JP2015142523A - Vitrification freezing preservation tool for cell or tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vitrification freezing preservation tool which can easily and reliably perform vitrification freezing work for a cell or a tissue.SOLUTION: A vitrification freezing preservation tool for a cell or a tissue comprises a porous sintered formation as a vitrification liquid absorbent.

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-resistant 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 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. A high concentration of antifreeze has chemical toxicity, and it is preferable that the vitrification solution is not more than necessary when cryopreserving cells or tissues. Moreover, it is preferable that the time during which the cells or tissues are exposed to the vitrification solution, that is, the time until freezing is short. Furthermore, it is necessary to dilute the vitrification solution immediately after thawing.

  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, 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 3 and the like, an egg or an embryo is vitrified and frozen in a straw filled with a vitrification solution, and quickly when thawed. Attempts have been made to improve the regeneration rate by contacting with a diluent.

  In Patent Document 4, an excellent viability is obtained by removing an excess vitrification solution adhering to the periphery of an ovum or an embryo by placing the ovum or embryo together with a vitrification solution on a removal material for vitrification preservation and sucking from the lower part. A method of cryopreserving is proposed. In addition, as a removal material for vitrification preservation | save, what has a through-hole by the film-form thing which consists of natural products and synthetic resins, such as a wire net and paper, is described.

  Patent Document 5 proposes a method of cryopreserving with an excellent survival rate by absorbing excess vitrification solution adhering to the periphery of an egg or embryo with an absorbent such as filter paper.

  In Patent Document 6 and Patent Document 7, a strip-like flexible, colorless and transparent film is used as a strip for holding eggs attached by the so-called cryotop method used in the field of human infertility treatment. 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 8, a method for producing a frozen cell sheet by vitrification is proposed. On a pedestal made of a mesh-like net or the like, a transport auxiliary film (manufactured by Cellseed Co., Ltd., which is a sheet mainly made of cellulose). A method is described in which a cell sheet is placed together with a cell shifter or a PVDF membrane, and the cell sheet is cryopreserved after the excess vitrification solution is sucked out or dropped.

Japanese Patent No. 3443323 JP 2008-5846 A JP-A-10-248860 International Publication WO2011 / 077093 JP 2005-40073 A JP 2002-315573 A JP 2006-271395 A JP 2013-1111017 A

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

  The method proposed in Patent Document 3 has a problem that the time until freezing is long because the vitrification liquid is filled in the straw. In addition, since the size of cells or tissues that can be cryopreserved is limited to the inner diameter of the straw, it is difficult to preserve a sheet-shaped tissue such as a cell sheet.

  The method proposed in Patent Document 4 proposes a method for cryopreserving these germ cells with an excellent survival rate by removing excess vitrification solution adhering to the periphery of the ovum or embryo. However, the method described in the above publication requires a suction operation from the bottom when removing the excess vitrification solution, which is a complicated operation and is not suitable for a vitrification cryopreservation operation in a short time. is there. Furthermore, if the suction from the lower part is insufficient, there is a problem that excess vitrification liquid remains.

  Patent Document 5 proposes a method of cryopreserving these germ cells with an excellent survival rate by absorbing excess vitrification solution adhering to the periphery of an egg or embryo into an absorbent such as filter paper. However, when the volume of cells or tissues to be cryopreserved is large, there is a problem that excess vitrification solution remains because the absorption rate of the vitrification solution is not sufficient. Furthermore, when the filter paper is composed of general cellulose, the strength is insufficient when immersed in liquid nitrogen.

  In the methods proposed in Patent Document 6 and Patent Document 7, a method of cryopreserving an egg or embryo with a small amount of vitrification solution is described by limiting the width of the film on which the egg or embryo is placed. . In this method, an egg or embryo is placed on the film together with a very small amount of vitrification solution by the operator's operation, but there is a problem that the operation is difficult. In the cryotop method based on this method, in order to cryopreserve the egg or embryo with a smaller amount of vitrification solution, once the egg or embryo is placed on the film together with the vitrification solution, excess vitrification is performed. A complicated operation of sucking the liquid and removing it from the film may be performed. For example, it is not suitable for application to cryopreservation of a tissue having a large area in a sheet shape such as a cell sheet.

