JP2022151006A - Freezing preservation jig for cell or tissue - Google Patents

Abstract

To provide a freezing preservation jig for cells or tissues, comprising excellent visibility at which a user can easily visually confirm a cell or a tissue, in a freezing step for mounting the cell or the tissue on a mounting part of the freezing preservation jig, for freezing and preserving the same, and a melting step for melting the frozen cell or tissue, and in the melting step, capable of exhibiting preferable peeling properties at which the cell or the tissue can be quickly and surely collected.SOLUTION: There is provided a freezing preservation jig for cells or tissues comprising at least a mounting part for mounting the cell or the tissue with preservation liquid. The mounting part comprises: a light permeable support body having an irregular structure; and a layer including a water soluble high molecular compound on the light permeable support body, in which a pitch of the irregular structure provided on the light permeable support body is equal to or less than 1.3 μm.SELECTED DRAWING: Figure 4

Description

The present invention relates to jigs for cryopreservation of cells or tissues.

Excellent preservation techniques for cells or tissues are required in various industrial fields. For example, in bovine embryo transfer techniques, embryos are cryopreserved, thawed in accordance with the estrus cycle of the recipient cow, and transferred into the body. In the field of human infertility treatment, eggs collected from the mother's body are fertilized in vitro, cultured in vitro to form embryos, and the embryos are cryopreserved in order to adjust the timing for transplantation. It is used after being melted. Furthermore, in human infertility treatment, eggs and ovarian tissues are collected and cryopreserved while maintaining their functions.

In general, cells or tissues collected from the living body gradually lose their activity or undergo changes in their characteristics even in a culture medium. culture is not preferred. Therefore, techniques for long-term preservation while maintaining bioactivity are important. Good preservation techniques allow for more accurate analysis of the harvested cells or tissues. In addition, excellent preservation techniques enable cells or tissues to be used for transplantation while maintaining higher bioactivity, and are expected to improve the engraftment rate after transplantation. Furthermore, it is also possible to sequentially produce artificial tissues for transplantation, such as cultured skin cultured in vitro and so-called cell sheets constructed in vitro, store them, and use them when necessary. , great benefits can be expected not only in the medical field but also in the industrial field.

For example, the slow freezing method is known as a cryopreservation method for cells or tissues. In this method, cells or tissues are first immersed in a preservation solution obtained by adding a cryoprotectant to a physiological solution represented by phosphate-buffered saline. Compounds such as glycerol, ethylene glycol, and dimethylsulfoxide are used as the antifreeze agent. After immersing the cells or tissue in the preservation solution, by cooling to -30 to -35°C at a relatively slow cooling rate (eg, 0.3 to 0.5°C/min), The internal and external solutions are sufficiently cooled and become highly viscous. In this state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (-196°C), the microscopic solution inside the cells or tissues and the surrounding surroundings both solidify while remaining amorphous. The phenomenon vitrification occurs. Vitrification solidifies the inside and outside of the cell or the inside and outside of the tissue, which substantially eliminates the movement of molecules. Therefore, vitrified cells or tissues can be stored semi-permanently by storing them in liquid nitrogen.

Vitrification is also known as a cryopreservation method for cells or tissues. The vitrification method is based on the principle that ice crystals are less likely to form even at subzero temperatures due to the freezing point depression of storage solutions containing large amounts of antifreeze agents such as glycerol, ethylene glycol, and dimethyl sulfoxide. If this stock solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. Such solidification is called vitrification freezing. A preservation solution containing a large amount of antifreeze agent is called a vitrification solution.

In the slow freezing method described above, since it is necessary to cool at a relatively slow cooling rate, the operation for cryopreservation takes time. There is also the problem of requiring a device or jig to control the cooling rate. In addition, in the slow freezing method, ice crystals are formed in the extracellular or extracellular preservation solution, and the cells or tissues may be physically damaged by the ice crystals. On the other hand, the vitrification freezing method described above requires a short operation time and does not require any special device or jig. In addition, the vitrification freezing method provides a high survival rate by not forming ice crystals.

Various methods of cryopreservation of cells or tissues using the vitrification method have been demonstrated using various types of cells or tissues. For example, Patent Document 1 shows that application of the vitrification freezing method to germ cells or somatic cells of animals or humans is extremely useful in terms of survival rate after cryopreservation and thawing.

The vitrification freezing method is a technique that has been developed mainly using human reproductive cells, but recently its application to iPS cells and ES cells has also been widely investigated. In addition, Non-Patent Document 1 shows that vitrification was effective in preserving Drosophila embryos. Furthermore, Patent Document 2 shows that the vitrification freezing method is effective in preserving cultured plant cells and tissues. Thus, the vitrification method is known to be useful for preserving a wide variety of cells and tissues.

It is known that the faster the freezing rate, the better, in order to achieve proper vitrification freezing. Furthermore, it is known that the faster the thawing rate is, the more preferable it is from the viewpoint of suppressing re-ice crystal formation in cells or tissues during the thawing step after cryopreservation.

Of the freezing rate and thawing rate, which are important factors for achieving proper vitrification freezing, the thawing rate is considered to be particularly important. For example, as described in Non-Patent Document 2, even rapidly frozen cells are known to have a low survival rate if the thawing rate is slow. Moreover, Patent Document 3 describes that the survival rate of human iPS cell-derived neural stem cells/progenitor cells after thawing is improved by thawing a frozen sample by a rapid thawing method.

In Patent Documents 4 and 5, a so-called cryotop method used in the field of human infertility treatment is used for egg cryopreservation using strip-shaped flexible and colorless and transparent films as strips for retaining egg attachment. A method has been proposed in which ova or embryos are placed on the film together with a very small amount of preservative solution under microscope observation using a tool and cryopreserved.

Patent Literatures 6 and 7 describe cryopreservation jigs having a plurality of cell-holding recesses provided in the longitudinal direction of an elongated biological cell adhesion-holding portion.

In Patent Document 8, eggs or embryos are placed on a storage solution removing material together with a storage solution containing a large amount of antifreeze agent, and the excess storage solution attached around the eggs or embryos is removed by sucking from the bottom. , a method of cryopreservation has been proposed. As the material for removing the preservative solution, a wire mesh, a film-like material made of natural materials such as paper or a synthetic resin and having through holes is described. In addition, as a cryopreservation jig that can remove excess preservation solution without requiring an operation such as aspiration and that can improve the visibility when placing cells, for example, Patent Document 9 specifies A jig for cryopreservation using a preservation liquid absorber having a haze value of is described, and Patent Documents 10 and 11, etc., disclose a porous sintered body or a specific refractive index as a preservation liquid absorber. A vitrification cryopreservation jig having a porous structure formed of a material comprising:

The cryopreservation jig capable of improving workability when placing cells or tissues has been described above, but it is possible to improve workability when detaching cells or tissues. As a jig for cryopreservation, for example, a jig for cryopreservation of cells or tissues having a layer containing a water-soluble polymer compound on the outermost surface of a mounting portion on which cells or tissues are mounted is described in Patent Document 12. The document describes that it is possible to form projections with inorganic fine particles on the surface layer. Further, Patent Document 13 describes a jig for cryopreservation in which the surface of a mounting portion on which cells or tissues are placed together with a preservation solution has convex portions for holding cells or tissues and concave portions for retaining a preservation solution. It is described that the jig for cryopreservation is excellent in workability during freezing and thawing of cells or tissues.

