JP2015002681A - Jig for vitrification cryopreservation of cells or tissues - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a jig for vitrification cryopreservation which can certainly maintain cells or tissues even if at the time of defrosting due to having an excellent vitrified liquid absorption performance.SOLUTION: A jig for vitrification cryopreservation of cells or tissues has a vitrified liquid absorber on a metal support and the vitrified liquid absorber has an adhesive line and a vitrified liquid absorption layer in this order from a position near a metal support.

Description

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

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

  In general, since cells or tissues collected from within a living body gradually lose their activity even in a culture solution, long-term culture of cells or tissues in vitro is not preferable. Therefore, a technique for storing for a long time without losing biological activity is important. Superior storage techniques allow for more accurate analysis of harvested cells or tissues. Moreover, it can be used for transplantation while maintaining higher biological activity by an excellent storage technique, and it can be expected that the survival rate after transplantation is improved. Furthermore, artificial tissue for transplantation, such as cultured skin cultured in vitro or so-called cell sheets constructed in vitro, can be produced and stored sequentially, and can be used when necessary. Not only medical aspects but also industrial advantages can be expected.

  As a method for preserving cells or tissues, for example, a slow freezing method is known. In this method, cells or tissues are first immersed in a preservation solution obtained by adding a freezing agent to a physiological solution such as phosphate buffered saline. As the antifreeze, compounds such as glycerol and ethylene glycol are used. After immersing cells or tissues in the preservation solution, the cells or tissues are cooled or cooled to −30 to −35 ° C. at a relatively slow cooling rate (for example, 0.3 to 0.5 ° C./min), thereby allowing intracellular or extracellular or tissue The solution inside and outside is sufficiently cooled and the viscosity becomes high. In this state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (−196 ° C.), the micro solutions inside and outside the cells and the tissues remain solid while remaining amorphous. Vitrification occurs. When the inside or outside of a cell or tissue is solidified by vitrification, the molecular movement is substantially eliminated. Therefore, it is considered that the vitrified cell or tissue can be stored semipermanently by storing it in liquid nitrogen.

  However, in the slow freezing method, it is necessary to cool at a relatively slow cooling rate, so that an operation for cryopreservation takes time. In addition, there is a problem of requiring a device or jig for temperature control. In addition, in the slow freezing method, since ice crystals are formed in the preservation solution outside the cells or tissues, the cells or tissues may be physically damaged by the ice crystals.

  As a method for solving the problems in the slow freezing method, a vitrification preservation method has been proposed. The vitrification preservation method is based on the principle that it becomes difficult to form ice crystals even below freezing point due to the freezing point depression of an aqueous solution containing a large amount of antifreezing agents such as glycerol, ethylene glycol and DMSO (dimethyl sulfoxide). When this aqueous solution is rapidly cooled in liquid nitrogen, it can be solidified without generating ice crystals. Such solidification is called vitrification freezing. An aqueous solution containing a large amount of antifreeze is called vitrification solution.

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

  When vitrification preservation method is used, ice crystals do not form inside or outside the cell, so physical damage (freezing damage) to cells during freezing and thawing can be avoided, but it is included in the vitrification solution. High-concentration antifreezes are chemically toxic, and it is preferable that the vitrification solution is low when the cells are cryopreserved, and the time that the cells are exposed to the vitrification solution, that is, the time until freezing is short. preferable. Furthermore, it is necessary to dilute the vitrification solution immediately after thawing.

  Regarding cryopreservation of cells or tissues using these vitrification preservation methods, examples using various types of cells or tissues are shown by various methods. For example, Patent Document 1 shows that application of a vitrification method to animal or human germ cells or somatic cells is extremely useful in terms of cryopreservation and survival after thawing.

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

  As a jig and operation method for performing the vitrification preservation method more efficiently, in Patent Document 2, Patent Document 3, etc., an egg or an embryo is vitrified and frozen while a straw is filled with a vitrification solution. Attempts have been made to improve the regeneration rate by rapidly contacting with a diluent at the time of thawing.

  Patent Document 4 proposes a method of cryopreserving with excellent survivability by absorbing excess vitrification solution adhering to the periphery of an egg or embryo with an absorbent such as filter paper.

  In Patent Document 5 and Patent Document 6, a strip-like flexible and colorless and transparent film is used as a strip for egg attachment and retention by a so-called cryotop method used in the field of human infertility treatment. There has been proposed a method in which an egg or embryo is attached under a microscope together with a very small amount of vitrification solution and cryopreserved.

  In patent document 7, it is a metal plate-shaped member, and has many holes through which a cooling medium can enter, and is frozen using a tissue piece cryopreservation plate for vitrifying and storing a collected tissue piece. A method of saving has been proposed.