  In Patent Document 8, for example, a cell sheet is placed on a pedestal such as a glass plate, a metal plate, a mesh-like net, and a non-woven fabric together with a transport auxiliary film (Cell Shifter or PVDF film manufactured by Cellseed Co., Ltd.). The method of cryopreserving after sucking or dropping the vitrification solution is described, but when the amount of vitrification solution dripped with the cell or tissue is large, vitrification attached to the outer periphery of the cell or tissue When the amount of the liquid is large, the absorption rate of the vitrification liquid is not sufficient, and there is a problem that excess vitrification liquid remains.

  The main object of the present invention is to provide a vitrification cryopreservation jig that enables easy and simple cryopreservation of cells or tissues. More specifically, after the cells or tissues are immersed in the vitrification solution, when placed on the vitrification storage jig, it is excellent for absorbing excess vitrification solution adhering to the outer periphery of the cells or tissues. Another object of the present invention is to provide a vitrification jig for freezing storage having excellent absorption performance.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the above-described various technical problems can be solved by a cell or tissue vitrification cryopreservation jig having the following configuration.
(1) A jig for vitrification cryopreservation of a cell or tissue having a porous sintered compact as a vitrification liquid absorber.

  According to the present invention, when a cell or tissue immersed in a vitrification solution is placed on the vitrification solution absorber together with the vitrification solution, the excess vitrification solution in which the vitrification solution adheres to the outer periphery of the cell or tissue Other operations to remove excess vitrification liquid (for example, suction removal operation from the lower part of the vitrification liquid absorber or direct suction from the periphery of cells or tissues using a micropipette etc. It is possible to provide a jig for cryopreserving cells or tissues that can be easily and simply subjected to cryopreservation of cells or tissues without particularly requiring a removal operation. Moreover, if the jig | tool for vitrification cryopreservation of this invention is used, the vitrification freezing operation | work of a cell or a structure | tissue can be performed efficiently.

FIG. 1 is an overall view showing an example of a jig for vitrification cryopreservation of cells or tissues of the present invention. FIG. 2 is an enlarged view of the vitrification liquid absorber in FIG. 1. FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used when a support is provided. FIG. 4 is a schematic view showing an example of a vitrification liquid absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. FIG. 5 is a schematic view showing another example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig.

  The vitrification cryopreservation jig of the present invention will be described in detail below. The cell or tissue vitrification cryopreservation jig of the present invention has a porous sintered formed body as a vitrification liquid absorber.

  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 vitrification cryopreservation jig of the present invention preferably has cells or tissues attached to the vitrification solution absorber together with the vitrification solution, and the jig to which the cells or tissues have adhered is immersed in a cooling substance such as liquid nitrogen. It is for freezing. By having the vitrification liquid absorber, cells or tissues can be easily held together with the vitrification liquid, and further, the operation of immersing the cells or tissues in liquid nitrogen can be easily and simply 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 quickly absorbs excess vitrification solution, the cells or tissues immersed in the vitrification solution are absorbed together with the vitrification solution. When placed on the body, even if the amount of vitrification solution attached to the cells or tissues is large, a stable cell or tissue survival rate can be achieved. Furthermore, the cells or tissues thus manipulated are covered with a very small amount of vitrification solution, and can be quickly frozen even when freezing. Furthermore, since the vitrification solution covering the cells or tissues is very small, the vitrification solution can be diluted immediately after thawing the cryopreserved cells or tissues.