On the other hand, Patent Document 14 describes a cell container having an inner surface on which fine protrusions are formed at an arrangement pitch of 60 nm or more for the purpose of preventing cells from adhering to the inner surface of the container.

Japanese Patent No. 3044323 JP-A-2008-5846 JP 2017-104061 A JP-A-2002-315573 JP 2006-271395 A Japanese Patent No. 5781621 Japanese Patent No. 5798633 International Publication No. 2011/070973 pamphlet JP 2014-183757 A JP 2015-142523 A International Publication No. 2015/064380 pamphlet JP 2017-60457 A International Publication No. 2018/230477 Pamphlet JP 2015-226497 A

Steponkus et al. , Nature 345:170-172 (1990) Hiroshi Sozu, "Mechanism of Injury and Protection by Freezing of Living Cells", Chemistry and Biology, Vol. 18 (1980), No. 2, p. 78-87 Published by Japan Society for Bioscience, Biotechnology and Agrochemistry

In Patent Documents 4 and 5, by limiting the width of the film on which the eggs or embryos are placed, the eggs or embryos are cryopreserved with a small amount of preservative solution, thereby speeding up the freezing and thawing speeds. Methods have been proposed to obtain improved survival rates. However, when the eggs or embryos are thawed after freezing, the eggs or embryos on the film often stick to the surface of the film depending on the condition of the frozen target, and a high level of skill is required to recover them. there were. Furthermore, there is a problem that the more the ova or embryos are cryopreserved by the operator with a smaller amount of preservative solution, the more the ova or embryos adhere firmly to the film surface when thawed after freezing, making recovery more difficult. rice field.

Patent Document 6 and Patent Document 7 propose a method of improving the dropout of cells during freezing operation by providing a concave portion in which cells are stored in the cell adhesion holding portion. 4 and Patent Document 5, there is a problem that a high skill is required when recovering.

Patent Document 8 proposes a method of cryopreserving germ cells by removing excess storage solution attached around eggs or embryos by aspirating from the bottom of a storage solution removing material. However, as in Patent Documents 4 and 5, when thawing after freezing, eggs or embryos adhere to the preservative solution-removing material, and a high level of skill is required to recover them. rice field. In Patent Documents 9 to 11, there is no need for the operator to remove the excess preservation solution attached around the egg or embryo as in Patent Document 8 described above, and good workability is obtained, but preservation solution absorption is not required. There has been the problem that a particularly high degree of skill is required when retrieving the ova or embryos, since the ova or embryos adhere particularly firmly to the storage fluid absorber when the body absorbs the storage fluid.

In Patent Document 12, a jig for cryopreservation that enables easy collection of cells or tissues by forming a layer containing a water-soluble polymer compound on a mounting portion on which cells or tissues are mounted. In addition, it has been proposed to provide convex portions by inorganic fine particles in the layer containing the water-soluble polymer compound, but improvement of the recoverability of cells or tissues during freezing and thawing is a permanent problem, Further improvement was desired.

In Patent Document 13, as in Patent Document 12, when cells or tissues are thawed after freezing, cells or tissues are thawed to avoid sticking to the mounting portion of a jig for cryopreservation. A method has been proposed in which a convex portion for holding and a concave portion for storing a storage solution are provided. However, in the thawing process of thawing frozen cells or tissues, there is a problem that the cells or tissues in the thawing solution are difficult to see, and an improvement has been desired.

In Patent Document 14, in a cell container filled with cells together with a storage solution, such as a so-called cell storage vial, adhesion of cells to the inner surface of the cell container is suppressed by forming a fine projection shape on the inner surface of the cell container. A method is proposed. However, this technique suppresses adhesion of cells, and is not a technique for positively detaching and collecting adhered cells. In the so-called cryotop method in which a very small number (often about 1 to 3 cells) of cells or tissues are placed on a mounting part together with a very small amount of preservation solution, not only can the cells be easily recovered when thawed, but also the cells can be frozen. It is also required that the cells or tissues be reliably retained on the cryopreservation jig during storage.

A main object of the present invention is to provide a jig for cryopreservation of cells or tissues, which allows easy and reliable cryopreservation of cells or tissues. More specifically, in a freezing step of placing cells or tissues on a mounting portion of a cryopreservation jig and cryopreserving them, and a thawing step of thawing the frozen cells or tissues, the cells or tissues are easily treated. An object of the present invention is to provide a jig for cryopreservation, which has excellent visibility that can be easily visually recognized, and has good detachability that enables rapid and reliable recovery of cells or tissues in a thawing process.

As a result of intensive studies to solve the above problems, the present inventors have found that a cell or tissue cryopreservation jig (hereinafter also referred to as "cryopreservation jig of the present invention") having the following configuration: , found that the above problems can be solved.

The present invention has at least a mounting portion for mounting cells or tissues together with a preservation solution, the mounting portion comprising a light-transmitting support having an uneven structure, and a water-soluble polymer on the light-transmitting support. A jig for cryopreservation of cells or tissues, comprising a layer containing a compound, wherein the light-transmitting support has an uneven structure with a pitch of 1.3 μm or less.

According to the present invention, in the freezing step of placing cells or tissues on the mounting portion of a cryopreservation jig and cryopreserving them, and the thawing step of thawing the frozen cells or tissues, the cells or tissues can be easily removed. It is possible to provide a jig for cryopreservation that has excellent visibility that can be visually recognized from the inside, and that has good detachability that allows cells or tissues to be quickly and reliably recovered in the thawing process.

1 is an overall view showing an example of a jig for cryopreservation of the present invention; FIG. FIG. 4 is an overall view showing another example of the jig for cryopreservation of the present invention; FIG. 4 is an overall view showing another example of the jig for cryopreservation of the present invention; It is a cross-sectional structure schematic which shows an example of the mounting part in the jig|tool for cryopreservation of this invention. It is a cross-sectional structure schematic which shows another example of the mounting part in the jig|tool for cryopreservation of this invention. It is a cross-sectional structure schematic which shows another example of the mounting part in the jig|tool for cryopreservation of this invention. It is a cross-sectional structure schematic which shows another example of the mounting part in the jig|tool for cryopreservation of this invention. 5 is a schematic top view and a schematic cross-sectional view of the uneven structure shown in FIG. 4. FIG. 7 is a schematic top view and a schematic cross-sectional view of the uneven structure shown in FIG. 6. FIG. FIG. 8 is a schematic top view and a schematic cross-sectional view of the concave-convex structure shown in FIG. 7; FIG. 5 is a schematic model diagram when cells and a preservation solution are dropped and adhered to the mounting portion shown in FIG. 4 ; FIG. 4 is a top structural schematic view showing another example of the mounting portion in the cryopreservation jig of the present invention. 13 is a cross-sectional structural schematic diagram showing an enlarged shape of a mounting region 8a and a mounting region 8b in FIG. 12; FIG.

