JP7232693B2 - Cryopreservation jig - Google Patents

Description

The present invention relates to a cryopreservation jig for cryopreserving 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 estrous cycle of the recipient cow, and transferred. Further, in the treatment of infertility in humans, a procedure is performed in which eggs or ovaries are collected from the mother's body, cryopreserved for appropriate timing for transplantation, and then thawed before transplantation.

In general, cells or tissues collected from within a living body gradually lose their activity even in a culture medium, and therefore long-term culture outside the body is not preferable. Therefore, techniques for long-term storage without loss of bioactivity are important. Good preservation techniques allow the harvested cells or tissues to be analyzed more accurately. In addition, an excellent preservation technique makes it possible to use cells or tissues for transplantation while maintaining higher bioactivity, and an improvement in the survival rate after transplantation can be expected. Furthermore, it is also possible to sequentially 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 in both medical and industrial fields.

For example, a slow freezing method is known as a method for preserving cells or tissues. In this method, cells or tissues are immersed in a preservation solution obtained by adding a cryoprotectant to a physiological solution such as phosphate-buffered saline. Compounds such as glycerol and ethylene glycol are used as the antifreeze agent. After immersing the cells or tissue in the preservation solution, cooling to −30 to −35° C. at a relatively slow cooling rate (eg, 0.3 to 0.5° C./min) will The stock solution cools sufficiently and becomes viscous. In such a state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (−196° C.), the microfluidic solution in and around the cells or tissues becomes amorphous. Vitrification occurs where the solid remains. 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, it is thought that vitrified cells or tissues can be preserved semi-permanently by storing them in liquid nitrogen.

For example, Patent Document 1 describes the cryopreservation of porcine embryos by the slow freezing method, and Patent Document 2 describes the preservation of bovine embryos by the slow freezing method.

However, since the slow freezing method cools cells or tissues at a relatively slow cooling rate, it takes time to operate for cryopreservation. There is also the problem of requiring a device or jig for controlling the cooling rate. In addition, since ice crystals are formed in the extracellular or extracellular preservation solution, the cells or tissue may be physically damaged by the ice crystals.

On the other hand, the vitrification freezing method is known as a preservation method that solves the problems of the slow freezing method described above. 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 DMSO (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 a cryoprotectant used for vitrification freezing is called a vitrification solution.

Vitrification involves immersing cells or tissues in a vitrification solution followed by rapid cooling to the temperature of liquid nitrogen (−196° C.). Since the vitrification freezing method is such a simple and quick process, it does not require a long time for cryopreservation, and it does not require a device or jig for temperature control. There is

Cryopreservation of cells or tissues using vitrification has been exemplified in a variety of ways and with different types of cells or tissues. For example, Patent Document 3 shows that the application of the vitrification freezing method to animal, human germ cells or somatic cells is extremely useful. Non-Patent Document 1 shows that vitrification is effective for preservation of Drosophila embryos. Furthermore, Patent Document 4 shows that the vitrification freezing method is effective in preserving cultured plant cells and tissues.

However, high concentrations of cryoprotectant contained in the vitrification fluid are chemically toxic. For this reason, during cryopreservation of cells or tissues, it is desirable that the amount of vitrification fluid that exists around the cells or tissues is small, and that the time during which cells are exposed to the preservation fluid, that is, the time until freezing is short. . Furthermore, it is necessary to dilute the stock solution immediately after thawing.

Patent Documents 5 and 6 describe cryopreservation jigs having strip-shaped, flexible, colorless and transparent films as strips for retaining adherence of eggs. Such documents limit the width of the strip to reduce the amount of vitrifying fluid present around the cells or tissue.

Patent Literature 7 describes a cryopreservation jig having a preservative solution removing material that is a natural product such as a wire mesh or paper, or a film-like product made of a synthetic resin and has through holes. In such documents, eggs or embryos are placed on a storage medium removing material together with a vitrification liquid, and excess vitrification liquid adhering around the eggs or embryos is removed by aspirating from the bottom of the storage medium removing material. .

As a cryopreservation jig that can remove excess vitrification fluid from the bottom without aspirating it and that has improved workability when placing cells, Patent Document 8 discloses a storage device with a specific haze value. A jig for cryopreservation using a liquid absorber is disclosed in Patent Document 9, and a jig for cryopreservation using a storage liquid absorber having a specific refractive index is disclosed in Patent Document 10. Each jig for cryopreservation is described, which has a porous structure formed of a material having as a preservation liquid absorber.