Japanese Patent No. 3443323 JP-A-5-176946 JP-A-10-248860 JP 2005-40073 A JP 2002-315573 A JP 2006-271395 A JP 2008-222640 A

Steponkus et al. , Nature 345: 170-172 (1990). Akira Sakai, Journal of Low Temperature Biotechnology 42: 61-68 (1996)

  Patent Document 4 proposes a method for cryopreserving these germ cells with excellent viability by absorbing excess vitrification solution adhering to the periphery of an egg or embryo into an absorbent such as filter paper. However, when this method is used for vitrification preservation of a group or tissue of cells in which cells overlap each other, there is a problem that a sufficient cooling rate cannot be obtained.

  In the methods proposed in Patent Document 5 and Patent Document 6, the egg or embryo is cryopreserved by dropping and attaching the egg or embryo together with a very small amount (0.1 μL) of vitrification solution onto a colorless transparent flat film. it can. However, in practice, it is very difficult to dripping cells together with a very small amount of glass liquid, and skill is required. In addition, when the cryopreservation jig is used for vitrification of a cell population or tissue consisting of a large number of cells, the vitrification solution adhering to the cells or tissue also increases, so that the excess vitrification solution is removed from the egg. Or it may remain around the embryo and reduce viability of the egg or embryo due to the toxicity of the vitrification solution itself. Further, as with the problem in Patent Document 4, there is a problem that a sufficient cooling rate cannot be obtained.

  The method proposed in Patent Document 7 uses a metal plate having excellent thermal conductivity and further has a large number of holes into which liquid nitrogen, which is a cooling medium, can invade. Ingenuity has been made to obtain However, similar to the problems in Patent Document 5 and Patent Document 6, excess vitrification liquid remains around the tissue and the cells constituting the tissue, and due to the toxicity of the vitrification liquid, the cells constituting the tissue and tissue Viability may be reduced.

  The main object of the present invention is to provide a vitrification jig for cryopreservation capable of easily and reliably carrying out cryopreservation of cells or tissues. More specifically, when dripping the vitrification solution together with cells or tissues, it has excellent absorption performance to absorb excess vitrification solution, and securely holds cells and tissues even during freezing and thawing. Provided is a vitrification cryopreservation jig that can be used.

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

  (1) A cell or tissue glass having a vitrification solution absorber on a metal support, and the vitrification solution absorber having an adhesive layer and a vitrification solution absorption layer in this order from the side closer to the metal support. Freezing storage jig

  According to the present invention, when dripping the vitrification solution together with cells or tissues, it has excellent absorption performance for absorbing excess vitrification solution, and reliably holds cells and tissues even during freezing and thawing. Can be provided.

FIG. 1 is an overall view showing an example of a jig for vitrification cryopreservation of cells or tissues. FIG. 2 is an enlarged view of the vitrification liquid absorber in FIG. 1. FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. FIG. 4 is a schematic view showing another example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. FIG. 5 is a schematic view of a vitrification liquid absorber having vitrification liquid absorption layers disposed on both sides of a metal support. FIG. 6 is a schematic view of a vitrification liquid absorber in which a hole structure is provided in a part of a metal support. FIG. 7 is a schematic view showing a cut surface structure in a direction perpendicular to the surface where the vitrification liquid absorption layer and the adhesive layer are in contact with each other in the region including the pore structure portion in the vitrification absorber illustrated in FIG. 6. is there. FIG. 8 is a schematic view when the vitrification liquid absorber illustrated in FIG. 6 is viewed from the metal support side opposite to the vitrification liquid absorption layer.

  The jig for vitrification cryopreservation of the present invention is used when cryopreserving biological cells or tissues. In the present specification, the cell includes not only a single cell but also a cell population composed of a plurality of cells. The cell population composed of a plurality of cells may be a cell population composed of a single type of cell or a cell population composed of a plurality of types of cells. The tissue may be a tissue composed of a single type of cell or a tissue composed of a plurality of types of cells, and includes non-cellular substances such as an extracellular matrix in addition to cells. Things can be used. The jig for cryopreservation of vitrification of the present invention is preferably such that cells or tissues are attached to a vitrification solution absorber together with vitrification liquid, and the jig to which the cells or tissues are attached is immersed in a cooling solvent such as liquid nitrogen. For freezing. By having the above vitrification solution absorber, the cells or tissues can be easily retained together with the vitrification solution, and the cells and tissues can be reliably retained even during freezing and thawing. The immersion work in can also be easily performed. The vitrification cryopreservation jig of the present invention can be restated as a cell or tissue cryopreservation tool or a cell or tissue vitrification storage tool.

  In the vitrification cryopreservation jig of the present invention, since the vitrification solution absorber absorbs excess vitrification solution, the cell or tissue is stable even if the amount of the vitrification solution dropped together with the cell or tissue is large. Viability can be achieved. In addition, cells and tissues can be securely held even during freezing and thawing, and cells and tissues that have been so manipulated are covered with a very small amount of vitrification solution, so that even in freezing operations, they can be quickly frozen. Can be. Furthermore, since the vitrification solution adhering to the periphery of the cell or tissue is extremely small, the vitrification solution can be diluted immediately after thawing the cryopreserved cell or tissue.

