JP2018121581A - Tool for cryopreservation of cell or tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a tool for cryopreservation that makes it possible to cryopreserve a cell or tissue easily and reliably.SOLUTION: A tool for cryopreservation of a cell or tissue has a layer containing at least a water-soluble polymer compound and glycerol on the outermost surface of a mounting part for mounting a cell or tissue on.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cryopreservation jig used for cryopreserving cells or tissues.

  Excellent preservation technology for cells or tissues is required in various industrial fields. For example, in the bovine embryo transfer technique, the embryo is cryopreserved, and the embryo is thawed and transferred in accordance with the estrous cycle of the recipient cow. Moreover, in human infertility treatment, after collecting an egg or ovary from a mother body, it is stored frozen in order to match the timing suitable for transplantation, and thawed at the time of transplantation.

  In general, since cells or tissues collected from within a living body gradually lose their activity even in a culture solution, long-term culture of cells or tissues outside a living body is not preferable. Therefore, a technique for storing for a long time without losing biological activity is important. Excellent preservation techniques allow for more accurate analysis of harvested cells or tissues. In addition, excellent storage technology makes it possible to use cells or tissues for transplantation while maintaining higher biological activity, and it can be expected that the survival rate after transplantation is improved. Furthermore, it becomes possible to produce and store artificial tissues for transplantation, such as cultured skin cultured in vitro and so-called cell sheets constructed in vitro, and use them when necessary. Great benefits can be expected not only in the medical field but also in the industrial field.

  As a method for preserving cells or tissues, for example, a slow freezing method is known. In this method, cells or tissues are first immersed in a preservation solution obtained by adding a freezing-resistant agent to a physiological solution such as phosphate buffered saline. As the antifreeze agent, compounds such as glycerin and ethylene glycol are used. After immersing 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), or inside or outside the cells or tissues. The solution inside and outside is sufficiently cooled and the viscosity becomes high. In this 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 without being amorphous. Vitrification occurs. When the inside or outside of the cell or the inside or outside of the 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.

  However, in the slow freezing method, it is necessary to cool at a relatively slow cooling rate, so that an operation for cryopreservation takes time. In addition, there is a problem of requiring a device or a jig for controlling the cooling rate. 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.

  As a method for solving the problems in the slow freezing method, a vitrification preservation method has been proposed. The vitrification preservation method is based on the principle that ice crystals are hardly formed even when the temperature is below freezing point due to the freezing point depression of an aqueous solution containing a large amount of antifreezing agents such as glycerin, ethylene glycol and DMSO (dimethyl sulfoxide). 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 (preservation solution) containing a large amount of antifreezing agent is called vitrification solution.

  As a specific operation of the vitrification method, cells or tissues are immersed in a vitrification solution, and then cooled at the temperature of liquid nitrogen (−196 ° C.). Since the vitrification method is such a simple and rapid process, it does not require a long time for the operation for cryopreservation, and also has the advantage of not requiring a device or jig for temperature control. is there.

  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. High concentrations of antifreeze are chemically toxic. For this reason, when the cells or tissues are cryopreserved, it is preferable that there is less vitrification solution around the cells or tissues, and the time for which the cells are exposed to the vitrification solution, that is, the time until freezing is short. preferable. Furthermore, it is necessary to dilute the vitrification solution immediately after thawing.

  Regarding cryopreservation of cells or tissues using the vitrification preservation method, examples using various types of cells or tissues are shown by various methods. For example, Patent Document 1 shows that application of vitrification preservation methods 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 technology 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, it is known that the vitrification preservation method is useful for preservation of a wide variety of cells and tissues.

  In Patent Documents 3 and 4, the so-called cryotop method used in the field of human infertility treatment is a method for cryopreserving eggs using a strip-like flexible, colorless and transparent film as a strip for holding eggs attached. There has been proposed a method in which an egg or embryo is placed on the film together with a very small amount of vitrification solution under a microscope, and cryopreserved using a tool.

  In patent document 5, an ovum or an embryo is placed on a removal material for vitrification preservation together with a vitrification solution, and an extra vitrification solution adhering to the periphery of the ovum or embryo is removed by aspiration from the lower part, which is excellent. A method of cryopreserving the survival rate has been 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. Further, as a cryopreservation jig capable of removing excess vitrification liquid and improving workability when placing cells, for example, a vitrification liquid absorber having a specific haze value is disclosed in Patent Document 6. Further, Patent Document 7, Patent Document 8 and the like describe vitrification cryopreservation having a porous sintered body or a porous structure formed of a material having a specific refractive index as a vitrification liquid absorber. The jig for use is described.

  Furthermore, in Patent Document 9, after cells or tissues are placed in a cryopreservation jig together with a preservation solution and cryopreserved, the cells or tissues are easily detached and collected when the cells or tissues are thawed. As a cryopreservation jig that can be used, a cryopreservation jig in which a layer containing a water-soluble polymer compound is provided on the outermost surface of a placement part on which cells or tissues are placed is described.

Japanese Patent No. 3443323 JP 2008-5846 A JP 2002-315573 A JP 2006-271395 A International Publication No. 2011/077093 Pamphlet JP 2014-183757 A JP-A-2015-142523 International Publication No. 2015/064380 Pamphlet International Publication No. 2016/063806 Pamphlet

Steponkus et al. , Nature 345: 170-172 (1990).

  According to the cryopreservation jig of Patent Document 9 described above, even when the placement part for placing the cell or tissue has a vitrified liquid absorber, when the cell or tissue is thawed. In addition, it is possible to easily collect cells or tissues. However, sufficient peelability may not be obtained depending on the cell or tissue to be cryopreserved. Further, according to Patent Document 9, when the layer containing a water-soluble polymer compound further contains inorganic fine particles, it has a good releasability when the cells or tissues are melted, and the cells or tissues are easily Can be recovered. However, depending on the obstruction of the visual operation due to mixing of the dropped inorganic fine particles in the melt, or depending on the method of using the cells or tissues after freezing / thawing, the inorganic fine particles adhering to the cells or tissues are subsequently washed. May be required. Under these circumstances, further improvements have been demanded.