  The vitrification cryopreservation jig of the present invention is a vitrification cryopreservation jig having at least a vitrification solution absorber that absorbs a vitrification solution, and the vitrification solution absorber is a porous sintered compact. It is characterized by being. A porous sintered compact is a porous structure obtained by heating and sintering thermoplastic resin solid powder and metal oxide particles with a three-dimensional structure, and fusing the powder particle surfaces. Is the body. The porous structure in the present invention is a structure having pores (pores) on the surface, preferably a structure having continuous pores on the surface and inside. The jig for vitrification cryopreservation of the present invention has the porous sintered compact as a vitrified liquid absorber, and without the support, the porous sintered compact itself is made of glass. You may have as a chemical | medical solution absorber. Or the vitrification liquid absorber which has an above-mentioned porous sintered compact on a support body may be sufficient. When laminating a porous sintered compact on a support, an adhesive layer can be provided between the support and the porous sintered compact. Furthermore, in addition to the porous sintered body and the adhesive layer, for example, an undercoat layer may be provided for the purpose of obtaining a uniform adhesive layer on the support.

  The vitrification cryopreservation jig of the present invention passes through pores on the surface of the porous sintered compact when a cell or tissue immersed in the vitrification solution is placed on the vitrification solution absorber together with the vitrification solution. The vitrification solution is absorbed by the vitrification solution absorber, and excess vitrification solution can be removed from the periphery of the cell or tissue.

  When the porous sintered compact of the present invention is made of a resin, the thermoplastic resin used to obtain the resin is low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene, polypropylene, polymethyl methacrylate, polystyrene, Fluorine resin, ethylene-vinyl acetate copolymer, polyamide, styrene-acrylonitrile copolymer, styrene-butadiene-acrylonitrile terpolymer, polycarbonate, polyvinyl chloride and the like. The porous sintered compact of the resin contained in the vitrified liquid absorber of the present invention may be a porous sintered compact containing two or more kinds of resins. Moreover, when the porous sintered compact of this invention consists of a metal oxide, as a metal oxide used in order to obtain this, a silica, an alumina, a zirconium, quartz glass etc. are mentioned. Since processing is easy, Preferably, polyethylene and quartz glass are mentioned.

  As a method for producing a porous sintered compact contained in the vitrification liquid absorber of the present invention, a generally known method can be used. When the porous sintered body is made of a resin, for example, a method described in JP-A-2009-235417 can be used. More specifically, a solid powder of a thermoplastic resin obtained by a method such as emulsion polymerization or pulverization is filled in a mold, heated and sintered to fuse the powder particle surface, and cooled, A porous sintered compact can be produced. The temperature at the time of heating and sintering varies depending on the type of thermoplastic resin to be sintered, but in order to maintain a high porosity, the temperature at which the particles are sufficiently fused in the vicinity of the melting point of each resin, A temperature that does not fill the particle gap is selected. For example, in the case of polyethylene, the heating temperature is preferably 110 ° C to 180 ° C, more preferably 120 ° C to 150 ° C. When the porous sintered body is made of a metal oxide, for example, methods described in JP2009-29692A and JP2002-160930A can be used. The produced porous sintered compact may be processed into an arbitrary shape by secondary processing such as cutting, cutting, and punching.

  The surface of the vitrification liquid absorber of the present invention is preferably subjected to a hydrophilic treatment in order to enhance the absorption performance of the vitrification liquid. Hydrophilic treatment methods include graft modification methods, coating methods using hydrophilic polymer compounds, corona discharge, plasma treatment, general surface modification methods using various electron beams such as excimer laser, Can use surface modification methods such as surface roughening. The hydrophilic treatment may be performed on the resin powder particles before the sintering step, or may be performed on the porous sintered body after the sintering step.

