vitrification liquid absorber 2b is formed on the support 4 with a porous sintered body or a porous metal body. 3. The vitrification liquid absorber 2b shown in FIG. 3 is an example of a form having a porous sintered body or a porous metal body on the entire surface of the vitrification liquid absorber.
　図１の把持部１とガラス化液吸収体２の接続方法について説明する。把持部１が樹脂の場合、例えば、成形加工する時にインサート成形によりガラス化液吸収体２を把持部１に接続することができる。更に、把持部１に図示しないガラス化液吸収体挿入部を作製して接着剤にてガラス化液吸収体２を接続することができる。接着剤は様々なものが使用できるが、低温に強いシリコン系やフッ素系の接着剤が好適に用いることができる。 A method for connecting the grip 1 and the vitrification liquid absorber 2 in FIG. 1 will be described. When the holding part 1 is resin, for example, the vitrification liquid absorber 2 can be connected to the holding part 1 by insert molding at the time of molding. Furthermore, the vitrification liquid absorber insertion part which is not shown in figure in the holding part 1 can be produced, and the vitrification liquid absorber 2 can be connected with an adhesive agent. Although various adhesives can be used, a silicon-based or fluorine-based adhesive that is resistant to low temperatures can be suitably used.
　本発明におけるガラス化液保存用治具で細胞または組織を長期凍結保存する場合、図１に示す治具本体に安全のため、外界と遮断するためにキャップを被せることも可能である。更に、通常液体窒素は滅菌されておらず、直接液体窒素に接触させて凍結させる場合、ガラス化液保存用治具が滅菌されていても無菌状態を保証できない場合がある。よって凍結前に細胞または組織を付着させたガラス化液吸収体にキャップをして、直接液体窒素に接触させないで凍結させることがある。また、米国・ＥＵなど海外先進諸国では前記のように液体窒素に直接接触させない凍結方法が主流となってきている。このような理由からキャップは耐液体窒素性のある素材である各種金属、各種樹脂、ガラス、セラミックなどで作製することが好ましい。形状としては、鉛筆用のキャップや円柱状のストローキャップなどガラス化液吸収体と接触せず、外界と遮断できるような形状ならどのような形状でもよい。 When the cell or tissue is cryopreserved for a long time with the vitrification solution storage jig according to the present invention, the jig body shown in FIG. 1 can be covered with a cap to shut off from the outside for safety. Furthermore, liquid nitrogen is usually not sterilized, and when frozen by direct contact with liquid nitrogen, sterility may not be guaranteed even if the vitrification solution storage jig is sterilized. Therefore, a vitrified liquid absorber to which cells or tissues are attached before freezing may be capped and frozen without being in direct contact with liquid nitrogen. In the advanced countries such as the US and EU, the freezing method that does not directly contact liquid nitrogen as described above has become mainstream. For this reason, the cap is preferably made of various metals, various resins, glass, ceramic, etc., which are liquid nitrogen resistant materials. The shape may be any shape, such as a pencil cap or a cylindrical straw cap, as long as the shape can be cut off from the outside without being in contact with the vitrification liquid absorber.
　図４、図５に、本発明におけるガラス化液吸収体の別の態様の例を示す。図１に示す細胞または組織のガラス化凍結保存用治具において、ガラス化液吸収体を図４、図５に示すものとすることもできる。図４は、複数個の細胞または組織を１つの当該ガラス化凍結保存用治具で凍結保存させる場合に用いるガラス化液吸収体の一例を示す概略図を示す。また、図５は複数個の細胞または組織を１つの当該ガラス化凍結保存用治具で凍結保存させる場合に用いるガラス化液吸収体の別の一例を示す概略図である。図４及び図５では多孔質焼結形成体あるいは多孔質金属体３が支持体４上に、不連続であって複数が配置されている。 4 and 5 show examples of other embodiments of the vitrification liquid absorber in the present invention. In the cell or tissue vitrification cryopreservation jig shown in FIG. 1, the vitrification solution absorber may be as shown in FIGS. FIG. 4 is a schematic view showing an example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. FIG. 5 is a schematic view showing another example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. 4 and 5, the porous sintered body or porous metal body 3 is discontinuous and arranged on the support 4.