The jig for cryopreservation of cells or tissues of the present invention (hereinafter also referred to as the jig for cryopreservation of the present invention) is used for cryopreservation of cells or tissues, and is used for so-called vitrification freezing of cells or tissues. It is suitably used when cryopreserving in a preservation method. In the present invention, a cell includes not only a single cell but also a cell population of an organism consisting of a plurality of cells. A 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. In addition, the tissue may be a tissue composed of a single type of cells, or a tissue composed of a plurality of types of cells, and includes non-cellular substances such as extracellular matrices in addition to cells. Anything is fine. The cryopreservation jig of the present invention is particularly suitable for cryopreservation of eggs or embryos.

The jig for cryopreservation of cells or tissues of the present invention includes a freezing step in which cells or tissues are placed and then frozen using a cryogenic refrigerant, a cooling step in which the frozen state is maintained, and a freezing step in which the cells or tissues are thawed. It is used in a series of operations in the melting process of melting and thawing in a liquid.

Specifically, the cryopreservation jig of the present invention is for freezing by immersing a cryopreservation jig having a mounting portion on which cells or tissues are mounted in a cooling solvent such as liquid nitrogen. Further, the cryopreservation jig of the present invention is for holding frozen cells or tissues under cryogenic conditions, and furthermore, the cells or tissues mounted on the mounting part are cooled with a cooling solvent. It is for melting by immersing it in the melting liquid.

The jig for cryopreservation of cells or tissues of the present invention can be called a cell or tissue preservation instrument, a cell or tissue cryopreservation instrument, a cell or tissue preservation instrument, or the like.

The jig for cryopreservation of the present invention will be described in detail below.

The cryopreservation jig of the present invention has at least a mounting portion for mounting cells or tissues together with a preservation solution, the mounting portion having a light-transmitting support having an uneven structure, and the light-transmitting support. It has a layer containing a water-soluble polymer compound thereon. Here, the placement portion on which cells or tissues are placed together with a preservation solution actually corresponds to a region where cells or tissues are placed together with a preservation solution. In the present invention, the mounting portion may be formed on the entire light-transmitting support, or may be formed on a part of the light-transmitting support. , the mounting portion 3 is formed on a part of the light-transmissive support 2 .

In the jig for cryopreservation of the present invention, the shape of the placing portion is not particularly limited as long as the shape allows cells or tissues to be placed together with the preservation solution. Preferably, the placement section has a substantially sheet-like shape. A cryopreservation jig having a substantially sheet-shaped mounting portion is one of preferred embodiments of the present invention. The term “substantially sheet-like” in the present invention means a shape having a flat surface from a macroscopic point of view, and examples thereof include a planar sheet-like shape, a sheet-like shape having a curvature, a corrugated sheet-like shape, a V-shaped sheet-like shape, and the like. be.
Moreover, it is preferable that the mounting part is strip-shaped. If the placement section is strip-shaped, it is easy to cover and protect the placement section with a cap member, which will be described later.

The mounting portion of the cryopreservation jig of the present invention has a light-transmissive support having an uneven structure.
As the concave-convex structure, a projection assembly in which substantially conical projection structures are arranged in a substantially triangular lattice is preferable, and a projection assembly in which the structures are arranged in a substantially triangular lattice and are two-dimensionally close-packed is more preferable. When focusing on one substantially conical projection structure, its shape is a convex structure.
The substantially conical protrusion structure includes, in addition to the cone, a structure having a circular base and a curved generatrix, and a structure having a curved apex.
The substantially conical projection structure may have a truncated cone shape with a flat uppermost portion.
In this specification, the uppermost part of the convex structure is called a convex part, and the part between the convex structures is called a concave part or a concave structure.

The uneven structure of the light-transmissive support has a pitch of 1.3 μm or less. In this specification, the pitch of the concavo-convex structure is the average distance between the centers of the convex structures adjacent to each other when viewed from above. The "pitch of the uneven structure" can also be said to be the average of the intervals (distances) between the centers of the two closest convex structures when viewed from above. If the pitch of the concave-convex structure is larger than 1.3 μm, the visibility of the cells or tissue during the melting operation is lowered, and good workability cannot be obtained. Further, in the present invention, the pitch of the concave-convex structure of the light-transmitting support constituting the mounting portion is preferably 50 nm or more. If it is smaller than 50 nm, the layer containing the water-soluble polymer compound cannot sufficiently cover the concave-convex structure of the light-transmitting support, and the cells or tissues are quickly exfoliated (recovered) in the melting step. ) can be difficult. More preferably, the pitch is 100 nm or more, and preferably 1 μm or less.

When the mounting portion is formed on a part of the light-transmitting support, the concave-convex structure may or may not be formed on a portion of the light-transmitting support other than the mounting portion. It doesn't have to be. When the mounting portion is formed on a part of the light-transmitting support, the layer containing the water-soluble polymer compound may or may not exist in a portion other than the mounting portion. Also good.

The concave-convex structure of the light-transmitting support constituting the mounting portion of the jig for cryopreservation of the present invention has a height of the concave-convex structure (the difference between the height of the concave portion and the convex portion, for example, H1 in FIG. 4) of 50 nm or more. is preferred. If the height of the uneven structure is less than 50 nm, the effect of improving the detachability of cells during the melting operation may not be sufficient. The height of the uneven structure is more preferably 100 nm or more. Moreover, the height of the uneven structure is preferably 3 μm or less. If the height of the uneven structure is higher than 3 μm, the visibility during the melting operation may be insufficient. The height of the uneven structure is more preferably 1 μm or less.

The light-transmissive support having the concave-convex structure of the mounting portion of the jig for cryopreservation of the present invention may be made of one type of material, or may be made of two or more types of materials. As a material for forming the light-transmitting support, a thermoplastic resin having light-transmitting properties is suitable. Thermoplastic resins are preferably used in the present invention because they have little or no cytotoxicity, which is a problem with thermosetting resins and photocurable resins. Examples of such thermoplastic resins include polystyrene resins, acrylic resins, ABS resins, polycarbonate resins, polydimethylsiloxane resins, polyvinyl chloride resins, polysulfone resins, polyolefin resins, cyclic polyolefin resins, and polyurethane resins.

The mounting portion of the jig for cryopreservation of the present invention has a layer containing a water-soluble polymer compound on a light-transmitting support. The layer containing the water-soluble polymer compound is formed on the uneven structure of the light-transmitting support. In the present invention, the water-soluble polymer compound is preferably a compound having a solubility of 0.2% by mass or more in a melt at 25°C.
In the present invention, examples of water-soluble polymer compounds include cellulose derivatives such as hydroxyethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose, starch and its derivatives, gelatin, casein, alginic acid and its salts, polyvinyl alcohol, polyvinylpyrrolidone, styrene- Examples include maleic acid copolymer salts, styrene-acrylic acid copolymer salts, and the like. These water-soluble polymer compounds may be used alone or in combination of two or more. Above all, when the layer containing the water-soluble polymer compound contains hydroxypropylcellulose and/or polyvinyl alcohol, it is preferable because good solubility in the storage solution and appropriate film-forming action can be obtained. In addition, the layer containing the water-soluble polymer compound can contain a cross-linking agent within a range where the solubility in the melt does not fall below 10% by mass.