  The main object of the present invention is to provide a cell or tissue cryopreservation jig capable of easily and reliably performing a cell or tissue cryopreservation operation. More specifically, the cell or tissue is immersed in a vitrification solution, and the cell or tissue is placed on the vitrification cryopreservation jig together with the vitrification solution and cryopreserved, and then the cell or tissue is thawed. In this case, it is a first object to provide a cryopreservation jig that can easily peel and collect cells or tissues. Another object is to provide a cryopreservation jig having excellent absorbability of vitrification liquid in addition to the ability to easily collect the cells or tissues described above.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have found that a cell or tissue cryopreservation jig having the following configuration (in this specification, “cell or tissue cryopreservation jig” It was found that the above-mentioned problem can be solved simply by “a jig for cryopreservation”).

(1) A jig for cryopreservation of a cell or tissue, comprising a layer containing at least a water-soluble polymer compound and glycerin on the outermost surface of a placement portion on which the cell or tissue is placed.
(2) The cell according to (1) above, wherein the placement part has a preservation solution absorber, and has a layer containing at least a water-soluble polymer compound and glycerin on the preservation solution absorber. Or a jig for cryopreservation of tissue.

  According to the invention of (1) above, cells or tissues are immersed in a vitrification solution, and the cells or tissues are placed on the vitrification cryopreservation jig together with the vitrification solution and cryopreserved. When the tissue is thawed, it is possible to provide a cryopreservation jig that can easily collect cells or tissues. Furthermore, according to the invention of the above (2), in addition to being able to easily peel and collect the cells or tissues described above, it is possible to provide a cryopreservation jig having an excellent absorbability of vitrification solution.

FIG. 1 is an overall view showing an example of a jig for cryopreservation of cells or tissues of the present invention. FIG. 2 is a schematic sectional view of the mounting portion in FIG. FIG. 3 is a schematic cross-sectional structure diagram when the mounting portion shown in FIG. 1 has a storage liquid absorber. FIG. 4 is a schematic cross-sectional structure diagram illustrating another example in the case where the placement unit illustrated in FIG. 1 includes a storage liquid absorber. FIG. 5 is a schematic cross-sectional structure diagram illustrating another example in the case where the placement unit illustrated in FIG. 1 includes a storage liquid absorber.

  The cryopreservation jig of the present invention is used when cryopreserving cells or tissues. In this specification, the cell includes not only a single cell but also a cell population of an organism 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 the present invention is used for cryopreservation work, and is preferably used for vitrification cryopreservation work. Specifically, the cryopreservation jig of the present invention is for immersing a cryopreservation jig on which cells or tissues are placed in a cooling solvent such as liquid nitrogen to freeze it. When the cells or tissues placed on the cryopreservation jig are thawed, the cells or tissues are taken out of the cooling medium together with the cryopreservation jig and immersed in a melting solution to be thawed. When the jig for cryopreservation according to the present invention is used, the cell or tissue can be securely held when placed, and the cell or tissue can be easily recovered at the time of thawing. Can be easily and reliably performed. The jig for cryopreservation of the present invention can be restated as a cell or tissue cryopreservation tool, a cell or tissue preservation tool, a cell or tissue cryopreservation instrument, or a cell or tissue preservation instrument.

  The jig for cryopreservation of the present invention comprises a layer containing at least a water-soluble polymer compound and glycerin (hereinafter referred to as the surface layer or simply the surface layer of the present invention) on the outermost surface of the placement part for placing cells or tissues. Description). Such a surface layer is preferably soluble in the melt. Here, the term “soluble” means that the solubility of the surface layer in the melt at 25 ° C. is 0.2% by mass or more. As described above, the cryopreservation jig of the present invention is immersed in a refrigerant (for example, liquid nitrogen) and frozen after placing cells or tissues together with the preservation solution on the surface layer of the cryopreservation jig during the freezing operation. At the time of thawing, frozen cells or tissues are taken out together with a cryopreservation jig and immersed in a thawing solution to be thawed. In the present invention, when cells or tissues are thawed, all or part of the surface layer dissolves in the melt, so that the cells or tissues can be easily fixed without being fixed to the placement part of the cryopreservation jig. It is possible to peel off and collect.

  In the case where the placement portion of the cryopreservation jig of the present invention has a preservation solution absorber and further has the surface layer of the present invention on the outermost surface of the placement portion, in addition to the above effects, the preservation solution Since the absorbent body absorbs the extra storage solution, other operations for removing the extra storage solution are not particularly required, and the workability is remarkably improved. In addition, the cells or tissues thus manipulated are covered with a very small amount of preservation solution, and can be quickly frozen even when freezing. Furthermore, in the vitrification freezing method described above, chemical toxicity due to the preservation solution containing a large amount of antifreeze becomes a problem, but according to the cryopreservation jig in which the mounting portion has the preservation solution absorber. Since the vitrification solution around the placed cell or tissue becomes extremely small, the survival rate of the cell or tissue can be expected.

  Hereinafter, the configuration of the cryopreservation jig of the present invention will be described.

  The jig for cryopreservation of the present invention has a layer containing at least a water-soluble polymer compound and glycerin on the outermost surface of a placement part on which cells or tissues are placed. Here, the outermost surface of the placement portion on which the cells or tissues are placed corresponds to the surface portion on which the cells or tissues are placed together with the preservation solution. In the present invention, the mounting part preferably has the surface layer of the present invention on a non-absorbent support such as various resin films, metals, glass, rubber, etc., or on a preservation solution absorber, and in particular, a preservation solution absorber. It is preferable to have the surface layer of this invention on it. In the present invention, the surface layer does not need to form a uniform layer in the entire placement portion on which cells or tissues are placed, and may be, for example, a sea island shape or a stripe shape layer. That is, it is important that the surface layer of the present invention is present on the outermost surface of the place where the cell or tissue is placed together with the preservation solution, and the surface layer is present at the place where the cell or tissue is not placed. However, it does not have to exist.