　前述した図３のように多孔質焼結形成体あるいは多孔質金属体３が連続した形状である場合、複数の細胞または組織を多孔質焼結形成体あるいは多孔質金属体３に付着させようとすると、ガラス化液は多孔質金属体で横方向と厚み方向に広がるため、例えば２個目以降の細胞または組織を多孔質焼結形成体あるいは多孔質金属体３に付着させた場合、ガラス化液の吸収性が低下する場合がある。しかし、図４及び図５のように多孔質焼結形成体あるいは多孔質金属体３が支持体４上に、不連続で複数設けられているとそのような心配がなく、ガラス化液と共に細胞または組織を各多孔質焼結形成体あるいは多孔質金属体３に１個ずつ確実に付着させることができる。図４及び図５では、一例として、升目状の多孔質金属体３を複数配置した。図４及び図５に示すガラス化液吸収体２ｃ及びガラス化液吸収体２ｄは、支持体上の一部に多孔質焼結形成体あるいは多孔質金属体を有するガラス化液吸収体の一例でもある。 When the porous sintered body or the porous metal body 3 has a continuous shape as shown in FIG. 3 described above, an attempt is made to attach a plurality of cells or tissues to the porous sintered body or the porous metal body 3. Then, since the vitrification liquid is a porous metal body and spreads in the lateral direction and the thickness direction, for example, when the second or subsequent cells or tissues are attached to the porous sintered body or the porous metal body 3, vitrification is performed. The liquid absorbency may decrease. However, if a plurality of porous sintered bodies or porous metal bodies 3 are provided on the support 4 in a discontinuous manner as shown in FIGS. Alternatively, one structure can be reliably attached to each porous sintered body or porous metal body 3. 4 and 5, a plurality of grid-like porous metal bodies 3 are arranged as an example. The vitrification liquid absorber 2c and the vitrification liquid absorber 2d shown in FIGS. 4 and 5 may be an example of a vitrification liquid absorber having a porous sintered body or a porous metal body on a part of the support. is there.
　本発明のガラス化凍結保存用治具は、例えば、クライオトップ法において好適に用いられるものである。また、従来のクライオトップ法は、通常、単一の細胞または１０個未満の少数の細胞の保存に用いられるが、本発明のガラス化凍結保存用治具は、より多くの細胞の保存（例えば、１０～１００００００個の細胞の保存）においても好適に用いることができる。さらには、複数の細胞からなるシート状の細胞（所謂細胞シート）の保存にも好適に用いることができる。本発明のガラス化凍結保存用治具を用いると、細胞または組織の凍結時及び融解時に細胞外のガラス化液による損傷を受けにくく、細胞または組織を優れた生存率で凍結保存することができる。 The vitrification cryopreservation jig of the present invention is suitably used, for example, in the cryotop method. In addition, the conventional cryotop method is usually used for storage of a single cell or a small number of cells of less than 10, but the vitrification cryopreservation jig of the present invention can store more cells (for example, (Preservation of 10 to 1000000 cells) can also be suitably used. Furthermore, it can be suitably used for the preservation of sheet-like cells (so-called cell sheets) composed of a plurality of cells. When the jig for vitrification cryopreservation according to the present invention is used, it is difficult to be damaged by the vitrification solution outside the cell or tissue during freezing and thawing, and the cell or tissue can be cryopreserved with an excellent survival rate. .
　本発明のガラス化凍結保存用治具を用いて細胞または組織を凍結保存する方法は特に限定されず、例えば、まず、ガラス化液に浸漬した細胞または組織をガラス化液と共にガラス化液吸収体上に滴下し、該細胞または該組織の周囲に付着しているガラス化液をガラス化液吸収体に吸収させる。次いで、前記細胞または前記組織をガラス化液吸収体上に保持させたまま液体窒素等の中に浸漬することにより、細胞または組織を凍結することができる。ガラス化液は、通常卵子、胚等の細胞の凍結のために使用されるものを使用でき、例えば、上述したグリセロールやエチレングリコール、ＤＭＳＯ（ジメチルスルホキシド）などの耐凍剤を３０容量％以上、より好ましくは耐凍剤を３０～４０容量％含有する水溶液を好ましく使用できる。融解作業の際は、液体窒素等の冷却溶媒中から、該ガラス化凍結保存用治具をとり出し、凍結された細胞または組織を載せたガラス化液吸収体を融解液中に浸漬させることで、速やかに融解作業を行うことができる。 The method for cryopreserving cells or tissues using the vitrification cryopreservation jig of the present invention is not particularly limited. For example, first, a vitrification solution absorber comprising cells or tissues immersed in a vitrification solution together with the vitrification solution The vitrification solution adhering to the periphery of the cell or the tissue is dropped onto the vitrification solution absorber. Next, the cells or the tissues can be frozen by immersing them in liquid nitrogen or the like while being held on the vitrification liquid absorber. As the vitrification solution, those usually used for freezing cells such as eggs and embryos can be used. For example, the above-mentioned cryoprotectant such as glycerol, ethylene glycol, DMSO (dimethyl sulfoxide) or more is 30% by volume or more. Preferably, an aqueous solution containing 30 to 40% by volume of an antifreezing agent can be preferably used. At the time of thawing work, the vitrification cryopreservation jig is taken out from a cooling solvent such as liquid nitrogen, and the vitrified liquid absorber on which the frozen cells or tissues are placed is immersed in the melt. The melting operation can be performed quickly.