In addition, regarding a jig for cryopreservation having a storage fluid absorber as described above, Patent Document 11 describes an example in which the storage fluid absorber and a support are partially adhered without an adhesive layer. Are listed. Furthermore, in Patent Document 12, a strip-shaped flexible and colorless transparent film is provided as a strip for retaining adhered eggs, and a defect surrounded by a water-absorbing portion (storage liquid absorber) on a part of the film. An example of a jig for cryopreservation that does not require an operation for removing excess vitrification fluid by providing a part is described.

JP-A-9-122158 Japanese Patent Application Laid-Open No. 2005-261413 Japanese Patent No. 3044323 JP-A-2008-5846 JP-A-2002-315573 JP 2006-271395 A International Publication No. 2011/070973 pamphlet JP 2014-183757 A International Publication No. 2015/064380 pamphlet JP 2015-188405 A JP 2016-10359 A WO2019/004300 pamphlet

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

The jigs for cryopreservation described in Patent Documents 5 and 6 describe a method of cryopreserving eggs or embryos together with a small amount of vitrification liquid by limiting the width of the film on which the eggs or embryos are placed. It is In this method, an operator places ova or embryos on a film together with a very small amount of vitrification fluid. There was a problem that the difficulty of In addition, in the Cryotop (registered trademark) method based on this method, in order to cryopreserve eggs or embryos together with a smaller amount of vitrification fluid, once the eggs or embryos are placed on a film together with the vitrification fluid, In some cases, complicated operations such as sucking the excess vitrification liquid and removing it from the film have been performed.

The jig for cryopreservation described in Patent Document 7 removes excess vitrification fluid attached around the ovum or embryo with a vitrification fluid removal material, thereby freezing these reproductive cells with an excellent survival rate. Suggested ways to save. This method requires a suction operation from the bottom to remove the excess vitrification fluid, which is a complicated operation and difficult to perform the vitrification cryopreservation operation in a short time.

The jigs for cryopreservation described in Patent Documents 8 to 11 solve all of the above problems, and are effective in easily and reliably performing cryopreservation of cells or tissues. In these cryopreservation jigs, since the excess preservation liquid is absorbed by the preservation liquid absorber of the cryopreservation jig, there is no need for complicated operations as in the other cryopreservation jigs described above. . In addition, due to the inherent haze of the storage liquid absorber, the visibility of the placement location where cells or tissues are placed is excellent under observation with a transmission microscope. Since the total light transmittance of the preservation liquid absorber increases as the preservation liquid is absorbed, the visibility of the placed cells or tissues is also improved. However, the preservation liquid absorber used in these cryopreservation jigs is an image of the tip of a pipette used for dropping the cells or tissue together with the preservation liquid onto the placement site when the cells or tissues are placed. is hidden in the image of the storage liquid absorber, making it difficult to grasp the state of the tip of the pipette.

In the cryopreservation jig described in Patent Document 12, there is no preservation liquid absorber at the place where cells or tissues are placed, and although it is easy to check the state of the pipette tip, the placed cells or tissues are not Since the lower region does not have the ability to absorb the storage solution, there is a problem that excess vitrification solution tends to remain.

However, the present invention provides excellent visibility of the mounting portion and the tip of the pipette when dropping cells or tissues together with a preservation solution onto the mounting portion of a cryopreservation jig under microscopic observation, and Another object of the present invention is to provide a jig for cryopreservation that does not require the work of removing excess preservation liquid around tissue.

As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be solved by a jig for cryopreservation having the following configuration.

A jig for cryopreservation having a porous preservation liquid absorbent, wherein the preservation liquid absorbent includes a mounting section on which cells or tissues are mounted, and a mounting section on which the cells or tissues are not mounted. A jig for cryopreservation, characterized in that it has an adjoining portion having higher absorption than the portion, and the total light transmittance of the mounting portion is 40% or more.

According to the above invention, when dropping cells or tissues together with a preservation solution onto the mounting portion of a cryopreservation jig under microscope observation, the mounting portion and the tip of the pipette are highly visible, and the mounting portion It is possible to provide a jig for cryopreservation that does not require the work of removing excess preservation solution around cells or tissues.