  Below, the structure of the jig | tool for vitrification cryopreservation of this invention is demonstrated.

  The jig for vitrification cryopreservation of the present invention has a vitrification liquid absorber on a metal support, and the vitrification liquid absorber has an adhesive layer and a vitrification liquid absorption from the side closer to the metal support. Have layers in this order.

  Examples of the metal support included in the jig for cryopreservation of vitrification according to the present invention include metal supports such as copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, iron, and stainless steel. Can be used. The thickness of the metal support is preferably 10 μm to 50 mm. For the purpose of improving the adhesion to the adhesive layer, the surface of the metal support may be subjected to chemical or electrical easy adhesion treatment, or the surface may be roughened. In addition, an undercoat layer may be provided for the same purpose. The metal support may have a plurality of holes and internal cavities for the purpose of improving the cooling efficiency by intrusion of the cooling solvent and improving the handleability by reducing the weight. Since the metal support is excellent in thermal conductivity, not only the cooling efficiency is improved, but also the heat storage property is high, so that it is effective in maintaining a low temperature environment after vitrification freezing. For example, in the operation of removing cells and tissues after cryopreserving them and immersing them in a freeze-thaw solution, even if the cells or tissues are removed from the cryopreservation container due to unforeseen circumstances and left for a while, they remain It is possible to maintain a low temperature for a certain period of time, and it can be expected to increase the viability of cells or tissues.

  Examples of the adhesive layer of the vitrified liquid absorber of the present invention include water-soluble adhesive materials such as polyvinyl alcohol, hydroxycellulose, polyvinylpyrrolidone, starch paste, vinyl acetate adhesive materials, acrylic adhesive materials, and epoxy adhesives. Substances, urethane adhesives, hot melt adhesives, elastomer adhesives, instant adhesives typified by cyanoacrylic, silicone adhesives, nitrocellulose adhesives, nitrile rubber adhesives, styrene-butadiene adhesives A layer containing an adhesive substance such as a substance, a urea resin adhesive substance, a styrene resin adhesive substance, a phenol resin adhesive substance, or a photocurable adhesive substance can be preferably used, and one kind of adhesive substance may be used, A plurality of types of adhesive substances may be used.

From the viewpoint of adhesiveness, it is preferable that a part of the adhesive layer enters the vitrification solution absorbing layer to some extent, but if the adhering layer enters a large amount, the absorption performance of the vitrification solution absorption layer may be impaired. Solid content of the adhesive layer is preferably in the range of 0.3~50g / ^{m 2,} further range of 0.5 to 10 g / ^{m 2} is more preferable.

  In addition, the adhesive layer can be positively used as a part of the vitrification liquid absorbing layer. From such a viewpoint, it is preferable that the adhesive layer contains a water-soluble adhesive substance such as polyvinyl alcohol, hydroxycellulose, polyvinylpyrrolidone, starch paste. Furthermore, it is preferable to contain polyvinyl alcohol and polyvinyl pyrrolidone from the viewpoint of toxicity of dissolved components and partially released components to cells or tissues. The adhesive layer can contain various matting agents, surfactants, pH adjusters and the like.

  As the vitrification solution absorbing layer of the vitrification solution absorber of the present invention, paper or non-woven fabric can be preferably used. The main constituent of paper or non-woven fabric is fiber, which can quickly absorb the vitrification liquid by capillary action. Moreover, since many vitrification liquids can be hold | maintained in the space | gap between fibers, it is preferable.

  When the vitrification liquid absorption layer is paper, examples of the pulp fiber constituting the paper include pulp fibers produced from either hardwoods or conifers. The pulp fiber may be unbleached or bleached, but bleached virgin pulp fiber is preferably used. Further, the whiteness of the paper used as the vitrification liquid absorbing layer is preferably 60% or more, and more preferably 70% or more. It is preferable for the whiteness to be in such a range because it is easy to confirm the attached cells or tissues. Such whiteness paper can be used not only for the virgin pulp but also for non-wood pulp fibers such as bleached kenaf. Recycled paper pulp fibers with a large amount of miscellaneous components may affect the activity and viability of cells that make up cells or tissues. If the whiteness is 70% or more, it can be suitably used. In the present invention, various pulp fibers mentioned above can be mixed and used. The whiteness of the pulp fiber can be measured using JIS P8123.

  In the present invention, when the vitrification liquid absorption layer is paper, the vitrification liquid absorption layer preferably has a ratio of the binder component such as a binder to the entire paper of 10% by mass or less, more preferably 5% by mass. Or less, particularly preferably 3% by mass or less. Ordinary paper or the like contains about 5 to 20% by mass of an inorganic pigment in order to improve writability, and further contains various papermaking chemicals. The proportion of the inorganic pigment in the entire paper is preferably 5% by mass or less, and more preferably 3% by mass or less. Some paper chemicals, such as fluorescent whitening agents, dyes, and cationic sizing agents, are thought to affect the activity and viability of cells that make up cells or tissues. The proportion of the entire paper is preferably 1% by mass or less.