　本発明のガラス化凍結保存用治具を用いて凍結保存することができる細胞として、例えば、哺乳類（例えば、人（ヒト）、牛、豚、馬、ウサギ、ラット、マウス等）の卵子または胚、精子等の生殖細胞、ｉＰＳ細胞、ＥＳ細胞が挙げられる。また、初代培養細胞、継代培養細胞、及び細胞株細胞等の培養細胞が挙げられる。また、細胞は、一または複数の実施形態において、繊維芽細胞、膵ガン・肝ガン細胞等のガン由来細胞、上皮細胞、血管内皮細胞、リンパ管内皮細胞、神経細胞、軟骨細胞、組織幹細胞、胚性幹細胞、及び免疫細胞等の接着性細胞が挙げられる。さらに、凍結保存することができる組織として、同種または異種の細胞からなる組織、例えば、卵巣、皮膚、角膜上皮、歯根膜、心筋、軟骨等の組織が挙げられる。本発明のガラス化凍結保存用治具は、直接生体から採取した細胞または組織だけでなく、例えば、生体外で培養し増殖させた培養皮膚、生体外で構築した所謂細胞シート、特開２０１２－２０５５１６号公報に記載されている三次元構造を有する組織モデルのような人工の組織のガラス化凍結保存においても好適に用いることができる。 Examples of cells that can be cryopreserved using the vitrification cryopreservation jig of the present invention include, for example, mammals (eg, humans, cows, pigs, horses, rabbits, rats, mice, etc.) eggs or embryos. And germ cells such as sperm, iPS cells, and ES cells. Moreover, cultured cells, such as a primary cultured cell, a subcultured cell, and a cell line cell, are mentioned. In one or a plurality of embodiments, the cells are fibroblasts, cancer-derived cells such as pancreatic cancer / hepatoma cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, Examples include embryonic stem cells and adherent cells such as immune cells. Furthermore, examples of tissues that can be cryopreserved include tissues composed of allogeneic or heterogeneous cells, such as tissues such as ovary, skin, corneal epithelium, periodontal ligament, myocardium, and cartilage. The vitrification cryopreservation jig of the present invention is not limited to cells or tissues collected directly from a living body, but also, for example, cultured skin grown and grown in vitro, a so-called cell sheet constructed in vitro, It can also be suitably used in vitrified cryopreservation of an artificial tissue such as a tissue model having a three-dimensional structure described in JP-A-205516.
　以下に本発明を実施例によりさらに詳細に示し、本発明を具体的に説明するが、本発明は以下の実施例に限定されるものではない。なお、実施例１～４の細孔径は、表面及び断面の画像観察から測定した平均細孔直径を示す。また、比較例１、比較例４の細孔径は、ＪＩＳ　Ｋ３８３２に記載されるバブルポイント試験により測定される最も大きい細孔の直径を示す。 Hereinafter, the present invention will be described in more detail with reference to examples, and the present invention will be specifically described. However, the present invention is not limited to the following examples. The pore diameters of Examples 1 to 4 indicate average pore diameters measured from image observation of the surface and cross section. Moreover, the pore diameter of Comparative Example 1 and Comparative Example 4 indicates the diameter of the largest pore measured by the bubble point test described in JIS K3832.