The layer containing the water-soluble polymer compound of the jig for cryopreservation of the present invention preferably has a thickness that does not expose the protrusions of the uneven structure described above, and the solid content of the water-soluble polymer compound is It is preferably 0.01 to 100 g/m ² , more preferably 0.1 to 10 g/m ² .
The mounting portion is formed of a layer containing a light-transmitting support and a water-soluble polymer compound. The mounting portion may have a structure other than the layer containing the light-transmitting support and the water-soluble polymer compound, but only the layer containing the light-transmitting support and the water-soluble polymer compound may be used. It is more preferable to be formed. The mounting section preferably has a layer containing a water-soluble polymer compound on the outermost surface. When a layer containing a water-soluble polymer compound is present on the outermost surface of the mounting portion, all or part of the layer containing the water-soluble polymer compound is dissolved in the melt when the cells or tissues are melted. By doing so, the detachability of cells or tissues is synergistically improved in combination with the effect of the concave-convex structure of the light-transmitting support.

The mounting portion in the jig for cryopreservation of the present invention comprises a light-transmitting support having an uneven structure, and a layer containing a water-soluble polymer compound provided on the uneven structure of the light-transmitting support. It can also be said that it is formed by

The jig for cryopreservation of the present invention preferably has a holding part together with the above-described placing part, and preferably has a placing part at the tip of the holding part. It is preferable to have a grip part because workability during cryopreservation work and thawing work is improved. In the following description, a jig for cryopreservation having a holding portion as well as a placing portion is also referred to as a main body member.
The shape of the grip portion is not particularly limited, but a shape that is easy for a person to grip is preferable, and a rod shape is more preferable.
The grip is preferably made of a material that is resistant to cooling solvents such as liquid nitrogen. As such materials, various metals such as aluminum, iron, copper and stainless alloys, ABS resins, polypropylene resins, polyethylene resins, fluorine-based resins, various engineering plastics, glass and the like can be suitably used.

When cells or tissues are to be cryopreserved for a long period of time using the jig for cryopreservation of cells or tissues of the present invention, the mounting portion is covered with a cap member in order to block the cells or tissues from the outside world, or It is possible to house the jig in a container of arbitrary shape and to seal it. Liquid nitrogen is not sterilized, and when cells or tissues are directly brought into contact with liquid nitrogen and frozen, the sterilization condition may not be guaranteed even if the cryopreservation jig is sterilized. Therefore, it is possible to freeze the cells or tissues without direct contact with liquid nitrogen by covering the mount to which the cells or tissues are attached before freezing with a cap member, or sealing the cryopreservation jig in the container. be. In developed countries such as Europe, the freezing method that avoids direct contact with liquid nitrogen as described above is the mainstream.
In addition, even when cells or tissues are directly brought into contact with liquid nitrogen and frozen, the cells or tissues placed on the cryopreservation jig are protected from physical impacts in the liquid nitrogen tank to be stored. For this purpose, the mounting portion is sometimes covered with a cap member. For this reason, the cap member and the container are preferably made of liquid nitrogen-resistant materials such as various metals, various resins, glass, and ceramics. The shape of the cap member is not particularly limited. For example, the cap member may be semi-spindle-shaped or dome-shaped, such as a pencil cap, or a cylindrical straw cap, etc., to prevent cells or tissues from contacting with the mounting portion. Any shape can be used as long as it can be cut off from the outside world. The shape of the container is not particularly limited as long as it can cover or house the cryopreservation jig without contacting the placed cells or tissues.

In the present invention, a jig for cryopreservation can be used in combination with such a cap member or container as long as the effects of the present invention are not impaired. The present invention also includes a cryopreservation jig used in combination with such a cap member or container.

The cap member preferably included in the cryopreservation jig of the present invention has a lumen capable of covering and protecting the placement section. An example of such a cap member is a substantially cylindrical body having one end that is open and one end that is closed.

When the cryopreservation jig of the present invention has a cap member, the cap member is preferably detachable from the body member. Detachable means that the cap member can be easily fitted into and fixed to the body member, and that the cap member can be easily removed from the body member, without the need for special tools. The body member preferably has a fitting structure in order to be detachable, and the body member preferably has a tapered structure or a threaded structure as the fitting structure. In this case, it is preferable that the mounting portion described above be positioned at the tip of the fitting structure portion (the tip on the side without the grip portion). From the viewpoint of quick and easy attachment and detachment, it is particularly preferable that the fitting structure has a tapered structure.

If the mating structure of the body member has a threaded structure, the cap member also preferably has a threaded structure. Moreover, when the fitting structure portion of the main body member has a tapered structure, the cap member can also have a tapered structure in the above-described lumen from the viewpoint of more reliable fitting and fixing.

The cap member of the jig for cryopreservation of the present invention is preferably made of materials such as various resins and metals that are resistant to cooling solvents such as liquid nitrogen. The cap member may be made of one type of material, or may be made of two or more types of material. Among them, resin is preferable because a tapered structure or a threaded structure can be easily formed by a process such as injection molding. Specific examples of resins include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, acrylic resins, epoxy resins, silicone resins, polycarbonate resins, diacetate resins, triacetate resins, polyacrylate resins, polyvinyl chloride resins, polysulfone resins, Resins such as polyethersulfone resins, polyimide resins, polyamide resins, polyolefin resins, and cyclic polyolefin resins can be used. Further, when the total light transmittance of the cap member, more specifically, the total light transmittance of the side surface portion of the cap member is 80% or more, it is possible to easily check the state of the mounting portion after the cap member is fitted. Therefore, it is preferable.

FIG. 1 is an overall view showing an example of a jig for cryopreservation of the present invention. In FIG. 1, the cryopreservation jig 9a has a grip portion 1, a flat sheet-like light-transmitting support 2, and a flat sheet-like mounting portion 3 formed on the light-transmitting support 2. As shown in FIG.

In FIG. 1, the grip part 1 has a cylindrical shape, but its shape is arbitrary. Further, as described above, in order to prevent the cells or tissues from directly contacting liquid nitrogen or to protect the cells or tissues, the placing section 3 may be covered with a cap member (not shown). The part 1 preferably has a fitting structure part 13 on the side having the mounting part 3 . The fitting structure portion 13 in FIG. 1 has a tapered structure in which the cross section continuously decreases from the side not having the mounting portion 3 toward the side having the mounting portion 3 . In one aspect, the fitting structure 13 having such a structure improves workability when the cap member is put on.

FIG. 2 is an overall view showing another example of the jig for cryopreservation of the present invention. In FIG. 2, the cryopreservation jig 9b includes a grip portion 1, a V-shaped sheet-shaped light-transmitting support 2, and a V-shaped sheet-shaped support 2 formed on a part of the light-transmitting support 2. It has a mounting section 3 . By dripping the cells or tissue and the preservation solution onto the central portion of the V-shaped sheet-like mounting portion 3, workability is improved when excess preservation solution is removed with a pipette or the like.