  Examples of the water-soluble polymer compound of the surface layer of the present invention include cellulose derivatives such as hydroxyethyl cellulose and carboxymethyl cellulose, starch and derivatives thereof, gelatin, casein, alginic acid and salts thereof, polyvinyl alcohol, polyvinyl pyrrolidone, and styrene malee. Examples include acid copolymer salts and styrene / acrylic acid copolymer salts. Among them, polyvinyl alcohol, alginic acid and salts thereof, and gelatin are preferable because they can be dissolved in a preservation solution and have an appropriate film-forming action. Polyvinyl alcohol is a non-biological material and is also suitable for cells or tissues. Particularly preferred because of its low toxicity. These water-soluble polymer compounds may be used alone or in combination of two or more.

  The surface layer of the present invention contains glycerin. Since glycerin can be mixed with a water-soluble polymer compound with good compatibility, it can be easily peeled off from cells or tissues, and at the same time, can form a surface layer with excellent coating properties. The glycerin content of the surface layer of the present invention is preferably 5 to 50% with respect to the water-soluble polymer compound contained in the surface layer. When the content of glycerin in the surface layer exceeds 50%, a problem occurs in the formation of the film, and good peelability may not be stably obtained. On the other hand, if it is less than 5%, the solubility of the surface layer in the melt may be impaired, and the cells or tissues may not be easily detached when melted.

  The surface layer of the cryopreservation jig of the present invention can contain a crosslinking agent. In that case, it is desirable to use it in the range in which the solubility of the surface layer in the melt does not fall below 0.2% by mass.

  The surface layer possessed by the cryopreservation jig of the present invention can contain inorganic fine particles within a range where mixing into the melt is not remarkable. Specifically, the content is desirably 1% by mass or less with respect to the solid content mass of the surface layer.

Solid content of the surface layer cryopreservation jig has of the present invention is preferably 0.01 to 100 g / ^{m 2,} more preferably from 0.1 to 10 g / ^{m 2.} When the solid content of the surface layer exceeds 100 g / m ² , elution / mixing of components into the melt increases, which is not preferable. On the other hand, when the solid content of the surface layer is less than 0.01 g / m ² , the cells or tissues may not be easily detached when melting.

  In the present invention, the placing part has a support for the purpose of supporting cells or tissues when cryopreserved, and when the placing part does not have a preservation solution absorber, the support is a surface layer. Have Examples of such a support (non-absorbable support) include various resin films, metals, glass, rubber and the like. Such a support may be made of one kind of material or two or more kinds of materials. Among these, the resin film is preferably used from the viewpoint of handling. 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, polyacrylate resins, polychlorinated resins. Examples of the resin film include vinyl resin, polysulfone resin, polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, and cyclic polyolefin resin. In addition, it is preferable that the total light transmittance of the support is 80% or more because the cells or tissues placed on the placing portion can be easily confirmed using a transmission microscope. Furthermore, a metal support can be suitably used from the viewpoint of excellent temperature conductivity and enabling rapid freezing. Specific examples of the metal support include copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, iron, and stainless steel. The thickness of the above support is preferably 10 μm to 10 mm. Further, depending on the purpose, the surface of the support can be subjected to an easy adhesion treatment by an electrical method such as corona discharge treatment or a chemical method, and can also be roughened. Further, one or more other layers may be provided between the support and the surface layer, or one or more other layers may be provided on the back side of the support. Moreover, a surface layer can also be provided in the front and back of a support body. The support may be a storage liquid absorber described later.

  Next, the case where the cryopreservation jig of the present invention has a surface layer containing at least a water-soluble polymer compound and glycerin on the preservation solution absorber will be described.

  As described above, when the cell or tissue mounting portion of the present invention has a preservation solution absorber, the preservation solution absorber can remove the preservation solution even if the amount of the preservation solution attached to the cells or tissues is large. This eliminates the need for removing the preservative solution, and improves the workability. In addition, the cells or tissues thus manipulated are covered with a very small amount of a preservation solution, and can be quickly frozen even when freezing. Furthermore, in the vitrification freezing method described above, an aqueous solution containing a large amount of a freezing agent such as glycerin, ethylene glycol, DMSO (dimethyl sulfoxide) is used as a storage solution, and this storage solution has chemical toxicity due to a large amount of the freezing agent. However, according to the cryopreservation jig in which the placement part has a preservation solution absorber, the vitrification liquid around the placed cell or tissue becomes extremely small, and the viability of the cell or tissue is reduced. Improvement can be expected. Therefore, the cryopreservation jig having the surface layer on the preservation solution absorber is suitable as a vitrification cryopreservation jig.

  In the present invention, when the placement unit has a preservation solution absorber, it is preferable to provide a surface layer on the outermost surface of the placement unit without blocking the pores of the preservation solution absorber. In addition, a surface layer can be present inside the pores of the storage liquid absorber as long as the absorbency of the storage liquid absorber is not significantly reduced.

In the present invention, when the mounting portion has a storage liquid absorber, the larger the solid content of the surface layer, the narrower the pores of the storage liquid absorber, the tendency of the storage liquid absorbency to decrease. It is in. On the other hand, the peelability of the cells or tissues during the melting operation is more advantageous as the solid content of the surface layer increases. Although depending on the thickness, pore diameter, and porosity of the storage liquid absorber used, the solid content of the surface layer in the case where the mounting portion has the storage liquid absorber can be appropriately adjusted in consideration of the balance of the above performance, and preferably is set to a value of 0. 1 to 5 g / m ² .