（実施例１）
　多孔質焼結形成体として、ポリエチレン樹脂からなる多孔質焼結形成体である旭化成社製のサンファインＡＱ８００（細孔径３５μｍ、空隙率４３％、厚み５００μｍ）をガラス化液吸収体とし、このガラス化液吸収体５００ｍｍ２（１０ｍｍ×５０ｍｍ）をＡＢＳ樹脂製の把持部と接合させ、図１に示す形態で実施例１のガラス化凍結保存用治具を作製した。 Example 1
As the porous sintered compact, Sunfine AQ800 (pore diameter 35 μm, porosity 43%, thickness 500 μm) manufactured by Asahi Kasei Co., Ltd., which is a porous sintered compact made of polyethylene resin, is used as a vitrified liquid absorber. The vitrification solution absorber 500 mm 2 (10 mm × 50 mm) was joined to a gripping part made of ABS resin, and the vitrification cryopreservation jig of Example 1 was produced in the form shown in FIG.
（実施例２）
　多孔質焼結形成体として、セラミックスの多孔質焼結形成体であるコバレントマテリアル社製の石英ガラス多孔体（細孔径３．５μｍ、空隙率３３％、厚み２ｍｍ）をガラス化液吸収体とし、このガラス化液吸収体５００ｍｍ２（１０ｍｍ×５０ｍｍ）をＡＢＳ樹脂製の把持部と接合させ、図１に示す形態で実施例２のガラス化凍結保存用治具を作製した。 (Example 2)
As a porous sintered body, a quartz glass porous body (pore diameter 3.5 μm, porosity 33%, thickness 2 mm) manufactured by Covalent Materials, which is a ceramic porous sintered body, is used as a vitrified liquid absorber. This vitrified liquid absorber 500 mm 2 (10 mm × 50 mm) was joined to a gripping part made of ABS resin, and a vitrification cryopreservation jig of Example 2 was produced in the form shown in FIG.
（比較例１）
　多孔質焼結形成体にかえて、セルロースアセテートからなるアドバンテック社製セルロースアセテートメンブレン（細孔径０．５μｍ、空隙率６８％、膜厚１２５μｍ）をガラス化液吸収体とした。メンブレンが薄く、自立性が十分でないことから、メンブレンは厚み２５０μｍのＰＥＴフィルム上にウレタン系接着物質を用いて貼り付け、実施例１と同様にして比較例１のガラス化凍結保存用治具を作製した。 (Comparative Example 1)
Instead of the porous sintered body, a cellulose acetate membrane made of cellulose acetate (pore size 0.5 μm, porosity 68%, film thickness 125 μm) was used as a vitrification liquid absorber. Since the membrane is thin and the self-supporting property is not sufficient, the membrane is pasted onto a PET film having a thickness of 250 μm using a urethane-based adhesive substance, and the vitrification cryopreservation jig of Comparative Example 1 is used in the same manner as in Example 1. Produced.
（比較例２）
　濾紙であるアドバンテック社製濾紙Ｎｏ．５Ｃ（坪量１２０ｇ／ｍ２、密度０．５７ｇ／ｃｍ３）をガラス化液吸収体とした以外は比較例１と同様にして、比較例２のガラス化凍結保存用治具を作製した。 (Comparative Example 2)
Filter paper No. 1 manufactured by Advantech, which is a filter paper. A jig for vitrification cryopreservation of Comparative Example 2 was produced in the same manner as Comparative Example 1 except that 5C (basis weight 120 g / m 2 , density 0.57 g / cm 3 ) was used as the vitrification liquid absorber.
（比較例３）
　特許文献８に記載のある細胞の運搬補助膜であるセルロースからなるセルシード社製Ｃｅｌｌ　Ｓｈｉｆｔｅｒをガラス化液吸収体とした以外は比較例１と同様にして、比較例３のガラス化凍結保存用治具を作製した。 (Comparative Example 3)
The treatment for vitrification cryopreservation of Comparative Example 3 was performed in the same manner as Comparative Example 1 except that Cell Shifter made of Cellulose, which is a cell transport auxiliary membrane described in Patent Document 8, was used as a vitrification solution absorber. A tool was prepared.
