WO2015115313A1 - Gabarit pouvant être utilisé dans la cryoconservation par vitrification de cellules ou de tissus - Google Patents

Gabarit pouvant être utilisé dans la cryoconservation par vitrification de cellules ou de tissus 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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Prior art keywords
vitrification
cells
jig
cryopreservation
solution
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PCT/JP2015/051770
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English (en)
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/ja
Priority claimed from JP2014069295A external-priority patent/JP2015188404A/ja
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/fr

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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

Cette invention concerne un gabarit pouvant être utilisé dans la cryoconservation par vitrification et permet d'effectuer une opération de cryoconservation par vitrification de cellules ou de tissus de manière facile et fiable. Un gabarit pouvant être utilisé dans la cryoconservation par vitrification, qui comporte un corps moulé fritté poreux ou un corps métallique poreux à titre d'absorbeur de solution de vitrification, est en outre décrit. Selon la présente invention, une étape supplémentaire consistant à éliminer une partie excédentaire de la solution de vitrification n'est pas particulièrement requise quand les cellules ou les tissus immergés dans la solution de vitrification sont placés sur un absorbeur de solution de vitrification avec la solution de vitrification, et par conséquent l'opération de cryoconservation de cellules ou de tissus peut être effectuée de manière facile et simple.
PCT/JP2015/051770 2014-01-31 2015-01-23 Gabarit pouvant être utilisé dans la cryoconservation par vitrification de cellules ou de tissus WO2015115313A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016063806A1 (fr) * 2014-10-23 2016-04-28 三菱製紙株式会社 Outil pour la cryoconservation d'une cellule ou d'un tissu et procédé de cryoconservation
CN108925549A (zh) * 2018-08-10 2018-12-04 中国科学院深圳先进技术研究院 一种细胞冻存载杆及其制备方法和应用
CN109006806A (zh) * 2018-10-23 2018-12-18 南京艾维艾康生物技术有限公司 一种组织冷存管
WO2019004300A1 (fr) * 2017-06-30 2019-01-03 株式会社北里コーポレーション Outil de cryoconservation de cellules biologiques et outil pour cryoconservation de cellules biologiques
JP2021114963A (ja) * 2020-01-28 2021-08-10 三菱製紙株式会社 凍結保存用治具の固定具
CN113575572A (zh) * 2021-08-19 2021-11-02 上海理工大学 一种血管玻璃化保存的超快速复温方法
EP3796779A4 (fr) * 2018-05-22 2022-03-23 Mitegen, LLC Système de refroidissement et de réchauffement rapides de cellules et d'autres matériaux biologiques
WO2022065499A1 (fr) * 2020-09-28 2022-03-31 テルモ株式会社 Dispositif de transfert de greffon congelé et procédé de transfert de greffon congelé
CN115462370A (zh) * 2022-11-03 2022-12-13 能科达(上海)干细胞研究中心有限公司 一种用于保存脐带近全能干细胞的凝胶溶液及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003047464A (ja) * 2001-08-02 2003-02-18 Institute Of Tsukuba Liaison Co Ltd 動物細胞の保存方法
JP2005040073A (ja) * 2003-07-23 2005-02-17 Yoshinori Fukuda 生殖細胞の凍結保存方法及び凍結保存容器
JP2009195682A (ja) * 2008-01-21 2009-09-03 Gc Corp 多孔質細胞支持体及びその製造方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003047464A (ja) * 2001-08-02 2003-02-18 Institute Of Tsukuba Liaison Co Ltd 動物細胞の保存方法
JP2005040073A (ja) * 2003-07-23 2005-02-17 Yoshinori Fukuda 生殖細胞の凍結保存方法及び凍結保存容器
JP2009195682A (ja) * 2008-01-21 2009-09-03 Gc Corp 多孔質細胞支持体及びその製造方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIROMITSU SANO ET AL.: "Saibogai Yoeki o Kyushu suru Membrane Filter-jo deno Buta Taigai Seijuku Ranshi no Glass-ka Hozon", THE JAPANESE SOCIETY FOR ANIMAL SCIENCES DAI 106 KAI TAIKAI KOEN YOSHI, 20 March 2006 (2006-03-20), pages 83 *
TAKU ANAN ET AL.: "Direct Ishoku no Dekiru Ushi Haibanho no Gaishu Glass-ka Eki Kyushu Jokyo Glass-kaho", THE JAPANESE SOCIETY FOR ANIMAL SCIENCES DAI 112 KAI TAIKAI KOEN YOSHI, 28 March 2010 (2010-03-28), pages 88 *

Cited By (15)

* Cited by examiner, † Cited by third party
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WO2016063806A1 (fr) * 2014-10-23 2016-04-28 三菱製紙株式会社 Outil pour la cryoconservation d'une cellule ou d'un tissu et procédé de cryoconservation
US10624335B2 (en) 2014-10-23 2020-04-21 Mitsubishi Paper Mills Limited Tool for cryopreservation of cell or tissue and cryopreservation method
JPWO2019004300A1 (ja) * 2017-06-30 2020-04-30 株式会社北里コーポレーション 生体細胞凍結保存具および生体細胞凍結保存用具
JP7108318B2 (ja) 2017-06-30 2022-07-28 株式会社北里コーポレーション 生体細胞凍結保存具および生体細胞凍結保存用具
WO2019004300A1 (fr) * 2017-06-30 2019-01-03 株式会社北里コーポレーション Outil de cryoconservation de cellules biologiques et outil pour cryoconservation de cellules biologiques
EP3796779A4 (fr) * 2018-05-22 2022-03-23 Mitegen, LLC Système de refroidissement et de réchauffement rapides de cellules et d'autres matériaux biologiques
US11653644B2 (en) 2018-05-22 2023-05-23 Mitegen, Llc System for rapid cooling and warming of cells and other biological material
CN108925549B (zh) * 2018-08-10 2021-03-26 中国科学院深圳先进技术研究院 一种细胞冻存载杆及其制备方法和应用
CN108925549A (zh) * 2018-08-10 2018-12-04 中国科学院深圳先进技术研究院 一种细胞冻存载杆及其制备方法和应用
CN109006806A (zh) * 2018-10-23 2018-12-18 南京艾维艾康生物技术有限公司 一种组织冷存管
JP2021114963A (ja) * 2020-01-28 2021-08-10 三菱製紙株式会社 凍結保存用治具の固定具
JP7471836B2 (ja) 2020-01-28 2024-04-22 三菱製紙株式会社 凍結保存用治具の固定具
WO2022065499A1 (fr) * 2020-09-28 2022-03-31 テルモ株式会社 Dispositif de transfert de greffon congelé et procédé de transfert de greffon congelé
CN113575572A (zh) * 2021-08-19 2021-11-02 上海理工大学 一种血管玻璃化保存的超快速复温方法
CN115462370A (zh) * 2022-11-03 2022-12-13 能科达(上海)干细胞研究中心有限公司 一种用于保存脐带近全能干细胞的凝胶溶液及其应用

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