1 is an overall view showing an example of a jig for cryopreservation of the present invention; FIG. 1. It is the schematic which looked at the mounting part in the jig|tool for cryopreservation of FIG. 1 from the mounting surface side which mounts a cell or a tissue. FIG. 2 is a schematic cross-sectional view of a mounting portion in the cryopreservation jig of FIG. 1; It is another example of the mounting part in the jig for cryopreservation of the present invention. It is another example of the mounting part in the jig for cryopreservation of the present invention. It is another example of the mounting part in the jig for cryopreservation of the present invention. It is an example of the cross-sectional structure schematic of the mounting part in the jig|tool for cryopreservation of this invention.

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

The cryopreservation jig of the present invention is used for cryopreserving cells or tissues. Examples of such a cryopreservation protocol include the slow freezing method and the vitrification freezing method described above, but the present invention is particularly suitable for the vitrification freezing method.

As used herein, a cell includes not only a single cell but also a cell population 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, in the present specification, a tissue may be a tissue composed of a single type of cell, or a tissue composed of a plurality of types of cells. It may contain cellular substances.

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.). germ cells such as; 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 are fibroblasts, cancer-derived cells such as pancreatic cancer/hepatoma cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, and Adherent cells such as immune cells are included. Furthermore, tissues that can be cryopreserved include tissues composed of allogeneic or xenogeneic cells, such as ovary, skin, corneal epithelium, periodontal ligament, and myocardium. The cryopreservation jig of 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 with three-dimensional structure, can also be suitably used. The present invention is preferably used in the cryopreservation method for cells or tissues as described above.

The cryopreservation jig of the present invention has a porous preservation liquid absorbent. The term "porous" in the present invention means a structure having pores (pores), and it is more preferable to have continuous pores on the surface and inside of the storage liquid absorbent. Examples of the storage liquid absorber include various sheets such as a sheet made of fibers and a porous resin sheet.

In the present invention, a nonwoven fabric is exemplified as a sheet made of fibers used as a storage liquid absorbent. 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 made from cellulose fibers, polyester fibers, nylon fibers, acrylic fibers, and polypropylene. fibers, polyethylene fibers, polyvinyl chloride fibers, vinylidene fibers, polyurethane fibers, vinylon (polyvinyl alcohol) fibers, glass fibers, silk fibers, etc., and non-woven fabrics in which various types of these fibers are mixed can also be used. Among them, cellulose fibers, rayon fibers and cupra fibers, which are regenerated cellulose fibers derived from cellulose fibers, and acetate fibers, which are semi-synthetic fibers from cellulose fibers, are preferable.

The nonwoven fabric described above preferably has a density of 0.1 to 0.4 g/cm ³ and a basis weight of 10 to 130 g/m ² . In particular, nonwoven fabrics with a density of 0.12 to 0.3 g/cm ³ and a basis weight of 10 to 100 g/m ² are excellent in the absorption of storage solutions and have excellent visibility of cells or tissues. This is preferable because it makes it possible to provide a storage jig.

The nonwoven fabric preferably contains 10% by mass or less of a binder component such as a binder, more preferably 5% by mass or less, and more preferably 3% by mass or less. A nonwoven fabric containing no binder is particularly preferred.

Unlike paper, nonwoven fabrics have various manufacturing methods, but nonwoven fabrics with reduced binder components include nonwoven fabrics manufactured by the spunbond method and meltblown method, and also by arranging fibers by a wet method or a dry method. A nonwoven fabric manufactured by a hydroentangling method or a needle punching method can be preferably used. As described above, preferred fibers contained in the nonwoven fabric in the present invention include cellulose fibers, rayon fibers and cupra fibers that are regenerated cellulose fibers derived from cellulose fibers, and acetate fibers that are semi-synthetic fibers from cellulose fibers. However, when these fibers are used for production, a hydroentangling method or a needle punching method is suitable regardless of whether it is a wet method or a dry method.

The porous resin sheet used as the storage liquid absorber in the present invention includes, for example, those described in JP-B-42-13560 and JP-A-08-283447. A resin sheet having a porous structure 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. Examples include a resin sheet having a porous structure formed by filling a mold with solid powder, heating and sintering to fuse the surfaces of the powder particles, and then cooling. When a porous resin sheet is used as a preservation liquid absorber, it is possible to provide a jig for cryopreservation with excellent preservation liquid absorption and excellent visibility of cells or tissues, which is more preferable. .