（比較例４）
　特許文献８に記載のある細胞の運搬補助膜であるＰＶＤＦからなるメルクミリポア社製の親水性デュラポアメンブレン（細孔径０．１μｍ、空隙率７０％、膜厚１２５μｍ）をガラス化液吸収体とした以外は比較例１と同様にして、比較例４のガラス化凍結保存用治具を作製した。 (Comparative Example 4)
A hydrophilic durapore membrane (pore diameter 0.1 μm, porosity 70%, film thickness 125 μm) made of Merck Millipore, made of PVDF, which is a cell transport auxiliary membrane described in Patent Document 8, is used as a vitrification solution absorber. A jig for vitrification cryopreservation of Comparative Example 4 was produced in the same manner as Comparative Example 1 except that.
（比較例５）
　無色透明なフィルムである透明ＰＥＴフィルム（空隙率０％、厚さ２５０μｍ）をそのまま用いてガラス化液吸収体とした以外は実施例１と同様にして、比較例５のガラス化凍結保存用治具を作製した。 (Comparative Example 5)
The vitrification cryopreservation treatment of Comparative Example 5 was carried out in the same manner as in Example 1 except that a transparent PET film (porosity 0%, thickness 250 μm), which was a colorless transparent film, was used as it was to obtain a vitrification liquid absorber. A tool was prepared.
＜ガラス化液の吸収性の評価＞
　実施例１、実施例２及び比較例１～５の各ガラス化凍結保存用治具のガラス化液吸収体上に、ガラスビーズ（直径１００μｍ）を疑似細胞として複数個含むガラス化液２００μＬを滴下付着させた。なお、ガラス化液は、シグマアルドリッチ社製　修正ＴＣＭ１９９培地に、２０容積％血清、１５容積％ＤＭＳＯ、１５容積％エチレングリコール、０．２容積％スクロースが含まれる組成のものを用いた。滴下付着後、落射型正立光学顕微鏡（オムロン（株）製、ＶＣ４５００－Ｓ１）にて、ガラス化液吸収体上に載置された疑似細胞周辺のガラス化液が吸収される様子を観察し、以下の基準で評価した。これらの結果を、表１の「ガラス化液の吸収性の評価」の項目に示す。 <Evaluation of absorbability of vitrification solution>
200 μL of a vitrification solution containing a plurality of glass beads (diameter: 100 μm) as pseudo cells is dropped on the vitrification solution absorber of each vitrification cryopreservation jig of Example 1, Example 2 and Comparative Examples 1 to 5. Attached. The vitrification solution used was a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich. After dropping and adhering, the state where the vitrification solution around the pseudo cell placed on the vitrification solution absorber was absorbed with an epi-illuminated optical microscope (OM4, VC4500-S1) was observed. The evaluation was based on the following criteria. These results are shown in the item “Evaluation of absorbability of vitrification solution” in Table 1.
○：ガラス化液滴下付着後、１０秒以内にガラス化液が吸収された。
×：ガラス化液滴下付着後、１０秒以内にガラス化液の吸収が十分でなく、疑似細胞周辺にガラス化液の残存が確認された。 ○: The vitrification solution was absorbed within 10 seconds after adhering under the vitrification droplet.
X: Absorption of the vitrification solution was not sufficient within 10 seconds after adhering under the vitrification droplet, and the vitrification solution remained around the pseudo cell.
＜冷却溶媒への浸漬評価＞
　実施例１、実施例２及び比較例１～５の各ガラス化凍結保存用治具のガラス化液吸収体上に、ガラスビーズ（直径１００μｍ）を疑似細胞として複数個含むガラス化液１００μＬを滴下付着させた。なお、ガラス化液は、シグマアルドリッチ社製　修正ＴＣＭ１９９培地に、２０容積％血清、１５容積％ＤＭＳＯ、１５容積％エチレングリコール、０．２容積％スクロースが含まれる組成のものを用いた。滴下付着させて１０秒後に、ガラス化液吸収体を完全に液体窒素中に浸漬させた。１分間の浸漬後、ガラス化凍結保存用治具を液体窒素中からとり出し、室温環境下においた。液体窒素浸漬前と比較して、ガラス化液吸収体の変形や破損が無いかを目視で観察し、実用上の耐久性を以下の基準で評価した。これらの結果を、表１の「冷却溶媒への浸漬評価」の項目に示す。 <Evaluation of immersion in cooling solvent>
100 μL of a vitrification solution containing a plurality of glass beads (diameter: 100 μm) as pseudo cells is dropped on the vitrification absorber of each vitrification cryopreservation jig of Examples 1, 2 and Comparative Examples 1 to 5. Attached. The vitrification solution used was a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich. Ten seconds after dropping and adhering, the vitrification liquid absorber was completely immersed in liquid nitrogen. After immersion for 1 minute, the vitrification cryopreservation jig was taken out of liquid nitrogen and placed in a room temperature environment. Compared to before immersion in liquid nitrogen, the vitrification solution absorber was visually observed for deformation and breakage, and practical durability was evaluated according to the following criteria. These results are shown in the item “Evaluation of immersion in cooling solvent” in Table 1.