  The porous sintered compact used in the present invention preferably has a pore diameter of 1 μm or more from the viewpoint of absorbability of the vitrification solution. When the pore diameter is less than 1 μm, the absorption performance when the vitrified droplet is dropped is not sufficient, and it may take time until frozen storage, or a large amount of excess vitrified solution may remain around the cells or tissues. is there. On the other hand, in the range where the pore diameter exceeds 1 mm, a part of the cell or sheet-shaped tissue is trapped in the pore gap, and in the operation of thawing and thawing after cryopreservation and the subsequent operation, the vitrification solution absorber There may be a problem that cells and tissues do not peel off from the surface. From the above viewpoint, the pore diameter of the porous sintered compact used in the present invention is preferably 1 μm to 1 mm. Moreover, it is preferable that the porosity of the porous sintered compact used for this invention is 20%-80%, More preferably, it is 30%-60%. The pore diameter, thickness, and porosity of the porous sintered compact used in the present invention can be appropriately set according to the type of cells or tissues to be used, the dripping amount of the vitrification solution dripped with the cells or tissues, and the like. .

  The pore diameter represents an average diameter of pores measured from surface and cross-sectional image observations.

The porosity is defined by the following formula. Here, the void volume V can be measured using a mercury intrusion method.
P = (V / T) × 100 (%)
P: Porosity (%)
V: void volume (ml / m ² )
T: Thickness (μm)

The area of the porous sintered compact used as the vitrification liquid absorber is not particularly limited as long as it is appropriately set according to the size of the cell or tissue and the amount of the vitrification liquid adhering to the cell or tissue. When the cell or tissue to be cryopreserved is a sheet-like tissue, the area of the porous sintered formed body is preferably larger than the bottom area of the sheet-like tissue. A more preferable area of the porous sintered compact is 1.5 times or more with respect to the bottom area of the sheet-like structure. In the case of state cells cryopreserved are suspended in particulate is preferably in the per 2 mm ² or more in vitrification liquid 1μL dripping, and more preferably a 10~400mm ^2.

  When the vitrification liquid absorber which the jig | tool for vitrification cryopreservation of this invention has has a support body, generally well-known various support bodies can be used. Examples of such a support include various resin films, metals, glass, and rubber. Such a support may be made of one kind of material or two or more kinds of materials. Among them, the resin film is preferably used from the viewpoint of handleability. Specific examples of the resin film include polyester resins such as polyethylene terephthalate (PET) and polyethylene naphthalate (PEN), acrylic resins, epoxy resins, silicone resins, polycarbonate resins, diacetate resins, triacetate resins, polyacrylate resins, polychlorinated resins. Examples of the resin film include vinyl resin, polysulfone resin, polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, and cyclic polyolefin resin. In addition, a metal support can be suitably used from the viewpoint of excellent temperature conductivity and enabling rapid freezing. Specific examples of the metal support include copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, iron, and stainless steel. The thickness of the support is preferably 10 μm to 10 mm. In addition, in order to increase the adhesive strength with the adhesive layer, the surface of the support can be easily adhered by an electrical method such as corona discharge treatment or a chemical method, and further roughened. You can also.

When the vitrification liquid absorber of the jig for vitrification cryopreservation of the present invention has an adhesive layer, an instantaneous adhesive material, a hot-melt adhesive material, a photo-curing property as typified by a moisture-curable adhesive material Common adhesive methods using adhesive materials can be used, for example, water-soluble adhesive materials such as polyvinyl alcohol, hydroxycellulose, polyvinylpyrrolidone, starch paste, vinyl acetate adhesive materials, acrylic adhesive materials, epoxy adhesives Substances, urethane adhesives, elastomer adhesives, cyanoacrylate adhesives, fluorine adhesives, silicon adhesives, nitrocellulose adhesives, nitrile rubber adhesives, styrene-butadiene adhesives, urea resin adhesives Adhesive materials, styrene resin adhesive materials, phenol resin adhesive materials, polyimide adhesive materials, Polyamide-based adhesive material, a polyester adhesive material, bismaleimide adhesive material, an olefin-based adhesive material, a non-water soluble adhesive material, such as EVA-based adhesive materials can be preferably used. The adhesive layer may contain one kind of adhesive substance or may contain a plurality of kinds of adhesive substances. Solid content of the adhesive layer is preferably in the range of 0.01 to 100 g / ^{m 2,} further range of 0.1 to 50 g / ^{m 2} is more preferable.