  When the vitrification liquid absorption layer in this invention is a nonwoven fabric, it is preferable that the ratio for which binder components, such as a binder, occupy the whole nonwoven fabric is 20 mass% or less, More preferably, it is 10 mass% or less, Most preferably 5% by mass or less.

  The nonwoven fabric used for this invention is demonstrated in detail. Similar to the paper described above, there are fibers that are also preferably used for nonwoven fabrics. Among these fibers, cellulose fibers, rayon fibers and cupra fibers that are regenerated fibers from cellulose fibers, acetate fibers that are semi-synthetic fibers from cellulose fibers, polyester fibers, nylon fibers, acrylic fibers, polypropylene Examples thereof include fibers, polyethylene fibers, polyvinyl chloride fibers, vinylidene fibers, polyurethane fibers, vinylon fibers, glass fibers, and silk fibers. Among them, cellulose fibers, rayon fibers and cupra fibers that are regenerated fibers from cellulose fibers, and acetate fibers, vinylon fibers, and glass fibers that are semi-synthetic fibers from cellulose fibers are more preferable from the viewpoint of absorption performance. Used for.

  When the vitrification liquid absorption layer in this invention is a nonwoven fabric, the nonwoven fabric which mixed various above-mentioned suitable fibers can also be used. Furthermore, it is possible to use a non-woven fabric having a different fiber configuration in a two-layer configuration or a two-layer configuration product bonded together with an adhesive or the like using a wet papermaking method.

  Next, the suitable manufacturing method of the nonwoven fabric used for this invention is demonstrated. There are various production methods for nonwoven fabrics, unlike paper, but for example, the following production methods are preferably used. The production of nonwoven fabrics can be roughly divided into two steps: a step of arranging fibers and a step of entwining or binding fibers. As a method of arranging the fibers, there are a wet method and a dry method, and other methods include a spunbond method and a melt blow method in which fibers are directly produced from resin pellets and bound simultaneously. As the wet method, there are a thermal bond method, a chemical bond method, a hydroentanglement method, and the like as a method in which fibers are dispersed in water and then the fibers are rolled up and bound in the same manner as in the paper making process. In the dry method, fibers are arranged by a machine called a card, and then the fibers are entangled and bonded by a thermal bond method, a chemical bond method, a hydroentanglement method, and a needle punch method to produce a nonwoven fabric. Nonwoven fabrics produced by any of these methods can also be used in the present invention.

  Among these production methods, a nonwoven fabric produced by a spunbond method or a melt blow method, and a nonwoven fabric produced by a hydroentanglement method or a needle punch method after arranging fibers by a wet method or a dry method can be preferably used. These production methods can bind fibers without using a binder, and can produce a nonwoven fabric containing only 100% fibers. In particular, fibers that are most preferably used in the present invention are cellulose fibers, fibers derived from cellulose fibers, rayon fibers and cupra fibers that are cellulose regenerated fibers, and acetate fibers that are semi-synthetic fibers from cellulose fibers. In the case of production, the production method by the hydroentanglement method or the needle punch method is most suitable regardless of the wet method or the dry method.

From the viewpoint of the absorption capacity of the vitrification solution, the vitrification solution absorption layer of the present invention has a thickness of preferably 10 μm or more, more preferably 50 μm or more. The upper limit is preferably 1 cm or less. The basis weight is preferably 10 g / m ² or more, more preferably 20 g / m ² or more. The upper limit is preferably 1 kg / m ² or less. By adjusting the thickness within such a range according to the cells and tissues to be cryopreserved, the absorption capacity of the vitrification solution absorption layer can be easily adjusted according to the amount of the vitrification solution dripped.

It is preferable that the density of the vitrification liquid absorption layer of this invention is 0.25 g / cm < ³ > or less from a viewpoint of the absorption performance and cooling rate of vitrification liquid. In this density range, the vitrification volume absorption layer has a high volume of voids, so that the high absorption performance of the vitrification liquid absorption layer is exhibited.

In the vitrification liquid absorber of the present invention, when the vitrification liquid absorption layer is paper or non-woven fabric, in order to observe the cells or tissues on the vitrification cryopreservation jig using a transmission microscope, the cells or tissues In the part where the metal support is adhered to the vitrification liquid absorption layer, a void structure portion in which the metal support does not exist is provided below the vitrification liquid absorption layer in a direction perpendicular to the surface where the metal support and the adhesive layer contact each other. be able to. When the vitrification absorption layer has a density of 0.5 g / cm ³ or less and a basis weight of 100 g / m ² or less, a certain degree of total light transmittance can be obtained when the vitrification solution is adhered. Can transmit light irradiated from a microscope light source, and the visibility of cells or tissues on a vitrification cryopreservation jig is improved.

The area of the vitrification solution absorbing layer of the present invention is not particularly limited as long as it is appropriately set according to the dripping amount of the vitrification solution dripped together with the cells or tissues, but it is more preferably 40 to 40,000 mm ² , for example. preferable.