  Examples of the preservation solution absorber included in the cryopreservation jig of the present invention include various sheets such as a fiber sheet, a porous resin sheet, a porous metal sheet, and a porous metal oxide sheet. “Porosity” in the present invention means that the above-mentioned sheet is a structure having pores (pores) on the surface, and is more preferably a structure having continuous pores on the sheet surface and inside. preferable. Moreover, it is preferable that the thickness of a preservation | save liquid absorber (above-mentioned various sheets) is 10 micrometers-5 mm, More preferably, they are 20 micrometers-2.5 mm. When the preservation solution absorber is a thin sheet, the above-described support can be used as the reinforcing member.

  In the present invention, the sheet made of fibers used as the preservation solution absorber is exemplified by paper or non-woven fabric, and the paper is preferably a paper whose ratio of the binder component such as a binder is 10% by mass or less, More preferably, it is 5% by mass or less, and more preferably 3% by mass or less. Thereby, excellent absorbability of the preservation solution is obtained. Further, the ratio of the papermaking chemicals contained in the paper to the whole paper is preferably 1% by mass or less. Among paper-making chemicals contained in normal paper, for example, fluorescent whitening agents, dyes, and cationic sizing agents may have a concern about the effect on cells.

When the sheet | seat which consists of fibers is paper, it is preferable that a density is 0.1-0.6 g / cm < ³ > and a basic weight is 10-130 g / m < ² > paper. In particular, a paper having a density of 0.12 to 0.3 g / cm ³ and a basis weight of 10 to 100 g / m ² is excellent in the absorbability of the storage solution, and further on the main body using a transmission microscope. It is preferable because it is possible to provide a cryopreservation jig having excellent cell or tissue visibility so that the cells or tissues placed on can be observed.

  When the sheet made of fibers is a nonwoven fabric, the fibers contained in the nonwoven fabric include cellulose fibers, rayon fibers and cupra fibers that are regenerated fibers made of cellulose fibers, and acetate fibers that are semi-synthetic fibers from cellulose fibers, Polyester fiber, nylon fiber, acrylic fiber, polypropylene fiber, polyethylene fiber, polyvinyl chloride fiber, vinylidene fiber, polyurethane fiber, vinylon fiber, glass fiber, silk fiber, etc. it can. Among these, cellulose fiber, cellulose fiber-derived rayon fiber and cupra fiber that are cellulose regenerated fibers, and acetate fiber that is semi-synthetic fiber from cellulose fibers are preferable.

When the sheet | seat which consists of fibers is a nonwoven fabric, it is preferable that a density is 0.1-0.4 g / cm < ³ > and a basic weight is 10-130 g / m < ² > nonwoven fabric. In particular, a non-woven fabric having a density of 0.12 to 0.3 g / cm ³ and a basis weight of 10 to 100 g / m ² is excellent in absorbability of a preservation solution and further in freezing excellent in cell or tissue visibility. This is preferable because a storage jig can be provided.

  In the non-woven fabric used as the storage liquid absorber, the non-woven fabric is preferably 10% by mass or less, more preferably 5% by mass or less, as in the case of the paper described above, in the binder component such as the binder. Furthermore, the nonwoven fabric which is 3 mass% or less is preferable. In particular, a non-woven fabric containing no binder is preferable.

  Non-woven fabrics have a variety of manufacturing methods, unlike paper, but the non-woven fabrics with the above-mentioned binder components reduced include non-woven fabrics manufactured by the spunbond method and melt-blow method, and fibers arranged by wet or dry methods. Thereafter, a nonwoven fabric produced by a hydroentanglement method or a needle punch method can be suitably used. As described above, preferred fibers contained in the nonwoven fabric in the present invention include cellulose fibers, fibers derived from cellulose fibers, rayon fibers and cupra fibers that are cellulose regenerated fibers, and acetate fibers that are semi-synthetic fibers from cellulose fibers. However, when these fibers are used for production, a hydroentanglement method or a needle punch method is suitable regardless of a wet method or a dry method.

  In the present invention, the porous resin sheet used as the storage liquid absorber is, for example, described in Japanese Patent Publication No. 42-13560 and Japanese Patent Application Laid-Open No. 08-283447, and is stretched at least in the uniaxial direction and is equal to or higher than the melting point of the resin. A resin sheet in which a porous structure is formed by a fine fibrous structure obtained by heating and sintering, a thermoplastic resin obtained by a method such as emulsion polymerization or pulverization described in JP-A-2009-235417 The resin sheet etc. which formed the porous structure by filling the solid powder of this in a metal mold | die, heating and sintering, fuse | melting the powder particle surface, and cooling are mentioned. When a porous resin sheet is used as a preservation solution absorber, it is preferable because it is possible to provide a cryopreservation jig having excellent preservation solution absorbability and excellent cell or tissue visibility.

  Examples of the resin forming the porous resin sheet include various types of polyethylene such as low density polyethylene, high density polyethylene, and ultrahigh molecular weight polyethylene, and fluorine such as polypropylene, polymethyl methacrylate, polystyrene, polytetrafluoroethylene, and polyvinyl difluoride. Examples thereof include resins, ethylene-vinyl acetate copolymers, polyamides, styrene-acrylonitrile copolymers, styrene-butadiene-acrylonitrile terpolymers, polycarbonates, and polyvinyl chloride. Among these, fluororesins such as polytetrafluoroethylene and polyvinylidene difluoride increase the light transmittance of the porous resin sheet when a cell or tissue is placed on a placement portion together with a vitrification solution. The visibility of cells or tissues below is dramatically improved, and it is possible to provide a cryopreservation jig that is particularly excellent in the visibility of cells or tissues. In addition, as the porous resin sheet, a membrane filter for filtration, which is commercially available for physics and chemistry experiment use or research use, can also be used.