  The vitrification liquid absorber in the present invention has been described above. The structure of the vitrification cryopreservation jig using these will be described below. The vitrification cryopreservation jig of the present invention may be anything as long as it has a vitrification solution absorber as described above, but the vitrification solution absorber is connected to the gripping part. May be. Having a gripping portion is preferable because workability during freezing is good.

  FIG. 1 is an overall view showing an example of a jig for vitrification cryopreservation of cells or tissues of the present invention. In FIG. 1, a vitrification cryopreservation jig 5 includes a gripping portion 1 and a vitrification liquid absorber 2. The grip portion 1 is preferably 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 2 of FIG. The vitrification liquid absorber 2a of FIG. 2 is an example of a form that does not have a support and the porous sintered body 3 itself is a vitrification liquid absorber. FIG. 3 is a schematic view showing an example of a vitrification liquid absorber used in the case of having a support. The vitrification liquid absorber has a porous sintered body 3 on a support 4. The vitrification liquid absorber 2b shown in FIG. 3 is an example of a form having the porous sintered body 3 on the entire surface of the vitrification liquid absorber.

  A method for connecting the grip 1 and the vitrification liquid absorber 2 in FIG. 1 will be described. 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, a silicon-based or fluorine-based adhesive that is 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, sterility may not be guaranteed even if the vitrification solution storage jig is sterilized. Therefore, a vitrified liquid absorber to which cells or tissues are attached before freezing may be capped and frozen without being in direct contact with liquid nitrogen. Also, in the overseas advanced countries such as EU, the freezing method that does not directly contact liquid nitrogen as described above has become the mainstream. For this reason, the cap is preferably made of various metals, various resins, glass, ceramics, 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.

  4 and 5 show examples of another embodiment of the vitrification liquid absorber in the present invention. In the cell or tissue vitrification cryopreservation jig shown in FIG. 1, the vitrification solution absorber may be as shown in FIGS. FIG. 4 is a schematic view showing an example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. FIG. 5 is a schematic view showing another example of a vitrification solution absorber used when a plurality of cells or tissues are cryopreserved with one vitrification cryopreservation jig. 4 and 5, the porous sintered body 3 is discontinuous and arranged on the support 4.

  When the porous sintered compact 3 has a continuous shape as shown in FIG. 2 and FIG. 3 described above, if a plurality of cells or tissues are attached to the porous sintered compact 3, the vitrification liquid becomes porous. For example, when the second or subsequent cells or tissues are attached to the porous sintered body 3, the absorbability of the vitrification liquid may be reduced. . However, as shown in FIGS. 4 and 5, there are no such concerns when a plurality of porous sintered compacts 3 are provided on the support 4 in a discontinuous manner. One by one can be reliably attached to the quality sintered body 3. 4 and 5, as an example, a plurality of grid-like porous sintered bodies 3 are arranged. The vitrification liquid absorber 2c and the vitrification liquid absorber 2d shown in FIGS. 4 and 5 are also examples of a vitrification liquid absorber having a porous sintered body on a part of the support.

  The vitrification cryopreservation jig of the present invention is suitably used, for example, in the cryotop method. In addition, the conventional cryotop method is usually used for storing a single cell or a small number of cells of less than 10, but the vitrification storage jig of the present invention can store more cells (for example, It can also be suitably used in the storage of 10 to 100,000 cells). Furthermore, it can be suitably used for the preservation of a sheet-like tissue (so-called cell sheet) composed of a plurality of cells. When the jig for vitrification cryopreservation according to the present invention is used, it is difficult to be damaged by the vitrification solution outside the cell or tissue during freezing and thawing, and the cell or tissue can be cryopreserved with an excellent survival rate. .