  The vitrification liquid absorber in the present invention has been described above. The structure of the vitrification cryopreservation jig using these will be described below. The vitrification cryopreservation jig of the present invention may be a vitrification solution absorber having an adhesive layer and a vitrification solution absorption layer in this order on a support, but the vitrification solution absorber is a gripping part. It may be connected to. Having a gripping portion is preferable because workability during freezing is good.

  FIG. 1 is an overall view showing an example of a jig for vitrification cryopreservation of cells or tissues in the present invention. In FIG. 1, a vitrification cryopreservation jig 5 includes a gripping portion 1 and a vitrification liquid absorber 2. The grip 1 is preferably made of a liquid nitrogen resistant material. As such a material, for example, various metals such as aluminum, iron, copper, and stainless alloy, ABS resin, polypropylene resin, polyethylene resin, fluorine resin, various engineer plastics, and glass can be preferably used. Basically, the vitrification liquid absorber 2 is preferably strip-shaped or sheet-shaped for handling.

  An example of the vitrification liquid absorber in the present invention is shown in FIG. FIG. 2 is an enlarged view of the vitrification liquid absorber in FIG. 1. The vitrification liquid absorber 2 a in FIG. 2 has a vitrification liquid absorption layer 3 on a metal support 4. The adhesive layer is not shown. The vitrification liquid absorber 2a shown in FIG. 2 is an example of a form having the vitrification liquid absorption layer 3 on the entire surface of the vitrification liquid absorber.

  A method of connecting the grip 1 and the vitrification liquid absorber 2 in FIG. When the holding part 1 is resin, for example, the vitrification liquid absorber 2 can be connected to the holding part 1 by insert molding at the time of molding. Furthermore, the vitrification liquid absorber insertion part which is not shown in figure in the holding part 1 can be produced, and the vitrification liquid absorber 2 can be connected with an adhesive agent. Although various adhesives can be used, silicone-based or fluorine-based adhesives that are resistant to low temperatures can be suitably used.

  When cells or tissues are cryopreserved for a long time using the jig for preserving cells or tissues in the present invention, it is possible to cover the jig body shown in FIG. is there. Furthermore, liquid nitrogen is usually not sterilized, and when frozen by direct contact with liquid nitrogen, the sterilized state may not be guaranteed even if the vitrification solution storage jig is sterilized. Therefore, a vitrified liquid absorber to which cells or tissues are attached before freezing may be capped and frozen without being in direct contact with liquid nitrogen. In the overseas advanced countries such as EU, the freezing method in which liquid nitrogen is not directly contacted as described above is the mainstream. For this reason, the cap is preferably made of various metals, various resins, glass, ceramic, etc., which are liquid nitrogen resistant materials. The shape may be any shape, such as a pencil cap or a cylindrical straw cap, as long as the shape can be cut off from the outside without being in contact with the vitrification liquid absorber.

  The example of another aspect of the vitrification liquid absorber in this invention is shown in FIGS. In the cell or tissue vitrification cryopreservation jig shown in FIG. 1, the vitrification solution absorber may be as shown in FIGS. 3-6, the adhesive layer is not shown in the same manner as in FIG. FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. FIG. 4 is a schematic view showing another example of a vitrification liquid absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. 3 and 4, the vitrification solution absorbing layer 3 is discontinuous and disposed on the support 4.

  When the vitrification liquid absorption layer 3 has a continuous shape as shown in FIG. 2 described above, when a plurality of cells or tissues are attached to the vitrification liquid absorption layer 3, the vitrification liquid is the vitrification liquid absorption layer. Since it spreads in the lateral direction and the thickness direction, for example, when a second cell or tissue is attached to the vitrification solution absorbing layer 3, the absorbability of the vitrification solution may be lowered. However, if a plurality of the vitrification solution absorption layers 3 are provided on the support 4 in a discontinuous manner as shown in FIGS. 3 and 4, there is no such concern, and cells or tissues are vitrified together with the vitrification solution. The liquid absorbing layer 3 can be securely attached one by one. 3 and 4, as an example, a plurality of grid-like vitrified liquid absorption layers 3 are arranged. The vitrification liquid absorber 2b and the vitrification liquid absorber 2c shown in FIGS. 3 and 4 are also examples of a vitrification liquid absorber having a vitrification liquid absorption layer on a part of a metal support.

  FIG. 5 is a schematic view of a vitrification liquid absorber 2d in which vitrification liquid absorption layers are arranged on both sides of the support in order to improve operability and quickness. By arranging the vitrification solution absorbing layer 3 on both surfaces of the support 4, cells or tissues can be quickly dropped and attached without worrying about the front and back.

  FIG. 6 is a schematic view of a vitrification liquid absorber 2e in which a hole structure portion 6 is provided in a part of a metal support in order to impart cell viewing performance with a transmission microscope. By providing the hole structure portion 6, cells or tissues on the vitrification solution absorbing layer 3 above the hole structure portion can be observed using a transmission microscope. FIG. 7 is a schematic view of a cut surface structure of a portion including the hole structure portion 6 of the vitrification liquid absorber 2e. FIG. 8 is a schematic view of the vitrification liquid absorber 2e as viewed from the metal support 4 side opposite to the vitrification liquid absorption layer.