As the resin for forming the porous resin sheet described above, various types of polyethylene such as low-density polyethylene, high-density polyethylene, and ultra-high-molecular-weight polyethylene; Resin, ethylene-vinyl acetate copolymer, polyamide, styrene-acrylonitrile copolymer, styrene-butadiene-acrylonitrile terpolymer, polycarbonate, polyvinyl chloride and the like. Among them, a porous resin sheet made of a fluororesin such as polytetrafluoroethylene or polyvinylidene difluoride, when cells or tissues are placed on the resin sheet together with a preservative solution, allows visual confirmation of the cells or tissues under transmission microscope observation. This is preferable because it enables provision of a jig for cryopreservation that is particularly excellent in the visibility of cells or tissues due to a dramatic increase in performance. As the porous resin sheet, a membrane filter for filtration, which is commercially available for physical and chemical experiments and research purposes, can also be used.

The jig for cryopreservation of the present invention has the above-described porous preservation liquid absorber, and the preservation liquid absorber includes a mounting section on which cells or tissues are mounted, and a mounting section on which cells or tissues are mounted. It has an adjoining portion that is less absorbent than the resting portion. The mounting portion for mounting cells or tissues in the present invention is a portion corresponding to a portion where cells or tissues are mounted together with a preservation solution. It is characterized by arranging an adjacent portion having a higher absorbency than the portion. The adjacent portion may be arranged so as to surround the placing portion, or may be arranged so as to be adjacent to a part of the placing portion. As a specific example in which the adjacent portion is adjacent to a part of the mounting portion, when the mounting portion has a polygonal shape when viewed from the mounting surface on which cells or tissues are mounted, one side of the polygon When the mounting portion is circular, the state where the adjacent portion is in contact with part of the circumference is exemplified. It is preferable that the parts are in contact with each other.

The porosity of the mounting portion is preferably 1% by volume or more, more preferably 5% by volume or more, from the viewpoint of absorption of the storage solution. The upper limit is preferably less than 35% by volume. Moreover, it is preferable that the pores inside the storage liquid absorber have a continuous structure not only in the thickness direction but also in the direction perpendicular to the thickness direction. With such a structure, the pores inside the porous body can be effectively used, resulting in high storage liquid absorption performance. The thickness of the storage liquid absorber of the mounting section and the void volume of the porous body can be appropriately selected according to the type of cells or tissues to be used, the amount of the storage liquid to be dropped together with the cells or tissues, and the like.

The above porosity is defined by the following formula. Here, the void volume V is the cumulative pore volume (ml/g ) by the dry solid content (g/m2) of the storage liquid absorber, it can be obtained as a numerical value per unit area (m2).
P = (V/T) x 100 (%)
P: porosity (%)
V: void volume (ml/m ² )
T: thickness (μm)

The pore diameter of the mounting portion is preferably 0.01 to 10 μm, more preferably 0.01 to 2 μm. In addition, in the case of a porous resin sheet, the pore diameter is the diameter of the largest pore measured by a bubble point test.

The mounting portion has a total light transmittance of 40% or more. When the total light transmittance of the mounting part is less than 40%, when dropping the cells or tissue together with the preservation solution onto the mounting part of the cryopreservation jig, the tip of the pipette can be well visualized under observation with a transmission microscope. It may not be possible to obtain sexuality. The total light transmittance of the mounting portion can be measured according to JIS-K-7361-1:1997 using, for example, a haze meter HZ-V3 (manufactured by Suga Test Instruments Co., Ltd.).

The jig for cryopreservation of the present invention has an adjoining portion having a higher absorbency with respect to the storage solution around the mounting portion described above.

The porosity of the adjacent portion is preferably 35% by volume or more, more preferably 50% by volume or more, from the viewpoint of absorption of the storage solution. The upper limit is preferably less than 90% by volume. It is preferable that the pores inside the storage liquid absorbent used in the adjacent portion also have a continuous structure not only in the thickness direction but also in the direction perpendicular to the thickness direction. The thickness of the preservation liquid absorbent in the adjacent portion and the porosity of the porous body can also be appropriately selected according to the type of cells or tissues to be used and the amount of the preservation liquid to be dropped together with the cells or tissues. The porosity of the adjacent portion can also be obtained by the same method as for the porosity of the mounting portion described above.

The pore diameter of the adjacent portion is preferably 0.02 to 20 μm, more preferably 0.02 to 5 μm.

The area of the mounting part and adjacent parts may be appropriately set according to the amount of the preservation solution to be dropped together with the cells or tissue, and is not particularly limited, but for example, 1 mm ² or more per 1 μl of the preservation solution to be dropped. and more preferably 2 to 400 mm ² . The mounting portion may be located anywhere on the storage liquid absorbent, and its area conforms to the area of the storage liquid absorbent.