  In the present invention, the porous metal sheet used as the storage liquid absorber includes copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, tin, zinc, lead, titanium, nickel, stainless steel, etc. Examples thereof include a porous metal sheet made of metal. As the porous metal oxide sheet, a porous metal oxide sheet made of a metal oxide such as silica, alumina, zirconium, quartz glass, or the like can be preferably used. The porous metal sheet and the porous metal oxide sheet may be a porous sheet containing two or more kinds of the above-described metals and metal oxides. Among these, a porous metal oxide sheet is preferable because it can provide a cryopreservation jig having excellent cell or tissue visibility.

  In the present invention, a generally known method can be used as a method for producing a porous metal sheet and a porous metal oxide sheet used as a preservation solution absorber. When the preservation solution absorber is a porous metal sheet, a method 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. 2006/041118 pamphlet or Japanese Patent No. 45788062 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, Metal sheet 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. Moreover, after heating metal powder at high temperature, the manufacturing method of metal porous 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, the manufacturing method like the slurry foaming method which manufactures a metal porous body using a foaming agent can also be used. When the preservation solution absorber is a porous metal oxide sheet, for example, methods described in JP2009-29692A and JP2002-160930A can be used.

  The surface of the porous body such as the porous resin sheet, the porous metal sheet, and the porous metal oxide sheet described above can be subjected to a hydrophilic treatment in order to increase the affinity with the surface layer. Hydrophilic treatment methods include graft modification methods, coating methods by applying non-eluting hydrophilic substances, general surface modification methods using various energies such as corona discharge, plasma treatment, and excimer laser. Can be used.

  When the preservation solution absorber is a porous body such as the above-described porous resin sheet, porous metal sheet, and porous metal oxide sheet, the pore diameter of the porous body is 0.02 to 130 μm. And more preferably 0.05 to 60 μm. When the pore diameter is less than 0.02 μm, the storage solution may not have sufficient absorption performance when the storage solution is dropped. Moreover, there exists a problem that manufacture of a porous sheet is difficult. On the other hand, when the pore diameter exceeds 130 μm, the absorption performance of the preservation solution may not be sufficient. In the case of a porous resin sheet, the pore diameter of the porous body is the diameter of the largest pore measured by a bubble point test. In the case of a porous metal sheet and a porous metal oxide sheet, the average pore diameter is measured from image observation of the surface and cross section of the porous body.

  The porosity of the storage liquid absorber is preferably 20% by volume or more, more preferably 30% by volume or more. Further, when the preservation solution absorber is a porous body such as the above-described porous resin sheet, porous metal sheet, and porous metal oxide sheet, the pores inside the porous body are not only in the thickness direction, A continuous structure is also preferred in a direction perpendicular to the thickness direction. With such a structure, since the pores inside the porous body can be used effectively, high absorption performance of the preservation solution can be obtained. The thickness of the preservation solution absorber and the porosity of the porous body can be appropriately selected according to the type of cells or tissues used, the amount of the preservation solution dripped together with the cells or tissues, and the like.

The above porosity is defined by the following formula. Here, the void volume V was measured and processed using a mercury porosimeter (measuring instrument name: Autopore II 9220 manufacturer micromeritics instrument corporation), and the cumulative pore volume (3 ml / g) of the pore diameter in the preservation solution absorber from 3 nm to 400 nm. ) Multiplied by the dry solid content (g / sq.m.) Of the storage liquid absorber can be obtained as a numerical value per unit area (sq.m.). Further, the thickness T of the preservation solution absorber can be obtained by photographing the cross section of the preservation solution absorber with an electron microscope and measuring the length.
P = (V / T) × 100 (%)
P: Porosity (%)
V: void volume (ml / m ² )
T: Thickness (μm)

In the present invention, when the mounting portion has the above-described support (non-absorbent support) as a reinforcing member in addition to the storage liquid absorber, between the storage liquid absorber and the non-absorbent support, An adhesive layer can be provided. The adhesive layer can contain an instantaneous adhesive composition, a hot melt adhesive composition, a photocurable adhesive composition, and the like represented by a moisture curable adhesive substance. For example, polyvinyl alcohol, hydroxy Water-soluble polymer compounds such as cellulose, polyvinyl pyrrolidone, starch paste, vinyl acetate resin, acrylic resin, epoxy resin, urethane resin, elastomer resin, cyanoacrylate resin, fluorine resin, silicon resin, Nitrocellulose resin, nitrile rubber resin, styrene-butadiene resin, urea resin, styrene resin, phenol resin, polyimide resin, polyamide resin, polyester resin, bismaleimide resin, olefin resin, EVA A composition containing a water-insoluble resin such as a resin is preferable. You can use. The adhesive layer may contain one type of resin or may contain a plurality of types of resins. Solid content of the adhesive layer is preferably in the range of 0.01 to 100 g / ^{m 2,} further range of 0.1 to 50 g / ^{m 2} is more preferable.

The area of the mounting part of the cryopreservation jig of the present invention may be appropriately set according to the amount of the preserving solution dripped together with the cells or tissues, and is not particularly limited. it is preferably set to 1 mm ² or more per 1 [mu] L, and more preferably a 2~400mm ^2. Further, in the same cryopreservation jig, when the placement unit has a plurality of storage liquid absorbers on the non-absorbent support, one continuous storage liquid absorber portion is It is preferable that it is an area.

  As described above, the placement portion of the cryopreservation jig of the present invention has been described. The cryopreservation jig of the present invention may have a grip portion together with the placement portion. Having a gripping portion is preferable because workability at the time of cryopreservation work and melting work is improved.

  FIG. 1 is an overall view showing an example of a jig for cryopreservation of cells or tissues of the present invention. In FIG. 1, the cryopreservation jig 7 includes a gripping portion 1 and a placement portion 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. In FIG. 1, the grip portion 1 has a cylindrical shape, but its shape is arbitrary. Further, as will be described later, for the purpose of preventing cells or tissues from coming into direct contact with liquid nitrogen, a cap may be placed on the mounting portion 2 to which the cells or tissues have been attached before freezing. 1 is made into a shape in which the diameter of the cylinder continuously decreases from the side not having the placement portion 2 toward the side having the placement portion 2, thereby improving workability when covering the cap. It is also possible. The shape of the mounting portion 2 is preferably a strip shape or a sheet shape in terms of handling.