  The method for cryopreserving cells or tissues using the vitrification cryopreservation jig of the present invention is not particularly limited. For example, first, a vitrification solution absorber comprising cells or tissues immersed in a vitrification solution together with the vitrification solution The vitrification solution adhering to the periphery of the cell or the tissue is put on the top and absorbed by the vitrification solution absorber. Next, the cells or the tissue can be frozen by being immersed in liquid nitrogen or the like while being held on the vitrification liquid absorber or cooled in a vapor such as liquid nitrogen. As the vitrification solution, those usually used for freezing germ cells such as eggs and embryos, and those used for cryopreservation of primate iPS cells and ES cells can be used. An aqueous solution containing various antifreezing agents such as ethylene glycol and DMSO (dimethyl sulfoxide) can be used. When thawing and thawing the cryopreserved cells or tissues, the vitrified solution absorber and the cells or tissues placed on the vitrified solution absorber are immersed in the melt and immediately thawed and thawed. can do.

  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, myocardium, and cartilage. The jig for vitrification cryopreservation of the present invention is not limited to cells or tissues collected directly from a living body, for example, cultured skin grown and grown in vitro, a so-called cell sheet constructed in vitro, It can also be suitably used in vitrified cryopreservation of an artificial tissue such as a tissue model having a three-dimensional structure described in JP-A-205516.

  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. In addition, the pore diameter of Example 1 and Example 2 shows the average pore diameter measured from the image observation of the surface and the cross section. Moreover, the pore diameter of the comparative example 1 and the comparative example 4 shows the diameter of the largest pore measured by the bubble point test described in JIS K3832.

Example 1
As the porous sintered compact, Sunfine AQ800 (pore diameter 35 μm, porosity 43%, thickness 500 μm) manufactured by Asahi Kasei Co., Ltd., which is a porous sintered compact made of polyethylene resin, is used as a vitrified liquid absorber. The vitrification solution absorber 500 mm ² (10 mm × 50 mm) was joined to a gripping part made of ABS resin, and the vitrification cryopreservation jig of Example 1 was produced in the form shown in FIG.

(Example 2)
As a porous sintered body, a quartz glass porous body (pore diameter 3.5 μm, porosity 33%, thickness 2 mm) manufactured by Covalent Materials, which is a ceramic porous sintered body, is used as a vitrified liquid absorber. This vitrified liquid absorber 500 mm ² (10 mm × 50 mm) was joined to a gripping part made of ABS resin, and a vitrification cryopreservation jig of Example 2 was produced in the form shown in FIG.

(Comparative Example 1)
Instead of the porous sintered body, a cellulose acetate membrane made of cellulose acetate (pore size 0.5 μm, porosity 68%, film thickness 125 μm) was used as a vitrification liquid absorber. Since the membrane is thin and the self-supporting property is not sufficient, the membrane is pasted onto a PET film having a thickness of 250 μm using a urethane-based adhesive substance, and the vitrification cryopreservation jig of Comparative Example 1 is used in the same manner as in Example 1. Produced.

(Comparative Example 2)
Filter paper No. 1 manufactured by Advantech, which is a filter paper. A jig for vitrification cryopreservation of Comparative Example 2 was produced in the same manner as Comparative Example 1 except that 5C (basis weight 120 g / m ² , density 0.57 g / cm ³ ) was used as the vitrification liquid absorber.

(Comparative Example 3)
The treatment for vitrification cryopreservation of Comparative Example 3 was performed in the same manner as Comparative Example 1 except that Cell Shifter made of Cellulose, which is a cell transport auxiliary membrane described in Patent Document 8, was used as a vitrification solution absorber. A tool was prepared.

(Comparative Example 4)
A hydrophilic durapore membrane (pore diameter 0.1 μm, porosity 70%, film thickness 125 μm) made of Merck Millipore made of PVDF, which is a cell transport auxiliary membrane described in Patent Document 8, is used as a vitrified liquid absorber. A jig for vitrification cryopreservation of Comparative Example 4 was produced in the same manner as Comparative Example 1 except that.