The thickness of the storage liquid absorbent constituting the mounting portion and the adjacent portion is preferably 10 μm to 5 mm, more preferably 20 μm to 2.5 mm. The thickness of the storage liquid absorber is the maximum thickness of the storage liquid absorber that either the placement section or the adjacent section has.

In the jig for cryopreservation of the present invention, the part having the storage fluid absorber can be composed only of the storage fluid absorber. It is preferred to have Such a light-transmitting support preferably does not reduce the total light transmittance of the mounting portion when laminated with the storage liquid absorbent, and preferably has a total light transmittance of 80% or more. Examples of such a light-transmitting support include various resin films, glass, rubber, and the like, and two or more light-transmitting supports may be used in combination as long as the effects of the present invention are not impaired. Among the above, the resin film is easy to handle and is preferably used. Specific examples of resin films include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, polycarbonate resins, acrylic resins such as polymethyl methacrylate, methyl methacrylate-styrene copolymers, and methyl methacrylate-polycarbonate copolymers, low-density Various types of polyethylene such as polyethylene, high-density polyethylene, ultra-high molecular weight polyethylene, polypropylene, polystyrene, fluororesins such as polytetrafluoroethylene and polyvinyl difluoride, polyvinyl chloride, epoxy resins, silicone resins, diacetate resins, triacetate resins, Examples include polyarylate resins, polysulfone resins, polyethersulfone resins, polyimide resins, polyamide resins, polyolefin resins, and the like. The above-mentioned total light transmittance can be measured according to JIS-K-7361-1:1997, for example, by the above-mentioned haze meter HZ-V3 manufactured by Suga Test Instruments Co., Ltd.

It is preferable to provide an adhesive layer between the storage liquid absorbent and the support. The adhesive layer preferably contains a water-soluble polymer compound or a water-insoluble resin, which will be described later, or an instant adhesive composition, a hot-melt adhesive composition, or a photocurable adhesive typified by a moisture-curable adhesive substance. It is also possible to contain compositions and the like. Examples of water-soluble polymer compounds include polyvinyl alcohol, hydroxycellulose, polyvinylpyrrolidone, and starch glue. Examples of water-insoluble resins include vinyl acetate-based resins, acrylic-based resins, epoxy-based resins, urethane-based resins, Elastomer resin, cyanoacrylate resin, fluorine resin, silicone resin, nitrocellulose resin, nitrile rubber resin, styrene-butadiene resin, urea resin, styrene resin, phenol resin, polyimide resin, polyamide resins, polyester resins, bismaleimide resins, olefin resins, EVA resins, and the like. The adhesive layer may contain one type of resin, or may contain a plurality of types of resin. The solid content of the adhesive layer is preferably in the range of 0.01-100 g/m ² , more preferably in the range of 0.1-50 g/m ² .

The adhesive layer described above is preferably provided at a location (adjacent portion) outside the placement portion. If an adhesive layer is present between the mounting portion and the support, it may adversely affect the visibility of cells or tissue under microscopy. As a specific example for providing the adhesive layer outside the mounting portion, the support is masked with a masking tape, and after the adhesive layer is laid, the masking tape is peeled off, and the position corresponding to the adjacent portion on the support is removed. An adhesive layer is provided on the substrate, a portion where no adhesive layer is present is provided on a portion corresponding to the mounting portion on the support, and the storage liquid absorber is adhered onto the adhesive layer.

A method of forming a mounting portion and an adjacent portion on a storage liquid absorber will be exemplified. For example, a square metal plate having smaller long and short sides than the storage liquid absorber is hot-pressed on one side of the storage liquid absorber, and some of the pores of the storage liquid absorber are uniaxially pressed. By compressing in the pressure direction, a mounting portion having a total light transmittance of 40% or more and having a low absorption with respect to the storage solution and an adjacent portion having a higher absorption than the mounting portion are formed (Method 1) is exemplified. In addition, a storage liquid absorber having two or more types of porosity with different storage liquid absorbability is arranged adjacent to one side of a support, and a storage liquid absorber having a total light transmittance of 40% or more and a low absorption is provided. is used as the mounting portion, and the storage liquid absorber with higher absorbency is used as the adjacent portion (Method 2). Another method is to infiltrate a part of the pores of the storage liquid absorber with a resin or a water-soluble polymer compound that has excellent permeability and fluidity, and with some of the pores blocked, the resin or highly water-soluble A method (Method 3) of forming a mounting portion having a total light transmittance of 40% or more and an adjacent portion having a higher absorption than the mounting portion by immobilizing a molecular compound is exemplified. As another method, a partially embossed nonwoven fabric is trimmed, the embossed part of the nonwoven fabric is used as a mounting part having a total light transmittance of 40% or more, and the unprocessed part is a highly absorbent material. A method (Method 4) using it as an adjacent portion is exemplified.