  A method for connecting the grip portion 1 and the placement portion 2 in FIG. 1 will be described. When the gripping portion 1 is made of resin, for example, the mounting portion 2 can be connected to the gripping portion 1 by insert molding when molding. Furthermore, a structure insertion portion (not shown) can be produced in the grip portion 1 and the mounting portion 2 can be connected with an adhesive. Although various adhesives can be used, a silicon-based or fluorine-based adhesive that is resistant to low temperatures can be suitably used.

  FIG. 2 is a schematic sectional view of the mounting portion in FIG. In FIG. 2, the mounting portion 2 a has a structure having the surface layer 3 on the support 4. In order to obtain the mounting portion 2a having such a structure, for example, a coating solution containing at least a water-soluble polymer compound and glycerin is applied on a support by a slide hopper method or by dip coating. A manufacturing method such as a method can be used.

  FIG. 3 is a schematic cross-sectional structure diagram when the mounting portion shown in FIG. 1 has a storage liquid absorber. In FIG. 3, the mounting portion 2 b has a structure having the storage liquid absorber 5 and the surface layer 3 on the storage liquid absorber 5. In order to obtain the mounting part 2b having such a structure, for example, a manufacturing method in which the above-described coating liquid is applied by a slide hopper method can be used.

  FIG. 4 is a schematic cross-sectional structure diagram illustrating another example in the case where the placement unit illustrated in FIG. 1 includes a storage liquid absorber. In FIG. 4, the mounting portion 2 c has a structure having the surface layer 3 over the entire porous body of the storage liquid absorber 5. Thus, when a surface layer exists on a preservation | save liquid absorber, the surface layer does not need to form the continuous layer in the preservation | save liquid absorber surface, and may exist in the hole surface of a preservation | save liquid absorber. This structure is preferable because it does not block the pores of the porous body, so that both the absorbability of the vitrification solution during freezing and the detachability of cells or tissues during melting can be achieved. Also in this aspect, since the surface of the present invention is present on the outermost surface of the mounting portion microscopically, it can be said that FIG. 4 shows an example of the present invention. In order to obtain the mounting part 2c having such a structure, for example, a manufacturing method of applying by dip coating can be used.

  FIG. 5 is a schematic cross-sectional structure diagram illustrating another example in the case where the placement unit illustrated in FIG. 1 includes a storage liquid absorber. In FIG. 5, the mounting portion 2 d has a support 4 and an adhesive layer 6 in addition to the form of FIG. 4 having the surface layer 3 over the entire porous body of the storage liquid absorber 5. In the case of this structure, for example, even when the porous material used for the preservation solution absorber 5 is poor in self-supporting property, the operation during freezing and thawing can be performed more reliably.

  When a cell or tissue is cryopreserved for a long period of time with the cell or tissue cryopreservation jig of the present invention, as described above, the cell or tissue is covered with a cap or the cryopreservation jig as described above. Can be sealed in a container of any shape. When liquid nitrogen is not sterilized and cells or tissues are frozen by direct contact with liquid nitrogen, the sterilized state may not be guaranteed even if the cryopreservation jig is sterilized. Therefore, the cell or tissue may be frozen without contacting the liquid nitrogen directly by capping the mounting part to which the cell or tissue is attached before freezing or by sealing the cryopreservation jig in the container. . Also, in the advanced countries such as Europe, freezing methods that do not directly contact liquid nitrogen as described above are the mainstream. For these reasons, the cap and the container are preferably made of various metals, various resins, glass, ceramics, etc., which are liquid nitrogen resistant materials. The shape is not particularly limited. For example, the cap does not contact the main body, such as a semi-spindle or dome-like cap such as a pencil cap, or a cylindrical straw cap, and blocks cells or tissues from the outside. Any shape is possible as long as it is possible. The container is not particularly limited as long as it can enclose or store the cryopreservation jig without being in contact with the cells or tissues placed thereon and can be sealed.

  In the present invention, the cryopreservation jig can be used in combination with a cap or a container capable of blocking the cells or tissues on such a placement portion from the outside unless the effects of the present invention are impaired. . Moreover, the cryopreservation jig | tool used in combination with such a cap or a container is also included by this invention.

  The 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 the preservation of a small number of cells or cell populations in which a single cell or cell population is less than 10, but the cryopreservation jig of the present invention has more cells. Or it can use suitably also in preservation | save (for example, preservation | save of 10-1 million cells or a cell population) of a cell population. 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 cryopreservation jig of the present invention is used, in the cryopreservation operation of cells or tissues, the cells or tissues can be easily recovered when thawing the cells or tissues after freezing. Further, in addition to the above-described effect, the mounting part has a preservation solution absorber, and when the cell or tissue is frozen, it absorbs excess vitrification liquid adhering to the outer periphery of the cell or tissue. When cells or tissues are frozen and thawed, they are not easily damaged by extracellular vitrification solution, and the cells or tissues can be cryopreserved with an excellent survival rate.