○：ガラス化液吸収体の変形や破損が一切認められなかった。
×：ガラス化液吸収体に変形または破損が認められた。または、一連の評価の工程の中で、変形または破損等の発生により、作業の不具合が認められた。 ○: No deformation or breakage of the vitrification liquid absorber was observed.
X: Deformation or breakage was observed in the vitrified liquid absorber. Or, in a series of evaluation processes, work defects were recognized due to the occurrence of deformation or breakage.
Figure JPOXMLDOC01-appb-T000001
  
Figure JPOXMLDOC01-appb-T000001
  
　本発明の実施例１、実施例２のガラス化凍結保存用治具は、いずれも優れたガラス化液吸収性能を示した。一方、比較例１～５のガラス化凍結保存用治具はガラス化液吸収性能が不十分であった。このうち比較例５を除いた比較例１～４のガラス化凍結保存用治具は、若干のガラス化液の吸収は見られたものの、疑似細胞周辺のガラス化液が１０秒以内には吸収されず、残存する様子が見られた。 The vitrification cryopreservation jigs of Examples 1 and 2 of the present invention both showed excellent vitrification solution absorption performance. On the other hand, the vitrification cryopreservation jigs of Comparative Examples 1 to 5 had insufficient vitrification solution absorption performance. Among these, the vitrification cryopreservation jigs of Comparative Examples 1 to 4 except Comparative Example 5 showed some absorption of the vitrification solution, but the vitrification solution around the pseudo cell was absorbed within 10 seconds. It was not seen, and it was seen that it remained.
　また、本発明の実施例１、実施例２、比較例２～５のガラス化凍結保存用治具は、液体窒素に浸漬した場合において変形や破損は認められず、一連の凍結保存操作において不具合は認められなかった。一方で、比較例１のガラス化凍結保存用治具では、液体窒素浸漬後にガラス化液吸収体に破損が認められた。 In addition, the vitrification cryopreservation jigs of Examples 1, 2 and Comparative Examples 2 to 5 of the present invention were not deformed or damaged when immersed in liquid nitrogen, and failed in a series of cryopreservation operations. Was not recognized. On the other hand, in the vitrification cryopreservation jig of Comparative Example 1, damage to the vitrification liquid absorber was observed after immersion in liquid nitrogen.
（実施例３）
　多孔質金属体として、太盛工業社製のステンレス多孔質体（細孔径１．５μｍ、空隙率６５容量％、厚み１ｍｍ）をガラス化液吸収体とし、このガラス化液吸収体５００ｍｍ２（１０ｍｍ×５０ｍｍ）をＡＢＳ樹脂製の把持部と接合させ、図１に示す形態で実施例３のガラス化凍結保存用治具を作製した。 Example 3
As the porous metal body, a stainless porous body (pore diameter 1.5 μm, porosity 65 volume%, thickness 1 mm) manufactured by Taisei Kogyo Co., Ltd. was used as the vitrification liquid absorber, and this vitrification liquid absorber 500 mm 2 (10 mm × 50 mm) was joined to a gripping part made of ABS resin, and the jig for vitrification cryopreservation of Example 3 was produced in the form shown in FIG.
（実施例４）
　多孔質金属体として、太盛工業社製のステンレス多孔質体（細孔径８μｍ、空隙率６５容量％、厚み１ｍｍ）をガラス化液吸収体とし、このガラス化液吸収体５００ｍｍ２（１０ｍｍ×５０ｍｍ）をＡＢＳ樹脂製の把持部と接合させ、図１に示す形態で実施例４のガラス化凍結保存用治具を作製した。 Example 4
As a porous metal body, a stainless porous body (pore diameter: 8 μm, porosity: 65 volume%, thickness: 1 mm) made by Taisei Kogyo Co., Ltd. was used as a vitrification liquid absorber, and this vitrification liquid absorber 500 mm 2 (10 mm × 50 mm). ) Was bonded to a gripping part made of ABS resin, and a jig for vitrification cryopreservation of Example 4 was produced in the form shown in FIG.