FIG. 3 is an overall view showing another example of the jig for cryopreservation of the present invention. In FIG. 3, the cryopreservation jig 9c includes a grip portion 1, a sheet-like light-transmitting support 2 having a curvature, and a sheet-like mount having a curvature formed on a part of the light-transmitting support 2. It has a placement section 3 . Similar to the cryopreservation jig of FIG. 2, the cryopreservation jig of FIG. Improves workability when removing excess storage solution.

FIG. 4 is a cross-sectional structural schematic diagram showing an example of the mounting portion in the cryopreservation jig of the present invention. In FIG. 4, the mounting portion 3a has a light-transmitting support 2 on which a concave-convex structure 11a composed of a plurality of substantially conical convex structures 10 and concave structures therebetween is formed. Further, a layer 12 containing a water-soluble polymer compound is provided on the concave-convex structure 11a of the light-transmitting support 2 . In the concave-convex structure 11a of the mounting portion 3a, the center-to-center distance between the two closest convex structures 10 is D1, and the height of the concave-convex structure is H1. In the uneven structure 11a, a concave portion 5 having a flat bottom surface is formed between two convex structures 10. As shown in FIG. The mounting portion 3a in FIG. 4 is covered with a layer 12 containing a water-soluble polymer compound so that the convex portion 4 is not exposed. outermost surface) is flat. When the outermost surface of the layer 12 containing the water-soluble polymer compound is flat, it is particularly preferable because a jig for cryopreservation with particularly excellent visibility of cells or tissues can be obtained.

FIG. 5 is a cross-sectional structural schematic diagram showing another example of the mounting portion in the cryopreservation jig of the present invention. The mounting portion 3b in FIG. 5 has a light-transmissive support 2 having an uneven structure 11b formed with a plurality of substantially conical convex structures 10 and concave structures therebetween. A layer 12 containing a water-soluble polymer compound is formed in a shape following the surface shape of the structure 11b. By having such a shape, the projections 4 of the light-transmitting support 2 are not exposed, and an uneven structure is formed on the outermost surface in contact with cells or tissues. In the uneven structure 11b, a concave portion 5 having a flat bottom surface is formed between two convex structures 10. As shown in FIG. In the concave-convex structure 11b of the mounting portion 3b, the center-to-center distance between the two closest convex structures 10 is D2, and the height of the concave-convex structure 11b is H2.

FIG. 6 is a cross-sectional structural schematic diagram showing another example of the mounting portion in the cryopreservation jig of the present invention. The mounting portion 3c in FIG. 6 has a light-transmissive support 2 in which the concave-convex structure 11c is formed more densely than in FIG. 5 described above. The concave-convex structure 11c does not have a flat bottom surface between the convex structures 10 and is in contact with the lowermost portion of the adjacent convex structure. A layer 12 containing a molecular compound is formed. By having such a shape, the projections 4 of the light-transmitting support 2 are not exposed, and an uneven structure is formed on the outermost surface in contact with cells or tissues. In the concave-convex structure 11c of the mounting portion 3c, the center-to-center distance between the two closest convex structures 10 is D3, and the height of the concave-convex structure is H3.

FIG. 7 is a cross-sectional structural schematic diagram showing another example of the mounting portion in the cryopreservation jig of the present invention. The mounting portion 3d in FIG. 7 has a light-transmitting support 2 on which an uneven structure 11d composed of a plurality of substantially truncated cone-shaped convex structures 10 and concave structures therebetween is formed. A light-transmitting support on which an uneven structure 11d is formed in which the lowermost portions of the adjacent projecting structures 10 are in contact with each other without having a flat bottom surface between the projecting structures 10, and the uppermost portions of the projecting structures 10 are flat. 2, and a layer 12 containing a water-soluble polymer compound is formed on the uneven structure 11d. The surface of the layer 12 containing the water-soluble polymer compound located on the uneven structure 11d is flat. In the concave-convex structure 11d of the mounting portion 3d, the center-to-center distance between the two closest convex structures 10 is D4, and the height of the concave-convex structure is H4.

8A and 8B are a schematic top view and a schematic cross-sectional view of the concavo-convex structure shown in FIG. In FIG. 8, the uneven structure 11a has a structure in which substantially conical convex structures 10 having a height H1 are arranged in a substantially triangular lattice, and the distance between the centers of the two nearest substantially conical convex structures 10 is D1. be. The concave portions 5 in the concave-convex structure 11a are formed between adjacent substantially conical convex structures 10 and are continuous.

9A and 9B are a schematic top view and a schematic cross-sectional view of the concavo-convex structure shown in FIG. In FIG. 9, the concave-convex structure 11c has a structure in which substantially conical convex structures 10 with a height H3 are two-dimensionally closely packed in a substantially triangular lattice arrangement. The center-to-center distance is D3. The concave portions 5 in the concave-convex structure 11c are separated from each other by substantially conical convex structures 10, and are independent of each other.

10A and 10B are a schematic top view and a schematic cross-sectional view of the concavo-convex structure shown in FIG. In FIG. 10, the concave-convex structure 11d has a structure in which substantially truncated conical convex structures 10 having a height H4 are two-dimensionally closely packed in a substantially triangular lattice arrangement, and the two nearest substantially truncated conical convex structures The center-to-center distance of 10 is D4. The concave portions 5 of the concave-convex structure 11d are separated from each other by the substantially truncated cone-shaped convex structures 10, and are independent of each other.

FIG. 11 is a schematic model diagram when cells and a preservation solution are dropped and adhered to the placement section having the structure shown in FIG. In FIG. 11, the mounting portion 3a has a layer 12 containing a water-soluble polymer compound having a flat surface on an uneven structure 11a having protrusions 4 and recesses 5. As shown in FIG. As shown in FIG. 11, the cells 6 placed together with the storage solution 7 on the placement portion 3a were placed on the layer 12 containing the water-soluble polymer compound without being in contact with the uneven structure 11a. It is cryopreserved in a cold solvent such as liquid nitrogen.

FIG. 12 is a top structural schematic view showing another example of the mounting portion in the cryopreservation jig of the present invention. In FIG. 12, the mounting portion 3e formed on the light-transmitting support 2 includes a mounting region 8a having an uneven structure and a mounting region 8b having an uneven structure different in shape from the mounting region 8a. It has a parallel and alternating structure. Note that FIG. 12 does not show the details of the uneven structure.