  The method for cryopreserving cells or tissues using the cryopreservation jig of the present invention is not particularly limited. For example, cells or tissues soaked in a preservation solution are first dropped onto the mounting portion together with the preservation solution, and the cells Alternatively, excess storage solution adhering around the tissue is removed as much as possible using a pipette or the like. At this time, when the placement part of the cryopreservation jig has a preservation solution absorber, the above operation is not necessary, and the excess preservation solution is automatically removed. Next, the cells or the tissues can be frozen by immersing them in liquid nitrogen or the like while the cells or the tissues are held on the placement portion. At this time, a cap capable of blocking the cell or tissue on the mounting unit from the outside world is attached to the mounting unit, or a cryopreservation jig is sealed in the container, and liquid nitrogen or the like is used. It can also be immersed in it. As the preservation solution, those usually used for freezing cells such as eggs and embryos can be used. For example, a cryoprotectant (glycerin, ethylene glycol, etc.) is added to a physiological solution such as the above-described phosphate buffered saline. A large amount of various antifreezes such as lysine, ethylene glycol, DMSO (dimethyl sulfoxide) and the like obtained by the inclusion of the storage solution (at least 10% by mass, more preferably 20% by mass with respect to the total mass of the storage solution) The above vitrification liquid can be used. At the time of thawing operation, the cryopreservation jig is taken out from a cooling solvent such as liquid nitrogen, and the placing portion on which the frozen cells or tissues are placed is immersed in the thawing solution. to recover. As the melting solution, those usually used for thawing cells such as eggs and embryos, and culture solutions can be used as the melting solution, for example, pH 5 to 5 such as the above-mentioned phosphate buffered saline. A physiological solution that is 9 can be used. In order to avoid cell membrane damage due to rapid volume changes of cells during freezing and thawing, the thawing solution may be adjusted by adding and dissolving saccharides such as sucrose and trehalose for the purpose of osmotic buffering. it can. Moreover, protein components, such as serum, can be contained.

  Examples of cells that can be cryopreserved using the jig for cryopreservation of the present invention include, for example, mammals (eg, humans, cows, pigs, horses, rabbits, rats, mice, etc.) eggs, embryos, and sperm. And pluripotent stem cells such as artificial pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). Moreover, cultured cells, such as a primary cultured cell, a subcultured cell, and a cell line cell, are mentioned. In one or more embodiments, the cell is a fibroblast, a cancer-derived cell such as pancreatic cancer / hepatoma, epithelial cell, vascular endothelial cell, lymphatic endothelial cell, nerve cell, chondrocyte, tissue stem cell, And adherent cells such as immune cells. Furthermore, examples of tissues that can be cryopreserved include tissues composed of the same or different types of cells, such as tissues such as ovary, skin, corneal epithelium, periodontal ligament, and myocardium. The present invention is particularly suitable for cryopreservation of tissues having a sheet-like structure (for example, cell sheets, skin tissues, etc.). The jig for cryopreservation of the present invention is not limited to tissues directly collected from a living body, for example, cultured skin grown and grown in vitro, a so-called cell sheet constructed in vitro, JP 2012-205516 A It can also be suitably used for cryopreservation of an artificial tissue such as a proposed tissue model having a three-dimensional structure. The cryopreservation jig of the present invention is suitably used as a cryopreservation jig for cells or tissues as described above.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, and the present invention will be specifically described. However, the present invention is not limited to the following examples.

Example 1
A transparent PET film (total light transmittance 91%, haze value 5.5%, thickness 200 μm) subjected to easy adhesion treatment as a support is used, and polyvinyl alcohol is made by Kuraray Co., Ltd. on the support. Slide hopper so that the solid content at the time of drying of the coating solution containing 1.8% by mass of PVA103 (degree of saponification 98.5 mol%, degree of polymerization 300) and 0.2% by mass of glycerin is 1 g / m ² After applying the coating method and drying at room temperature, a heat treatment was performed at 120 ° C. for 40 hours to produce a mounting portion having the form shown in FIG. 2 having a surface layer on the support. This placing part was cut into a size of 2 mm × 20 mm (40 mm ² ) and joined to a grip part made of ABS resin, and the cryopreservation jig of Example 1 was manufactured in the form shown in FIG.

(Example 2)
A transparent PET film (total light transmittance 91%, haze value 5.5%, thickness 200 μm) subjected to easy adhesion treatment as a support is used as an adhesive layer on the support, manufactured by Henkel Japan Co., Ltd. Hot-melt urethane resin Purmelt QR 170-7141P was applied so as to have a solid content of 30 g / m ² when dried. Before the adhesive layer is completely cured, a hydrophilic polytetrafluoroethylene porous material (pore diameter 0.2 μm, porosity 71%, thickness 35 μm) manufactured by Advantech Toyo Co., Ltd. is used as a preservation solution absorber. Pasted together. The laminate thus obtained was dip-coated by immersing it in a coating solution containing 1.8% by mass of PVA103 and 0.2% by mass of glycerin, dried at room temperature, and then at 120 ° C. for 40 hours. Heat treatment was performed to produce a placement portion having a surface layer having the form shown in FIG. The solid content of the surface layer is 1.6 g / m ² . In the same manner as in Example 1, this mounting part was cut into a size of 2 mm × 20 mm, and then joined to a grip part made of ABS resin to produce the cryopreservation jig of Example 2 in the form shown in FIG. did.

(Example 3)
A cryopreservation jig of Example 3 was prepared in the same manner as Example 2 except that dip coating was performed using a coating solution containing 1.3% by mass of PVA103 and 0.7% by mass of glycerin. did. Note that the solid content of the surface layer of the cryopreservation jig of Example 3 is 1.6 g / m ² .

Example 4
Using a coating liquid containing 1.8% by mass of GOHSENOL EG05P (degree of saponification 87.8 mol%, degree of polymerization 500) manufactured by Nippon Synthetic Chemical Industry Co., Ltd., which is polyvinyl alcohol, and 0.2% by mass of glycerin. A cryopreservation jig of Example 4 was produced in the same manner as Example 2 except that dip coating was performed. Note that the solid content of the surface layer of the cryopreservation jig of Example 4 is 1.6 g / m ² .

(Example 5)
The jig for cryopreservation of Example 5 was the same as Example 2 except that dip coating was performed using a coating solution containing 1.3% by mass of Gohsenol EG05P and 0.7% by mass of glycerin. Produced. Note that the solid content of the surface layer of the cryopreservation jig of Example 5 is 1.6 g / m ² .