＜ガラス化液の吸収性の評価＞
　実施例３、実施例４及び前記した比較例１～５の各ガラス化凍結保存用治具のガラス化液吸収体上に、先のガラス化液の吸収性の評価と同様にして、ガラスビーズ（直径１００μｍ）を疑似細胞として複数個含むガラス化液２００μＬを滴下付着させた。滴下付着後、落射型正立光学顕微鏡（オムロン（株）製、ＶＣ４５００－Ｓ１）にて、ガラス化液吸収体上に載置された疑似細胞周辺のガラス化液が吸収される様子を観察し、以下の基準で評価した。これらの結果を、表２の「ガラス化液の吸収性の評価」の項目に示す。 <Evaluation of absorbability of vitrification solution>
On the vitrification absorber of each of the vitrification cryopreservation jigs of Example 3, Example 4 and Comparative Examples 1 to 5 described above, glass beads were used in the same manner as in the evaluation of the absorptivity of the previous vitrification solution. 200 μL of a vitrification solution containing a plurality of (diameter: 100 μm) as pseudo cells was dropped and adhered. After dropping and adhering, the state where the vitrification solution around the pseudo cell placed on the vitrification solution absorber was absorbed with an epi-illuminated optical microscope (OM4, VC4500-S1) was observed. The evaluation was based on the following criteria. These results are shown in the item “Evaluation of Absorbability of Vitrification Solution” in Table 2.
○：ガラス化液滴下付着後、５秒以内にガラス化液が完全に吸収された。
×：ガラス化液滴下付着後、５秒以内にガラス化液の吸収が十分でなく、疑似細胞周辺にガラス化液の残存が確認された。 ○: The vitrification solution was completely absorbed within 5 seconds after adhering under the vitrification droplet.
X: Absorption of the vitrification solution was insufficient within 5 seconds after adhering under the vitrification droplet, and the vitrification solution remained around the pseudo cell.
＜冷却溶媒への浸漬評価＞
　実施例３、実施例４及び前記した比較例１～５について、先の冷却溶媒への浸漬評価と同様にして、各ガラス化凍結保存用治具のガラス化液吸収体上に、ガラスビーズ（直径１００μｍ）を疑似細胞として複数個含むガラス化液１００μＬを滴下付着させた。滴下付着させて１０秒後に、ガラス化液吸収体を完全に液体窒素中に浸漬させた。１０分間の浸漬後、ガラス化凍結保存用治具を液体窒素中からとり出し、室温環境下においた。液体窒素浸漬前と比較して、ガラス化液吸収体の変形や破損が無いかを目視で観察し、実用上の耐久性を、先の冷却溶媒への浸漬評価と同様にして評価した。これらの結果を、表２の「冷却溶媒への浸漬評価」の項目に示す。 <Evaluation of immersion in cooling solvent>
For Example 3, Example 4 and Comparative Examples 1 to 5 described above, glass beads (on the vitrification absorber of each vitrification cryopreservation jig were used in the same manner as in the previous immersion evaluation in the cooling solvent. 100 μL of a vitrification solution containing a plurality of pseudo-cells having a diameter of 100 μm was added dropwise. Ten seconds after dropping and adhering, the vitrification liquid absorber was completely immersed in liquid nitrogen. After the immersion for 10 minutes, the vitrification cryopreservation jig was taken out of liquid nitrogen and placed in a room temperature environment. Compared to before immersion in liquid nitrogen, the vitrification solution absorber was visually observed for deformation and breakage, and practical durability was evaluated in the same manner as in the previous immersion evaluation in a cooling solvent. These results are shown in the item “Evaluation of immersion in cooling solvent” in Table 2.
Figure JPOXMLDOC01-appb-T000002
  
Figure JPOXMLDOC01-appb-T000002
  
　本発明の実施例３、実施例４のガラス化凍結保存用治具は、いずれも優れたガラス化液吸収性能を示した。一方、比較例１～５のガラス化凍結保存用治具は、滴下したほとんどのガラス化液が残存したことから、ガラス化液吸収性能が不十分であった。 The vitrification cryopreservation jigs of Examples 3 and 4 of the present invention both showed excellent vitrification solution absorption performance. On the other hand, the vitrification cryopreservation jigs of Comparative Examples 1 to 5 were insufficient in vitrification solution absorption performance because most of the dripped vitrification solution remained.