  The vitrification cryopreservation jig of the present invention is suitable, for example, when cryopreserving cells or tissues by an operation similar to the cryotop method in an egg or embryo. When the vitrification cryopreservation jig of the present invention is used, it is difficult to be damaged by extracellular vitrification solution at the time of freezing and thawing of cells or tissues, and cells or tissues can be cryopreserved with an excellent survival rate. it can.

  The method for cryopreserving cells or tissues using the vitrification cryopreservation jig of the present invention is not particularly limited. For example, the cells or tissues immersed in the vitrification solution are first placed on the vitrification solution absorber together with the vitrification solution. The vitrification solution is dripped into the vitrification solution absorption layer, and the vitrification solution adhering to the periphery of the cell or tissue is absorbed by the vitrification solution absorption layer. Next, the cells can be frozen by immersing the cells or tissues in liquid nitrogen or the like while being held on the vitrification solution absorber. As the vitrification solution, those used for normal cell freezing can be used. For example, aqueous solutions containing various antifreezing agents such as glycerol, ethylene glycol, DMSO (dimethyl sulfoxide) can be used.

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

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

Example 1
<Production of vitrification liquid absorber>
A 5 mass% aqueous solution of Kuraray Co., Ltd. PVA617 (saponification degree 95-96 mol%, polymerization degree 1700) was applied to a copper support having a thickness of 300 μm at a dry mass of 4 g / m ² as an adhesive layer. Before the coating layer dries, as a vitrification solution absorption layer, Bemliese (registered trademark) SA14G (thickness 70 μm, basis weight 14 g / m) is a non-woven fabric manufactured by Asahi Kasei Fibers Co., Ltd. composed of cellulose (cupra) fibers. ² and a density of 0.20 g / cm ³ ) was overlaid on the coating layer and dried. As described above, the vitrification liquid absorber of Example 1 having the adhesive layer and the vitrification liquid absorption layer in this order on the metal support was produced.

Example 2
<Production of vitrification liquid absorber>
As a vitrification liquid absorption layer, Bemliese (registered trademark) SA28G (thickness 130 μm, basis weight 28 g / m ² , density 0.22 g / cm ³ ) made of Asahi Kasei Fibers Co., Ltd. composed of cellulose (cupra) fibers. The vitrification liquid absorber of Example 2 was produced like Example 1 except having used.

Example 3
<Production of vitrification liquid absorber>
As a vitrification liquid absorption layer, Bemliese (registered trademark) SA30G (thickness 250 μm, basis weight 30 g / m ² , density 0.12 g / cm ³ ) made of Asahi Kasei Fibers Co., Ltd. composed of cellulose (cupra) fibers. The vitrification liquid absorber of Example 3 was produced like Example 1 except having used it.

Example 4
<Production of vitrification liquid absorber>
As a vitrification liquid absorption layer, Bemliese (registered trademark) SA50U (thickness 500 μm, basis weight 50 g / m ² , density 0.10 g / cm ³ ) made of Asahi Kasei Fibers Co., Ltd. composed of cellulose (cupra) fibers. The vitrification liquid absorber of Example 4 was produced like Example 1 except having used it.

Example 5
<Production of vitrification liquid absorber>
As a vitrification liquid absorption layer, Bemliese (registered trademark) SE103 (thickness 540 μm, basis weight 100 g / m ² , density 0.19 g / cm ³ ) made of Asahi Kasei Fibers Co., Ltd. composed of cellulose (cupra) fibers. The vitrification liquid absorber of Example 5 was produced like Example 1 except having used it.

Example 6
<Production of vitrification liquid absorber>
As the vitrification solution absorbing layer, except that Erière (registered trademark) Pro-wipe soft micro wiper S220 (thickness 160 μm, basis weight 22 g / m ² , density 0.14 g / cm ³ ) manufactured by Daio Paper Co., Ltd., which is paper, was used. In the same manner as in Example 1, the vitrification liquid absorber of Example 6 was produced.

Example 7
<Production of vitrification liquid absorber>
As a vitrification liquid absorption layer, BFNNO. Which is a nonwoven fabric made of Kuraray Co., Ltd., which is composed of vinylon fibers. A vitrification liquid absorber of Example 7 was produced in the same manner as in Example 1 except that 1 (thickness 50 μm, basis weight 12 g / m ² , density 0.24 g / cm ³ ) was used.

Example 8
<Production of vitrification liquid absorber>
As a vitrification liquid absorption layer, D-17 (thickness 70 μm, basis weight 17 g / m ² , density 0.24 g / cm ³ ) which is a nonwoven fabric made of Miki Tokushu Paper Co., Ltd. composed of cellulose (rayon) fibers. A vitrification liquid absorber of Example 8 was produced in the same manner as Example 1 except that it was used.