FIG. 13 is a cross-sectional structural schematic diagram for explaining in detail the shapes of the mounting area 8a and the mounting area 8b in FIG. The concave-convex structure 11f of the mounting area 8a has a shape in which a plurality of substantially conical convex structures 10a having a center-to-center distance of D5 between the two nearest convex structures 10a and a height of H5 are arranged. It does not have a flat bottom surface between itself and the structure 10a, and the lowermost portions of the adjacent protruding structures 10a are in contact with each other. On the other hand, the concave-convex structure 11e of the mounting area 8b has a shape in which a plurality of substantially conical convex structures 10b having a center-to-center distance of D6 and a height of H6 between the two nearest convex structures 10b are arranged. A concave portion 5b having a flat bottom surface is formed between the convex structure 10b and the convex structure 10b. Then, the vertical length (height of the base portion) from the bottom surface of the light-transmitting support 2 to the concave portion 5a in the mounting region 8a on the light-transmitting support 2 is equal to the light-transmitting support in the mounting region 8b. By arranging so that the vertical length (height of the base portion) from the bottom surface of the body 2 to the concave portion 5b is small, the mounting regions 8a and 8b are arranged in parallel and alternately. A jig for cryopreservation having such a shape, that is, two types of stripe-shaped regions with different base heights on the light-transmitting support 2 facilitates cells and tissues in the thawing process after cryopreservation. This is preferable because it makes it possible to find out.

Cells or tissues to be cryopreserved in the present invention are placed on a layer containing a water-soluble polymer compound. As described above, when cells or tissues are firmly adhered to the mounting surface of a conventional cryopreservation jig, recovery after thawing may be difficult. In the aforementioned Patent Document 12, a layer containing a water-soluble polymer compound is provided on the surface of the mounting portion that is in contact with the placed cells or tissues, and the layer containing the water-soluble polymer compound dissolves in the melt. Accordingly, a technique for rapidly recovering placed cells or tissues is disclosed. However, as shown in Examples described later, when the surface of a light-transmitting support is smooth and a layer containing a water-soluble polymer compound is provided on the light-transmitting support, it can be placed in the melt. It was sometimes difficult to exfoliate the cells or tissue in a short period of time by gently shaking the placement site. Further, Patent Document 12 describes that the layer containing the water-soluble polymer compound can further contain inorganic fine particles. In some cases, the visibility of embryos and ova was reduced.

In contrast, the present invention can rapidly detach cells or tissues. When the surface of the mounting portion has an uneven structure, the visibility of cells or tissues is not a little affected. It is possible to peel off quickly. Although the action mechanism by which the effect of rapidly exfoliating cells or tissues is obtained in the present invention is not clear, by providing a layer containing a water-soluble polymer compound on an uneven structure composed of fine pitches, , the point at which the melt reaches the interface of the layer containing the water-soluble polymer compound and the light-transmitting support early increases, thereby increasing the peelability of the layer containing the water-soluble polymer compound, and the cells or It is presumed that the recoverability of the tissue is improved. In addition, since the pitch of the uneven structure of the light-transmitting support is as fine as 1.3 μm or less, cells or tissues can be visually recognized with good visibility even under microscope observation.

An example of the method for producing the concave-convex structure of the jig for cryopreservation of the present invention will be described below.
The concave-convex structure formed on the light-transmitting support of the jig for cryopreservation of the present invention is a projection assembly formed by a collection of convex structures as described above. The method for forming such a fine convex structure is not particularly limited. A method of forming a microstructure having a convex structure on a resin surface can be exemplified. An example of forming a concave-convex structure on the surface of a light-transmissive support by transferring a concave structure plate using a thermal nanoimprint method will be described below.

The structure surface of the concave structure plate has a concave structure corresponding to the surface convex structure of the light-transmitting support. The master mold, which is the base of the concave structure plate, uses a Si original plate, a quartz original plate, etc., and is subjected to microfabrication exemplified by photolithography, colloidal lithography, anodization, electron beam lithography, and interference exposure. formed by at least one microfabrication method selected from methods. The concave structure plate is produced from the obtained master mold by at least one mold transfer process, but the mold transfer process may be performed multiple times. A preferred concave structure plate to be produced in the mold transfer process is a Ni electroformed stamper obtained by a Ni electroformed stamper production method. Intermediate steps, such as transferring structures, may be provided.

As a method for manufacturing the above-described master mold, a colloidal lithography method in which fine processing is performed by dry etching using a monoparticle film as an etching mask is preferable. The reason for this is that the colloidal lithography method can easily fabricate microstructures, has the advantage of being able to quickly optimize structures and verify functions, and is suitable for forming large areas. Therefore, there is an advantage that a Ni electroforming stamper (concave structure plate) having a large area for production can be easily manufactured.

In the thermal nanoimprinting step, the structured surface of the concave structure plate is opposed to the surface of the thermoplastic resin for forming the placement section, and set in the pressure section of the nanoimprinting apparatus. The temperature of the concave structure plate and the base material is raised as it is, and a pressure of about 1 to 20 MPa is applied in a region where the temperature is higher than the glass transition point of the base material, and the fine structure of the concave structure plate is transferred to the surface of the thermoplastic resin. do. Next, after the temperature is lowered to a temperature range below the glass transition point of the substrate while the pressure remains, the concave structure plate is released from the surface of the thermoplastic resin. By the above operation, the fine structure of the concave structure plate is inverted and transferred to the surface of the thermoplastic resin, and the concave-convex structure on the surface of the light-transmitting support is obtained.

A method for forming a layer containing a water-soluble polymer compound on a light-transmitting support is not particularly limited. For example, a layer containing a water-soluble polymer compound is formed on a light-transmitting support by applying a coating liquid containing a water-soluble polymer onto the light-transmitting support and then drying the coating solution. be able to. The coating liquid may contain water, a solvent such as an organic solvent, and the like.

The grip part, the fitting structure part and the cap member of the jig for cryopreservation of the present invention can be produced by methods known to those skilled in the art.
An example of a method of connecting the grip portion and the light-transmissive support will be described. When the grip is made of resin, for example, the light-transmissive support can be connected to the grip by insert molding during molding. Furthermore, a structure inserting portion (not shown) can be prepared in the grip portion and the light-transmitting support can be connected with an adhesive. Various types of adhesives can be used, and silicone-based or fluorine-based adhesives that are resistant to low temperatures can be preferably used.

The cryopreservation jig of the present invention is suitable for use in, for example, the cryotop method. In addition, although conventional cryotop methods are commonly used for cryopreservation of embryos or eggs, and are usually used for the preservation of single cells or small numbers of cells, less than 10, the cryopreservation treatment of the present invention is The device can also be used favorably in preserving a larger number of cells (for example, preserving 10 to 1,000,000 cells). Furthermore, it can be suitably used for storage of sheet-like cells (so-called cell sheets) composed of a plurality of cells.

The method for cryopreserving cells or tissues using the cryopreservation jig of the present invention is not particularly limited. Alternatively, remove as much of the excess preservative solution around the tissue as possible with a pipette or the like. Next, the cells or tissues can be frozen by immersing them in liquid nitrogen or the like while holding them on the mounting portion. At this time, a cap member capable of blocking the cells or tissues on the mounting section from the outside world is attached to the mounting section, or the cryopreservation jig is sealed in the container, and liquid nitrogen or the like is used. can also be immersed in As the preservation solution, those commonly used for freezing cells such as eggs and embryos can be used. A large amount of various antifreeze agents such as glycerol, ethylene glycol, DMSO (dimethyl sulfoxide) and the like obtained by containing the storage solution (at least 10% by mass or more, more preferably 20% by mass with respect to the total mass of the storage solution above) can be used. During the thawing operation, the jig for cryopreservation was taken out from a cooling solvent such as liquid nitrogen, and the mounting section on which the frozen cells or tissues were placed was immersed in the thawing liquid and moved into the thawing liquid. Harvest cells or tissues.