The cryopreservation jig of the present invention may have a grip portion. It is preferable to have a grip part because workability during cryopreservation work and thawing work is improved. The grip is preferably made of liquid nitrogen resistant material. 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 cryopreservation jig of the present invention, the cryopreservation jig is capped to block the cells or tissue from the outside world, or the cryopreservation jig is closed. It can be sealed in a container of any shape. If the liquid nitrogen is not sterilized and the cells or tissues are frozen by direct contact with the liquid nitrogen, sterility may not be guaranteed even if the cryopreservation jig is sterilized. Therefore, the cells or tissues are frozen without direct contact with liquid nitrogen by capping the storage solution absorber to which the cells or tissues are attached before freezing, or by sealing the cryopreservation jig in the container. Sometimes. For the above reason, the cap and container are preferably made of liquid nitrogen-resistant materials such as various metals, various resins, glass, and ceramics. The shape of the cap is not particularly limited, and examples thereof include semi-spindle-shaped or dome-shaped caps such as pencil caps, cylindrical straw caps, and the like. Any shape may be used as long as it can block cells or tissues from the outside world. The shape of the container is not particularly limited as long as it can cover or house the cryopreservation jig and seal it without contacting the placed cells or tissues.

The cryopreservation jig of the present invention can be used in combination with the above caps and containers as long as the effects of the present invention are not impaired. The present invention also includes such a jig for cryopreservation that is used in combination with a cap or a container.

The cryopreservation jig of the present invention is suitable for use in, for example, the cryotop method. Also, while conventional cryotop methods are typically used for the preservation of single cells or a small number of cells, less than 10, the present invention can be used to preserve larger numbers of cells (e.g., 10-1,000,000 cells). storage) can also be suitably used. By using the present invention, it is possible to quickly remove excess preservation liquid present around cells or tissues with a preservation liquid absorber.

The procedure for cryopreserving cells or tissues using the cryopreservation jig of the present invention is not particularly limited. Examples are shown below. First, the cells or tissue immersed in the preservation solution are dropped onto the mounting part together with the preservation solution. Surrounding the cells or tissue is excess storage solution, but the storage solution absorber in the mounting section and the adjoining section (particularly by the highly absorbent adjoining section) automatically removes the excess preservation liquid. and promptly removed. Next, the cells or tissues are frozen by being immersed in liquid nitrogen or the like while being held on the mounting portion. At this time, a cap capable of blocking the cells or tissues on the mounting part from the outside world is attached to the preservation liquid absorber, or the cryopreservation jig is sealed in the container and placed in liquid nitrogen or the like. can also be immersed in Preservation solutions that are usually used for freezing cells such as ova and embryos can be used. and a large amount of various antifreeze agents such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide) (at least 10% by mass or more, more preferably 20% by mass of the total mass of the storage solution). % or more) can be used. During the thawing operation, the jig for cryopreservation is taken out from a cooling solvent such as liquid nitrogen, and the vicinity of the mounting part on which the frozen cells or tissues are placed is immersed in the thawing solution, and preserved as necessary. Cells or tissues are recovered by, for example, shaking the liquid absorber in the lysate.

The structure of the cryopreservation jig of the present invention will be described in more detail below with reference to the drawings.

FIG. 1 is an overall view showing an example of a jig for cryopreservation of the present invention. In FIG. 1, the cryopreservation jig 6 has a grip portion 1 to which a support 5 is connected. A mounting portion 2 surrounded by an adjacent portion 3 is provided in the vicinity of the portion.

In FIG. 1, the grip part 1 has a cylindrical shape, but its shape is arbitrary. In addition, as described above, in order to prevent the cells or tissues from directly contacting liquid nitrogen, the placement section 2 to which the cells or tissues are attached before freezing may be covered with a cap. 1 is formed into a shape (tapered shape) in which the diameter of the column continuously decreases from the side without the mounting portion 2 toward the side with the mounting portion 2, so that the cap is put on. It is also possible to improve the The shape of the mounting portion 2 is preferably strip-like or sheet-like in terms of handling.