Example 9
<Production of vitrification liquid absorber>
As a vitrification liquid absorption layer, S-17 (thickness 70 μm, basis weight 17 g / m ² , density 0. 1), which is a non-woven fabric made by Miki Tokushu Paper Co., Ltd., composed of mixed fibers of PET fibers and cellulose (rayon) fibers. The vitrification liquid absorber of Example 9 was produced like Example 1 except having used 24 g / cm < ³ >).

Example 10
<Production of vitrification liquid absorber>
A vitrification liquid absorber of Example 10 was produced in the same manner as in Example 3 except that a 300 μm thick stainless steel support was used instead of the 300 μm thick copper support.

Example 11
<Production of vitrification liquid absorber>
A vitrification liquid absorber of Example 11 was produced in the same manner as in Example 3 except that instead of the 300 μm thick copper support, a 300 μm thick aluminum support was used.

Comparative Example 1
<Production of vitrification liquid absorber>
A transparent PET film (thickness 250 μm), which is a colorless and transparent film, was used as it was to obtain a vitrification liquid absorber of Comparative Example 1.

Comparative Example 2
<Production of vitrification liquid absorber>
Advantech filter paper No. 5C (thickness 210 μm, basis weight 120 g / m ² , density 0.57 g / cm ³ ) was used as it was to obtain a vitrified absorber of Comparative Example 2.

Comparative Example 3
<Production of vitrification liquid absorber>
Advantech filter paper No. 2 (thickness 450 μm, basis weight 124 g / m ² , density 0.28 g / cm ³ ) was used as it was to obtain a vitrified absorber of Comparative Example 3.

Comparative Example 4
<Production of vitrification liquid absorber>
Advantech filter paper No. 7 (thickness 170 μm, basis weight 90 g / m ² , density 0.53 g / cm ³ ) was used as it was to obtain a vitrified absorber of Comparative Example 4.

Comparative Example 5
<Production of vitrification liquid absorber>
A comparative example using Bemliese (registered trademark) SA28G (thickness 130 μm, basis weight 28 g / m ² , density 0.22 g / cm ³ ) made of Asahi Kasei Fibers Co., Ltd. composed of cellulose (cupra) fibers. No. 5 vitrified liquid absorber.

Comparative Example 6
<Production of vitrification liquid absorber>
Bemliese (registered trademark) SA28G (thickness 130 μm, basis weight 28 g / m ² , density 0.22 g / cm ³ ) made of Asahi Kasei Fibers made of cellulose (cupra) fiber is made of copper with a thickness of 300 μm. This was placed on a support and used as a vitrification liquid absorber of Comparative Example 6 (the vitrification liquid absorption layer and the support were not bonded).

Comparative Example 7
<Production of vitrification liquid absorber>
A vitrified liquid absorber of Comparative Example 7 was obtained using a copper metal plate having a thickness of 300 μm as it was.

<Evaluation 1 of absorbability of vitrification solution>
The vitrification liquid absorbers of Examples 1 to 11 and Comparative Examples 1 to 7 were cut into strips having a width of 5 mm and a length of 50 mm. As the vitrification solution, a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich was used. In order to test the absorption rate of the vitrification liquid absorber, 2 μL of the vitrification liquid having the above composition was dropped on the vitrification liquid absorber cut into a strip shape and adhered thereto using a micropipette. After dripping adhesion, the state of the vitrification liquid droplet adhering on the vitrification liquid absorber was visually confirmed, and the absorbability was evaluated according to the following criteria.

Evaluation 1 of the absorptivity of the vitrification liquid was evaluated according to the following criteria.
A: All the vitrification liquid was absorbed in less than 10 seconds after adhering under the vitrification droplet.
○: The vitrification solution was almost absorbed in less than 10 seconds after adhering under the vitrification droplet.
X: After adhering under the vitrification droplet, it took 10 seconds or more to absorb the vitrification solution. Or was not absorbed.

<Evaluation 2 of absorbability of vitrification solution>
The vitrification liquid absorbers of Examples 1 to 11 and Comparative Examples 1 to 7 were cut into 50 mm squares. As the vitrification solution, a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich was used. In order to test the absorption capacity of the vitrification liquid absorber, 500 μL of the vitrification liquid was dropped onto a vitrification liquid absorber cut into 50 mm square using a micropipette. 10 seconds after dropping and adhering, when holding the vitrification liquid absorber with tweezers and inverting the front and back, observe whether the vitrification liquid held in the vitrification liquid absorber is dripping, according to the following criteria: evaluated.

Evaluation 2 of the absorbability of the vitrification liquid was evaluated according to the following criteria.
A: The vitrification liquid uniformly penetrates the entire vitrification liquid absorption layer, and no dripping or the like is observed.
○: The vitrification solution penetrates almost uniformly throughout the vitrification solution absorption layer, and a slight dripping is observed.
X: A clear dripping is observed when the front and back are reversed.