Cells that can be cryopreserved using the cryopreservation jig of the present invention include, for example, ova, embryos, and sperm of mammals (e.g., humans, cows, pigs, horses, rabbits, rats, mice, etc.). pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). Also included are cultured cells such as primary cultured cells, subcultured cells, and cell line cells. In one or more embodiments, the cells include fibroblasts, cancer-derived cells such as pancreatic cancer/hepatoma cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, and adherent cells such as immune cells. Furthermore, tissues that can be cryopreserved include tissues composed of allogeneic or heterologous cells, such as ovary, skin, corneal epithelium, periodontal ligament, and myocardium. The present invention is particularly suitable for cryopreservation of tissue having a sheet-like structure (eg, cell sheet, skin tissue, etc.). The jig for cryopreservation of the present invention can be used not only for tissues directly collected from a living body, but also for example, cultured skin cultured and grown in vitro, a so-called cell sheet constructed in vitro, and Japanese Unexamined Patent Application Publication No. 2012-205516. Cryopreservation of artificial tissue, such as the proposed tissue model having a three-dimensional structure, can also be suitably used. 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 below by way of examples, but the present invention is not limited to the following examples. In the examples, the height of the uneven structure and the pattern pitch were measured by scanning electron microscope observation.

(Example 1)
By the colloidal lithography method and the Ni electroforming stamper manufacturing method described in Japanese Patent Application Laid-Open No. 2009-034630, first, a Si surface microstructure master plate ( A convex type) was produced, and then a Ni electroforming stamper (concave type) was produced from this Si original plate. After that, using the Ni electroforming stamper (concave mold), the projecting structure 10 to be the mounting portion was transferred to the surface of the polystyrene sheet having a thickness of 150 μm by a thermal nanoimprint method to prepare the light-transmitting support 2 . Specifically, the structured surface of the Ni electroforming stamper (concave type) was opposed to the surface of a polystyrene sheet having a thickness of 150 μm, and the stamper was set in the pressure unit of the nanoimprinting apparatus. In this state, the temperature was started to rise, and a pressure of 4 MPa was applied at 130° C. for 5 minutes. After 5 minutes had passed, the pressure part was cooled to room temperature, and then the Ni electroforming stamper (concave mold) was released from the surface of the polystyrene sheet.

Through the above operations, a light-transmitting support 2 having the concave-convex structure 11a shown in FIG. 4 on the surface of the polystyrene sheet was obtained. The pitch of the uneven structure 11a was 300 nm, and the height H1 of the uneven structure was 500 nm. Thereafter, Gohsenex (registered trademark) WO-320R (manufactured by Mitsubishi Chemical Corporation), which is polyvinyl alcohol having an ethylene oxide group as a water-soluble polymer compound, is applied to the light-transmitting support 2 having the uneven structure 11a. and 0.5% by mass of Cellny (registered trademark) HPC-L (manufactured by Nippon Soda Co., Ltd.), which is hydroxypropyl cellulose as a water-soluble polymer compound. Solid content) of 1.6 g/m ² , and after drying at room temperature, heat drying was further performed at 40°C for 96 hours. The mounting part produced by the above operation had a flat surface as shown in FIG. 4 from observation of the cross-sectional structure after coating. After that, it was cut into strips of 1.5 mm×20 mm, and joined to the fitting structure part 13 of the ABS-made grip part 1 shown in FIG.

(Example 2)
Example 1 except that the pitch of the approximately conical projection aggregates of the Si surface microstructure master (convex type) used for producing the light-transmitting support 2 was changed to 700 nm, and the height was changed to 570 nm. In the same manner as above, a light-transmitting support 2 shown in FIG. 5 was obtained. The pitch of the uneven structure 11b of the light-transmissive support 2 was 700 nm, and the height H2 of the uneven structure 11b was 570 nm. After that, in the same manner as in Example 1 described above, an aqueous solution containing 2% by mass of Gohsenex WO-320R and 0.5% by mass of Cerny HPC-L was applied at a coating amount (solid content) of 1.6 g / m ² Dip-coating was performed to prepare the mounting portion 3 . The mounting portion 3 produced by the above operation had an uneven structure on the surface as shown in FIG. 5 from observation of the cross-sectional structure after coating. Thereafter, in the same manner as in Example 1, a jig for cryopreservation of Example 2 was produced.

(Example 3)
A microfabrication process was performed by the colloidal lithography method described in WO2019/039485 to produce a Si surface microstructure master plate (convex type) composed of an aggregate of approximately conical convex portions with a pitch of 600 nm. The microstructure of the obtained Si surface microstructure master (convex type) has two types of striped regions with different base heights (mounting regions 8a and 8b in FIG. 13). The vertical length from the bottom surface of the mounting area 8a to the recess 5a is longer than the vertical length of the mounting area 8b from the bottom surface to the recess 5b. Specifically, in the region (8b) with a low base height, an aggregate of approximately conical protrusions with a pitch of 600 nm and a height of 600 nm is formed, and in the region (8a) with a high base height, a pitch of 600 nm, A substantially conical protrusion assembly with a height of 750 nm is formed. The height difference between the base portions of the mounting regions 8a and 8b was 900 nm, and the mounting regions 8a and 8b were arranged in parallel and alternately as shown in FIG. Further, the mounting area 8a and the mounting area 8b have a shape extending linearly by several millimeters or more in a first direction (vertical direction in FIG. 12), and a second direction (vertical direction in FIG. 12) perpendicular to the first direction. 12), and the width (distance in the second direction) of the mounting regions 8a and 8b was 50 μm.

Next, a Ni electroforming stamper (concave mold) was produced from this Si surface fine structure master by the method described in Japanese Patent Laid-Open No. 2009-034630. After that, by a thermal nanoimprint method using the electroforming stamper made of Ni (concave mold), convex portions were transferred and formed on the surface of a polystyrene sheet having a thickness of 150 μm. Specifically, the structured surface of the Ni electroforming stamper (concave type) was opposed to the surface of a polystyrene sheet having a thickness of 150 μm, and the stamper was set in the pressure unit of the nanoimprinting apparatus. In this state, the temperature was started to rise, and a pressure of 4 MPa was applied at 130° C. for 5 minutes. After 5 minutes had passed, the pressure part was cooled to room temperature, and then the Ni electroforming stamper (concave mold) was released from the surface of the polystyrene sheet. By the above operation, on the surface of the polystyrene sheet, a substantially conical projection assembly region (mounting region 8a) with a pitch of 600 nm and a height of 600 nm and a substantially conical projection assembly region (mounting region) with a pitch of 600 nm and a height of 750 nm A sheet having a surface structure in which 8b) was alternately arranged in stripes with a height difference of 900 nm at the base (the height of the mounting region 8a>the mounting region 8b) was obtained. As a result, on the surface of the polystyrene sheet, a structure was formed in which two kinds of uneven structures with different base heights and convex heights were arranged in parallel and alternately. Body 2 was produced. Thereafter, in the same manner as in Example 1, an aqueous solution containing 2% by mass of Gohsenex WO-320R and 0.5% by mass of Cerny HPC-L was dipped at a coating amount (solid content) of 1.6 g/m ² . It coated and produced the mounting part. Observation of the cross-sectional structure after coating of the mounting portion prepared by the above operation revealed that both of the two types of uneven structure portions had an uneven structure on the surface, as shown in FIG. 13 . Thereafter, in the same manner as in Example 1, a jig for cryopreservation of Example 3 was produced.