A method of connecting the grip portion 1 and the support 5 shown in FIG. 1 will be described. If the grip portion 1 is made of resin, for example, the support 5 can be connected to the grip portion 1 by insert molding during molding. Furthermore, it is also possible to prepare a structure insertion portion (not shown) in the grip portion 1 and connect the support 5 with an adhesive. Various types of adhesives can be used, and silicon-based or fluorine-based adhesives that are resistant to low temperatures can be preferably used.

FIG. 2 is a schematic view of the mounting portion of the cryopreservation jig in FIG. A mounting portion 2a is provided in a storage liquid absorbent adhered to a support 5 (not shown). 3 is a schematic cross-sectional view of the mounting portion of the jig for cryopreservation of FIG. 1, and is a schematic cross-sectional view of the cross-sectional structure taken along line AA' of FIG. As a method for obtaining a jig for cryopreservation having the mounting portion 2a of such a structure, the mounting portion 2a and the adjacent portion 3a are formed in the storage liquid absorbent by the method 1 described above, and the storage liquid absorbent is formed. For example, after trimming into strips, the storage liquid absorber and strip-shaped support 5 are pasted together via an adhesive layer 4 formed on the support 5 .

FIG. 4 is another example of the mounting portion in the jig for cryopreservation of the present invention, and is a schematic diagram in which adjacent portions 3b are provided in two directions facing each other around the mounting portion 2b. For example, in Method 1 described above, the mounting portion 2b having such a structure is formed by using the metal plate used for hot pressing with one side longer than the short side of the storage liquid absorber and the other side longer than the long side of the storage liquid absorber. obtained by making it short.

FIG. 5 shows still another example of the mounting portion in the jig for cryopreservation of the present invention, in which a plurality of square mounting portions 2c surrounded by adjacent portions 3c are attached to a support 5 (not shown). It is provided in a preservative liquid absorbent. The mounting portion 2c having such a structure can be obtained, for example, by hot-pressing the metal plate in the method 1 described above while shifting the position a plurality of times in the long side direction of the storage liquid absorber.

FIG. 6 is still another example of the mounting portion in the jig for cryopreservation of the present invention, and is a schematic diagram in which a single mounting portion 2d of a perfect circle surrounded by adjacent portions 3d is provided. The mounting portion 2d having such a structure can be obtained, for example, by using the metal plate used for hot pressing in Method 1 described above as a circular plate having a smaller diameter than the short side of the storage liquid absorber.

FIG. 7 is an example of a schematic cross-sectional view of the mounting portion of the jig for cryopreservation of the present invention. 1. It is a schematic diagram of the cross-sectional structure between BB' in FIG. The mounting portion 2e having such a structure can be obtained, for example, by Method 2 described above.

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

(Example 1)
As a storage liquid absorber, a part of one side of a polytetrafluoroethylene porous body (pore diameter 0.2 μm, porosity 71%, thickness 35 μm, total light transmittance 28.4%) manufactured by Advantech Toyo Co., Ltd. Flat nickel plates (1 mm×1 mm) were superimposed and hot pressed at a temperature of 250° C. using a roll-type embossing machine EMBOSTAR (registered trademark) manufactured by Saitowengineers. After hot-pressing, the nickel plate was removed, and then cut to 1.5 mm×10 mm to obtain a storage liquid absorber having a mounting portion 2a and an adjacent portion 3a formed in the form shown in FIG. On the other hand, as a support, Lumirror (registered trademark) T60 (short side: 1.5 mm, long side: 25 mm, thickness: 188 μm, total light transmittance: 88.9%), which is a polyethylene terephthalate film manufactured by Toray Industries, Inc., was used. A masking tape (thickness: 55 μm) manufactured by Teraoka Seisakusho Co., Ltd. is used on the polyethylene terephthalate film to attach the adhesive layer 4 (adjacent portion 3a on the support 5) as shown in FIG. The adhesive layer 4 is present only at the joint) is masked so that it can be formed, and hot-melt urethane resin Purmelt (registered trademark) QR manufactured by Henkel Japan Co., Ltd. is used as the adhesive layer 4 on the polyethylene terephthalate film after the masking. 170-7141P was applied to give a dry solids content of 30 g/m ² . After the adhesive layer 4 is applied, the masking tape is peeled off, and while the adhesive has fluidity, the hot-pressed storage liquid absorber is attached so that the non-coated portion of the adhesive layer 4 and the mounting portion 2a overlap. Matched. In order to increase the adhesion between the storage liquid absorber and the support 5, the nickel plate was lightly pressed against the mounting portion 2a again, and then aged at room temperature for 24 hours to accelerate the hardening of the adhesive layer 4. After that, one end of the support 5 on the side not having the storage solution absorber was joined to the gripping part 1 made of ABS resin to obtain the jig for cryopreservation of Example 1 in the form shown in FIG. rice field. The total light transmittance of the mounting portion 2a of Example 1 was measured according to JIS-K-7361-1:1997 with a haze meter HZ-V3 (manufactured by Suga Test Instruments Co., Ltd.). was 9%. Further, when the porosity of the placing portion 2a of Example 1 was measured by the method described above, it was 6%, which was lower than the adjacent portion 3a (porosity of 71%).