<Evaluation of immersion in cooling solvent>
The vitrification liquid absorbers of Examples 1 to 11 and Comparative Examples 1 to 7 were cut into strips having a width of 5 mm and a length of 50 mm. As the vitrification solution, a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich was used. 2 μL of the vitrification liquid having the above composition was dropped on a vitrification liquid absorber cut into strips using a micropipette. Ten seconds after the vitrification liquid was dropped, one end of the strip-shaped vitrification liquid absorber was held with tweezers and immersed in liquid nitrogen for 30 seconds. After immersion, take out the vitrification solution absorber, place it in a room temperature environment, and visually observe whether the vitrification solution absorber is not deformed or damaged compared to before immersion in liquid nitrogen. Evaluation was made according to the following criteria.

Evaluation of immersion in the cooling solvent was performed according to the following criteria.
○: No deformation or breakage of the vitrification liquid absorber is observed.
X: Deformation or breakage is observed in the vitrification liquid absorber, and it cannot be put into practical use.

<Evaluation of visibility>
In order to confirm the visibility of the cells on the vitrification liquid absorption layer with a transmission microscope, in the vitrification liquid absorbers of Examples 1 to 11 and Comparative Example 6 having a metal support, A vitrification liquid absorber was produced in the embodiment shown in FIG. In the evaluation, a glass bead having a diameter of 0.1 mm as a pseudo cell was dropped onto the vitrification solution absorption layer located above the pore structure portion together with the vitrification solution, and a transmission microscope (manufactured by Olympus Corporation) was used. In SZH-121), whether or not the glass beads on the vitrification liquid absorbing layer are visible was evaluated according to the following criteria. As the vitrification solution, a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich was used. In Comparative Examples 2 to 5 having no support, the glass beads dropped onto the vitrification solution absorption layer were dropped on the support in Comparative Example 1 or 7 having no absorption layer. The attached glass beads were observed with the same transmission microscope and evaluated.

Visibility was evaluated according to the following criteria.
○: The morphology of the pseudo cell can be easily confirmed.
X: It is difficult or impossible to confirm the presence of pseudo cells.

The vitrification liquid absorbers of Examples 1 to 11 of the present invention all showed excellent vitrification liquid absorption performance. Further, even when immersed in liquid nitrogen, no deformation or breakage was observed. On the other hand, the vitrification liquid absorbers of Comparative Example 1 and Comparative Example 7 do not have vitrification liquid absorption performance, and there is concern about the risk of toxicity to cells or tissues due to excess vitrification liquid. The vitrification liquid absorbers of Comparative Examples 2 to 6 have vitrification liquid absorption performance, but when cooled under liquid nitrogen after the vitrification liquid drops, in the process of absorbing and freezing the fibers of the vitrification liquid absorption layer, Deformation of the vitrification liquid absorption layer due to swelling / shrinkage of the vitrification liquid absorption layer was observed. Due to such deformation of the vitrification solution absorption layer, cells fall into the gaps between the fibers, and there is a problem in the re-thawing process at the time of freezing and thawing. There is a risk of falling. Therefore, it was judged that it could not stand the actual use. The vitrification liquid absorbers of Examples 1 to 11 can prevent the deformation of the fibers by the adhesion between the vitrification liquid absorption layer and the metal support, and it is possible to produce a vitrification absorber that can withstand actual use. I understand. Furthermore, in the vitrification liquid absorbers of Comparative Examples 2 to 5, handling is difficult because the strength of the vitrification liquid absorber cannot be supported independently. The same results as in Examples 1 to 11 were obtained when the adhesive layer was replaced with polyvinyl alcohol to form an adhesive layer having a dry mass of 4 g / m ² made of polyvinylpyrrolidone K-90 manufactured by Kishida Chemical Co., Ltd. was gotten.

  From the above results, the vitrification cryopreservation jig of the present invention has excellent vitrification solution absorption performance, thereby reducing the risk of toxicity due to excess vitrification solution in the vitrification / freezing operation of cells or tissues. It turns out that it is suitable for doing. It can also be seen that cells and tissues can be reliably retained even during freezing and thawing.

  The present invention can be used for testing or transplanting from iPS cells, ES cells, commonly used cultured cells, living organisms, etc. in addition to embryo transfer or artificial insemination of humans such as cattle and animals, artificial insemination to humans, etc. It can be used for cryopreservation of tissues and tissues constructed in vitro.

DESCRIPTION OF SYMBOLS 1 Holding part 2a Vitrification liquid absorber 2b Vitrification liquid absorber 2c Vitrification liquid absorber 2d Vitrification liquid absorber 2e Vitrification liquid absorber 3 Vitrification liquid absorption layer 4 Metal support 5 For vitrification freezing preservation Jig 6 Hole structure 7 Adhesive layer

Claims (1)

  Vitrification cryopreservation of cells or tissues having a vitrification solution absorber on a metal support, and the vitrification solution absorber having an adhesive layer and a vitrification solution absorption layer in this order from the side closer to the metal support Jig.