　また、本発明の実施例３、実施例４、比較例２～５のガラス化凍結保存用治具は、液体窒素に浸漬した場合において変形や破損は認められず、一連の凍結保存操作において不具合は認められなかった。一方で、比較例１のガラス化凍結保存用治具では、液体窒素浸漬後にガラス化液吸収体に破損が認められた。 In addition, the vitrification cryopreservation jigs of Examples 3 and 4 and Comparative Examples 2 to 5 of the present invention were not deformed or damaged when immersed in liquid nitrogen, and failed in a series of cryopreservation operations. Was not recognized. On the other hand, in the vitrification cryopreservation jig of Comparative Example 1, damage to the vitrification liquid absorber was observed after immersion in liquid nitrogen.
　以上の結果から、本発明のガラス化凍結保存用治具によって、細胞または組織のガラス化凍結作業を容易にかつ確実に行うことが可能であることが判る。 From the above results, it can be seen that the vitrification / freezing operation of the cells or tissues can be easily and reliably performed by the vitrification cryopreservation jig of the present invention.
　本発明は、牛などの家畜や動物の胚移植や人工授精、人への人工授精などの他、ｉＰＳ細胞、ＥＳ細胞、一般に用いられている培養細胞、生体から採取した検査用または移植用の細胞または組織、生体外で培養した細胞または組織などの凍結保存に用いることができる。 The present invention can be used for testing or transplanting from iPS cells, ES cells, commonly used cultured cells, living organisms, etc. in addition to embryo transfer or artificial insemination of humans such as cattle and animals, artificial insemination to humans, etc. It can be used for cryopreservation of cells or tissues, cells or tissues cultured in vitro, and the like.
１　把持部
２ａ　ガラス化液吸収体
２ｂ　ガラス化液吸収体
２ｃ　ガラス化液吸収体
２ｄ　ガラス化液吸収体
３　多孔質焼結形成体または多孔質金属体
４　支持体
５　ガラス化凍結保存用治具 DESCRIPTION OF SYMBOLS 1 Holding part 2a Vitrification liquid absorber 2b Vitrification liquid absorber 2c Vitrification liquid absorber 2d Vitrification liquid absorber 3 Porous sintering formation body or porous metal body 4 Support body 5 Vitrification jig for cryopreservation

Claims (6)

  1. 多孔質焼結形成体または多孔質金属体をガラス化液吸収体として有する、細胞または組織のガラス化凍結保存用治具。 A jig for freezing and preserving vitrification of cells or tissues, comprising a porous sintered body or a porous metal body as a vitrification liquid absorber.
  2. 前記多孔質焼結形成体が、熱可塑性樹脂の樹脂固体粉末または金属酸化物粒子を三次元構造を持たせたまま加熱、焼結し、該粉末表面または該粒子表面を融着させることによって得られる多孔質構造体である、請求項１に記載のガラス化凍結保存用治具。 The porous sintered compact is obtained by heating and sintering a resin solid powder or metal oxide particles of a thermoplastic resin with a three-dimensional structure, and fusing the powder surface or the particle surface. The jig for vitrification cryopreservation according to claim 1, which is a porous structure to be produced.
  3. 前記多孔質焼結形成体の細孔径が１μｍ以上である、請求項１または２に記載のガラス化凍結保存用治具。 The jig for vitrification cryopreservation according to claim 1 or 2, wherein the porous sintered compact has a pore diameter of 1 µm or more.
  4. 前記多孔質焼結形成体の空隙率が２０～８０％である、請求項１～３のいずれか一項に記載のガラス化凍結保存用治具。 The vitrification cryopreservation jig according to any one of claims 1 to 3, wherein the porosity of the porous sintered compact is 20 to 80%.
  5. 前記多孔質金属体の細孔径が０．０５～５００μｍである、請求項１に記載のガラス化凍結保存用治具。 The jig for vitrification cryopreservation according to claim 1, wherein the porous metal body has a pore diameter of 0.05 to 500 µm.
  6. 前記多孔質金属体の空隙率が２０％以上である、請求項１または５に記載のガラス化凍結保存用治具。
     
          The jig for vitrification cryopreservation according to claim 1 or 5, wherein the porosity of the porous metal body is 20% or more.

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