(Comparative Example 5)
The vitrification cryopreservation treatment of Comparative Example 5 was carried out in the same manner as in Example 1 except that a transparent PET film (porosity 0%, thickness 250 μm), which was a colorless transparent film, was used as it was to obtain a vitrification liquid absorber. A tool was prepared.

<Evaluation of absorbability of vitrification solution>
200 μL of a vitrification solution containing a plurality of glass beads (diameter: 100 μm) as pseudo cells is dropped on the vitrification absorber of each vitrification cryopreservation jig of Examples 1, 2 and Comparative Examples 1-5. Attached. The vitrification solution used was a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich. After dripping and adhering, the state where the vitrification solution around the pseudo cell placed on the vitrification solution absorber was absorbed with an epi-illuminated optical microscope (OM4, VC4500-S1) was observed. The evaluation was based on the following criteria. These results are shown in the item “Evaluation of absorbability of vitrification solution” in Table 1.

○: The vitrification solution was absorbed within 10 seconds after adhering under the vitrification droplet.
X: Absorption of the vitrification solution was not sufficient within 10 seconds after adhering under the vitrification droplet, and the vitrification solution remained around the pseudo cell.

<Evaluation of immersion in cooling solvent>
On the vitrification solution absorber of each vitrification cryopreservation jig of Example 1, Example 2 and Comparative Examples 1 to 5, 100 μL of vitrification solution containing a plurality of glass beads (diameter 100 μm) as pseudo cells was dropped. Attached. The vitrification solution used was a composition containing 20% by volume serum, 15% by volume DMSO, 15% by volume ethylene glycol, and 0.2% by volume sucrose in a modified TCM199 medium manufactured by Sigma-Aldrich. Ten seconds after dropping and adhering, the vitrification liquid absorber was completely immersed in liquid nitrogen. After immersion for 1 minute, the vitrification cryopreservation jig was taken out of liquid nitrogen and placed in a room temperature environment. Compared to before immersion in liquid nitrogen, the vitrification solution absorber was visually observed for deformation and breakage, and practical durability was evaluated according to the following criteria. These results are shown in the item “Evaluation of immersion in cooling solvent” in Table 1.

○: No deformation or breakage of the vitrification liquid absorber was observed.
X: Deformation or breakage was observed in the vitrified liquid absorber. Or, in a series of evaluation processes, work defects were recognized due to the occurrence of deformation or breakage.

  Both the vitrification cryopreservation jigs of Examples 1 and 2 of the present invention exhibited excellent vitrification solution absorption performance. On the other hand, the vitrification cryopreservation jigs of Comparative Examples 1 to 5 had insufficient vitrification solution absorption performance. Among these, the vitrification cryopreservation jigs of Comparative Examples 1 to 4 excluding Comparative Example 5 showed some absorption of the vitrification solution, but the vitrification solution around the pseudo cell was absorbed within 10 seconds. It was not seen, and it was seen that it remained.

  In addition, the vitrification cryopreservation jigs of Examples 1 and 2 and Comparative Examples 2 to 5 of the present invention are not deformed or damaged when immersed in liquid nitrogen, and are defective in a series of cryopreservation operations. Was not recognized. On the other hand, in the vitrification cryopreservation jig of Comparative Example 1, damage to the vitrification liquid absorber was observed after immersion in liquid nitrogen.

  From the above results, it can be seen that the vitrification / freezing operation of cells or tissues can be easily and reliably performed by the vitrification cryopreservation jig of the present invention.

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

DESCRIPTION OF SYMBOLS 1 Holding part 2a Vitrification liquid absorber 2b Vitrification liquid absorber 2c Vitrification liquid absorber 2d Vitrification liquid absorber 3 Porous sintered formation body 4 Support body 5 Vitrification jig for cryopreservation

Claims (1)

  A jig for vitrification cryopreservation of cells or tissues having a porous sintered compact as a vitrification liquid absorber.