(Comparative example 1)
A jig for cryopreservation of Comparative Example 1 was produced by using a transparent PET film (thickness: 100 μm) as it was as a mounting portion.

(Comparative example 2)
A transparent PS film (thickness: 150 μm) having no uneven structure was used, and the film contained 2% by mass of Gohsenex WO-320R and 0.5% by mass of Celny HPC-L in the same manner as in Example 1. The aqueous solution was dip-coated at a coating amount (solid content) of 1.6 g/m ² to prepare a mounting portion. Thereafter, in the same manner as in Example 1, a jig for cryopreservation of Comparative Example 2 was produced.

(Comparative Example 3)
A light-transmissive support having an uneven structure on the surface of a polystyrene sheet having a thickness of 150 μm was prepared in the same manner as in Example 1, except that the pitch of the approximately conical projection aggregates was 2 μm and the height was 1.8 μm. made the body. Thereafter, in the same manner as in Example 1, an aqueous solution containing 2% by mass of Gohsenex WO-320R and 0.5% by mass of Cerny HPC-L was dipped at a coating amount (solid content) of 1.6 g/m ² . It coated and produced the mounting part. Observation of the cross-sectional structure after coating showed that the mounting portion prepared by the above operation had an uneven structure on the surface as shown in FIG. Thereafter, in the same manner as in Example 1, a jig for cryopreservation of Comparative Example 3 was produced.

<Preparation of mouse zona pellucida-removed blastocyst>
A male ICR mouse and a female ICR mouse were subjected to in vitro fertilization to produce 2-cell stage embryos. After culturing the obtained 2-cell stage embryos to blastocysts, the zona pellucida was removed by acidic Tyrode treatment to prepare zona-free blastocysts. The diameter of the embryo is about 100 µm.

<Freezing operation of mouse embryos>
The mouse zona pellucida-free blastocyst (mouse embryo) prepared by the above method was collected and equilibrated solution [7.5% by volume dimethyl sulfoxide, 7.5% by volume ethylene glycol, 85% by volume 199 medium [Medium 199 (GE Health care)]]]. After that, the mouse embryos were collected from the equilibration solution, and the stock solution (using the above 199 medium as a base solution, in addition to the composition of the base solution, 15% by volume dimethyl sulfoxide, 15% by volume ethylene glycol, 14% by volume fetal bovine serum , solution containing 0.5 M sucrose) and then immersed for 30 seconds. Thereafter, using a stripper pipetter (manufactured by Orizio Japan), mouse embryos were collected together with a very small amount of preservation solution (approximately 0.4 μl), and the cryopreservation jigs of Examples 1 to 3 and Comparative Examples 1 to 3 were used. Each of them was dripped and deposited on the mounting part. After dripping and adhering, excess preservative solution was removed as much as possible from around the mouse embryo, then immersed in liquid nitrogen, and the mouse embryo on the mount was vitrified and cryopreserved. The frozen mouse embryo and cryopreservation jig were covered with a polypropylene cap (fixed to the fitting structure 13 of the grip 1) and stored in a liquid nitrogen storage container until thawed. .

In addition, the visibility of the mouse embryo on the mounting part during the freezing operation was good in all of the cryopreservation jigs of Examples 1 to 3 and Comparative Examples 1 to 3, and excellent operability was obtained. there were.

<Evaluation of Visibility of Cells or Tissues>
The cryopreservation jigs of Examples 1 to 3 and Comparative Examples 1 to 3 on which the mouse embryos were placed were removed from liquid nitrogen, and the thawed solution at 37 ° C. (the above 199 medium was used as the base solution, and the composition of the base solution was In addition, it was immersed in a solution containing 1M sucrose. After immersion in the lysate, the mouse embryos were observed under a transmission optical microscope, and the ease of observation was evaluated according to the following criteria. These results are shown in Table 1, "Visibility of Cells or Tissues".

The visibility of cells or tissues was evaluated according to the following criteria.
⊚: The mouse embryo on the mounting part was visually recognized particularly well in the lysate.
○: The mouse embryo on the mounting part was easily visible in the melt.
Δ: It was difficult to visually recognize the mouse embryo on the mounting part in the thawing solution.

<Evaluation of detachability of cells or tissues>
Detachability of mouse embryos during the melting operation was evaluated according to the following criteria. These results are shown in Table 1, "Peelability of Cells or Tissues."

○: The mouse embryo could be detached in less than 40 seconds by gently shaking the gripped portion after immersing the cryopreservation jig in the thawing solution.
Δ: The mouse embryo could not be detached within 40 seconds by gently shaking the grip, but the mouse embryo could be detached by shaking the grip slightly strongly.
×: The mouse embryo could not be detached within 60 seconds by gently shaking the gripped part, and the mouse embryo could not be detached even by slightly strongly shaking the gripped part after that. .

From the above results, the cryopreservation jig of the present invention, in the freezing step of placing cells or tissues on the mounting portion of the cryopreservation jig and cryopreserving them, or in the thawing step of thawing the cells or tissues, It can be seen that it has excellent workability that allows easy visual recognition of cells or tissues, and that it has excellent workability that allows rapid collection work.

In addition to embryo transfer and artificial insemination of livestock such as cows and animals, artificial insemination of humans, etc., the present invention also includes iPS cells, ES cells, commonly used cultured cells, and tests collected from living organisms containing embryos or eggs. It can be used for cryopreservation and thawing of cells or tissues for use or transplantation, cells or tissues cultured in vitro, and the like.

Reference Signs List 1 grip portion 2 light-transmitting supports 3, 3a, 3b, 3c, 3d, 3e mounting portions 4, 4a, 4b convex portions 5, 5a, 5b concave portions 6 cells 7 storage solutions 8a, 8b mounting regions 9a, 9b , 9c Cryopreservation jigs 10, 10a, 10b Convex structures 11a, 11b, 11c, 11d, 11e, 11f Concavo-convex structure 12 Layer containing water-soluble polymer compound 13 Interlocking structure part

Claims (1)

It has at least a mounting portion for mounting cells or tissues together with a preservation solution, and the mounting portion comprises a light-transmitting support having an uneven structure and a water-soluble polymer compound on the light-transmitting support. A jig for cryopreservation of cells or tissues, comprising a layer, wherein the light-transmitting support has an uneven structure with a pitch of 1.3 μm or less.

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