(Example 6)
Dip coating is performed using a coating solution containing 1.8% by mass of PVA505 (saponification degree: 73.5 mol%, polymerization degree: 500) and 0.2% by mass of glycerin manufactured by Kuraray Co., Ltd., which is polyvinyl alcohol. A cryopreservation jig of Example 6 was produced in the same manner as Example 2 except that. Note that the solid content of the surface layer of the cryopreservation jig of Example 5 is 1.6 g / m ² .

(Example 7)
A jig for cryopreservation of Example 7 was produced in the same manner as Example 2 except that dip coating was performed using a coating solution containing 1.3% by mass of PVA505 and 0.7% by mass of glycerin. did. Note that the solid content of the surface layer of the cryopreservation jig of Example 7 is 1.6 g / m ² .

(Example 8)
Example 8 was carried out in the same manner as Example 2 except that dip coating was carried out using a coating solution containing 1.8% by mass of PN505 made by Gelais Co., Ltd. and 0.2% by mass of glycerin. A cryopreservation jig was prepared. Note that the solid content of the surface layer of the cryopreservation jig of Example 8 is 1.6 g / m ² .

(Comparative Example 1)
After obtaining a laminated body in the same manner as in Example 2, without coating, the laminated body was used as a placement portion, cut into a size of 2 mm × 20 mm, and then joined to a gripping portion made of ABS resin. The jig for cryopreservation of Comparative Example 1 was produced in the form shown in FIG.

(Comparative Example 2)
A cryopreservation jig of Comparative Example 2 was produced in the same manner as Example 2 except that dip coating was performed using a coating solution containing 2% by mass of PVA103. The solid content of the surface layer of the cryopreservation jig of Comparative Example 2 is 1.6 g / m ² .

<Spheroid adjustment>
Spheroids used for evaluation of cell or tissue detachability were adjusted as follows. HepG2 cells, which are hepatoma-derived cell lines, were cultured on a culture dish and then detached and collected by trypsin treatment. Thereafter, spheroid formation was induced by seeding a PrimeSurface 96U plate manufactured by Sumitomo Bakelite Co., Ltd. at a cell number of 100 cells / well, followed by suspension culture. Spheroids having a diameter of about 100 μm were obtained after 3 days of culture.

<Freezing spheroids>
Spheroids prepared by the above method were collected and vitrified (15% by volume DMSO (dimethyl sulfoxide), 15% by volume ethylene glycol, 14% by volume fetal calf serum, 56% by volume modified phosphate buffer (137 mM NaCl, 2 7 mM KCl, 0.9 mM CaCl ₂ .2H ₂ O, 0.5 mM MgCl ₂ .6H ₂ O, 1.5 mM KH ₂ PO ₄ , 8 mM Na ₂ HPO ₄ , 5.6 mM glucose, 0.3 mM pyruvate Na, 65 μg / ml dibekacin sulfate, 1 mg / ml polyvinylpyrrolidone, 14.8 mM L-proline, 200 mM trehalose), 0.5 M sucrose) for 20 seconds, and then spheroids of Examples 1 to 8 or Comparative Examples 1 and 2 were used. Place them on the placement part of the cryopreservation jig and observe under a transmission microscope. The excess vitrification solution was removed. At this time, for Example 1, the excess vitrification solution around the spheroid was removed as much as possible using a pipette. In Examples 2 to 8 and Comparative Examples 1 and 2, excess vitrification liquid was spontaneously absorbed by the vitrification liquid absorber. Thereafter, the spheroids were vitrified and frozen by immersion in liquid nitrogen. The frozen cryopreservation jig was stored in a liquid nitrogen storage container until thawed.

<Evaluation of cell or tissue detachability>
The cryopreservation jigs of Examples 1 to 8 and Comparative Examples 1 and 2 on which spheroids were placed were respectively taken out from liquid nitrogen, and were melted at a temperature of 37 ° C. ). While observing the state of the spheroids after immersion with a transmission optical microscope, the spheroids were collected using a pipette and evaluated according to the following criteria. These results are shown in the item “Peelability of cells or tissues” in Table 1. In addition, the solubility with respect to the melt of the surface layer of the cryopreservation jigs of Examples 1 to 8 and Comparative Example 2 was 0.2% or more.

The peelability of cells or tissues was evaluated according to the following criteria.
○: When the cryopreservation jig was immersed in the melt, the spheroids could be recovered by shaking the gripping part or performing a suction operation using a pipette.
△: When the cryopreservation jig was immersed in the melt, it could not be peeled off by shaking the gripping part or by a suction operation using a pipette, but collecting the spheroids by touching with the tip of the pipette I was able to.
X: The spheroids were fixed so that they could not be easily recovered, and the cells constituting the spheroids fell apart during the recovery.

  From the results in Table 1, it can be seen that the cryopreservation jig of the present invention exhibits excellent peelability upon melting.

  The present invention is not limited to livestock such as cattle and animal embryo transfer, artificial insemination, artificial insemination to humans, iPS cells, ES cells, commonly used cultured cells, for inspection or transplantation collected from living organisms. It can be used for cryopreservation of cells or tissues, cells or tissues cultured in vitro, and the like.

DESCRIPTION OF SYMBOLS 1 Holding part 2, 2a-2d Mounting part 3 Surface layer 4 Support body 5 Preservation liquid absorber 6 Adhesive layer 7 Freezing preservation jig

Claims (2)

  A jig for cryopreservation of a cell or tissue, comprising a layer containing at least a water-soluble polymer compound and glycerin on the outermost surface of a placement part for placing the cell or tissue.   2. The cell or tissue freezing according to claim 1, wherein the placement part has a preservation solution absorber, and has a layer containing at least a water-soluble polymer compound and glycerin on the preservation solution absorber. Storage jig.

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