(Example 2)
A jig for cryopreservation of Example 2 was obtained in the same manner as in Example 1, except that the temperature condition during hot pressing was 150°C. The total light transmittance of the mounting portion 2a of the cryopreservation jig of Example 2 was 53.9%, and the porosity was 18%.

(Example 3)
A jig for cryopreservation of Example 3 was obtained in the same manner as in Example 1, except that the temperature condition during hot pressing was 40°C. The total light transmittance of the mounting portion 2a in the cryopreservation jig of Example 3 was 43.1%, and the porosity was 30%.

(Comparative example 1)
In Example 1, the polytetrafluoroethylene porous material manufactured by Advantech Toyo Co., Ltd. (pore size: 0.2 μm, porosity: 71%, thickness: 35 μm, total light transmittance: 27.3%) was stored without hot pressing. A jig for cryopreservation of Comparative Example 1 was obtained in the same manner as in Example 1 except that it was used as a liquid absorber. The mounting portion of the jig for cryopreservation of Comparative Example 1 had a total light transmittance of 27.3% and a porosity of 71%.

(Comparative example 2)
In Example 1, a jig for cryopreservation of Comparative Example 2 was obtained by removing the storage liquid absorber of the mounting portion 2a with a cutter knife to expose the polyethylene terephthalate film of the support. The mounting portion of Comparative Example 2 had a total light transmittance of 88.9% and a porosity of 0%.

<Freezing operation of mouse ova>
For each of the cryopreservation jigs of Examples 1 to 3 and Comparative Examples 1 and 2, Vit Kit glass manufactured by Irvine Scientific, which is commonly used for vitrified preservation of cells or tissues, was used as the preservation solution. Using the vitrifying liquid, 0.2 μL of the vitrifying liquid and mouse ova having a diameter of about 100 μm were dripped onto the mounting portion with a micropipette under observation with a stereoscopic microscope. In this operation, the visibility of the mounting portion and the tip of the pipette under microscope observation, and the absorbability of the storage fluid absorber for excess vitrification fluid around mouse ova were evaluated according to the following criteria.

<Evaluation Criteria for Visibility of Mounting Unit and Pipette>
◯: Under microscope observation, the mounting portion and the tip of the pipette could be clearly observed.
x: Under microscope observation, the mounting portion could be clearly observed, but the tip of the pipette could not be seen well.

<Evaluation Criteria for Absorbency of Storage Liquid Absorber>
◯: Excess vitrification fluid around the mouse ovum was absorbed into the storage fluid absorber in less than 10 seconds.
x: The excess vitrification fluid around the mouse ovum was not absorbed by the storage fluid absorber even after 10 seconds or more, and the operator felt the need to remove the excess vitrification fluid by aspiration.

From the results in Table 1, the jig for cryopreservation of the present invention has good visibility of the mounting portion and the tip of the pipette under microscope observation, and removes excess preservation solution around cells or tissues in the mounting portion. It can be seen that it is a jig for cryopreservation that does not require

In addition to embryo transfer and artificial insemination of livestock such as cows and animals, and artificial insemination of humans, the present invention also provides iPS cells, ES cells, commonly used cultured cells, and test or transplantable cells collected from living organisms. It can be used for cryopreservation of cells or tissues, cells or tissues cultured in vitro, and the like.

1 Gripping portion 2, 2a to 2e Mounting portion 3, 3a to 3e Adjacent portion 4 Adhesive layer 5 Support body 6 Cryopreservation jig

Claims (1)

A jig for cryopreservation having a porous preservation liquid absorbent, wherein the preservation liquid absorbent includes a mounting section on which cells or tissues are mounted, and a mounting section on which the cells or tissues are not mounted. A jig for cryopreservation, characterized in that it has an adjoining portion having higher absorption than the portion, and the total light transmittance of the mounting portion is 40% or more.

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