JP7093280B2 - Jig for cryopreservation of cells or tissues - Google Patents

Description

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

Excellent cell or tissue preservation techniques are sought after in various industrial fields. For example, in a bovine embryo transfer technique, embryos are cryopreserved, thawed and transplanted in accordance with the estrous cycle of a recipient cattle. Further, in human infertility treatment, after collecting an egg or an ovary from a mother, it is cryopreserved in order to adjust to a timing suitable for transplantation, and thawed at the time of transplantation for use.

In general, cells or tissues collected from a living body gradually lose their activity even in a culture medium, and therefore long-term culture of cells or tissues in vitro is not preferable. Therefore, a technique for long-term storage without losing biological activity is important. Superior preservation techniques allow for more accurate analysis of harvested cells or tissues. Further, the excellent preservation technique makes it possible to use cells or tissues for transplantation while maintaining higher biological activity, and it is expected that the engraftment rate after transplantation will be improved. Furthermore, it is also possible to sequentially produce and store artificial tissues for transplantation such as cultured skin cultured in vitro and so-called cell sheets constructed in vitro, and use them when necessary. , Not only in terms of medical care, but also in terms of industry, great merits can be expected.

As a method for preserving cells or tissues, for example, a slow freezing method is known. In this method, first, cells or tissues are immersed in a preservation solution obtained by containing a freezing agent in a physiological solution such as phosphate buffered saline. As the antifreeze agent, compounds such as glycerol and ethylene glycol are used. After immersing the cells or tissues in the preservation solution, the cells or tissues are cooled to −30 to −35 ° C. at a relatively slow cooling rate (for example, a rate of 0.3 to 0.5 ° C./min) to the inside and outside of the cells or the tissues. The inner and outer solutions are sufficiently cooled and become more viscous. In such a state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (-196 ° C.), the minute solutions inside and around the cells or tissues remain amorphous and solid. Vitrification occurs. When the inside and outside of the cell or the inside and outside of the tissue are solidified by vitrification, the movement of the molecule is substantially stopped. Therefore, it is considered that the vitrified cell or tissue can be stored semi-permanently 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 the operation for cryopreservation takes time. Further, there is a problem that an apparatus or a jig for controlling the cooling rate is required. In addition, in the slow freezing method, ice crystals are formed in the storage solution outside the cells or tissues, so that the cells or tissues may be physically damaged by the ice crystals.

A vitrification freezing method has been proposed as a method for solving the problems in the slow freezing method. The vitrification freezing method uses the principle that ice crystals are less likely to form even below freezing point due to the freezing point depression of a storage solution containing a large amount of antifreezing agents such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide). .. When this preservation solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. This solidification is called vitrification freezing. A preservative solution containing a large amount of antifreeze is called a vitrification solution.

As a specific operation of the vitrification freezing method, cells or tissues are immersed in a storage solution containing a large amount of antifreezing agent, and then cooled at the temperature of liquid nitrogen (-196 ° C.). Since the vitrification freezing method is such a simple and rapid process, it does not require a long time for operation for cryopreservation and has an advantage that it does not require a device or jig for temperature control. There is.

In principle, the vitrification freezing method can avoid physical damage (freezing damage) to cells during freezing and thawing because ice crystals do not form inside or outside the cells, but it is appropriate. In order to achieve stable vitrification freezing, the concentration of the antifreezing agent contained in the preservative solution used for vitrification must be high. On the other hand, the high concentration of antifreeze contained in the preservation solution is highly chemically toxic to cells.

From the above background, in the cryopreservation of cells or tissues, the time during which the cells or tissues are exposed to the preservation solution (that is, frozen) from the viewpoint of avoiding toxicity due to the high concentration of the antifreeze agent contained in the preservation solution. It is preferable that the time until the test is short. Further, from the viewpoint of increasing the freezing rate, it is preferable that the amount of the preservative solution present around the cells or tissues is small during the cryopreservation of the cells or tissues. It can be said that the smaller the preservative solution present around the cells or tissues, the smaller the heat capacity of the object to be frozen and the faster the freezing rate of the cells or tissues, which is preferable for vitrification freezing. Furthermore, the fact that the amount of the preservation solution present around the cells or tissues is small means that the preservation solution is rapidly diluted in the thawed solution even during thawing after freezing, and the formation of re-ice crystals in the cells or tissues is suppressed. Is preferable. Further, since the concentration of the antifreeze agent mixed in the thawed liquid at the time of thawing can be lowered, it is preferable from the viewpoint of toxicity derived from the antifreeze agent.

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

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

In Patent Documents 3 and 4, the so-called cryotop method used in the field of human infertility treatment is used to freeze and store eggs using a strip-shaped flexible and colorless transparent film as a strip for holding egg adhesion. A method has been proposed in which an egg or an embryo is placed on the film with a very small amount of a preservative solution under a microscope observation using a tool and cryopreserved.

In Patent Document 5, an egg or embryo is placed on a preservative solution removing material together with a preservative solution containing a large amount of antifreeze agent, and the excess preservative solution adhering to the periphery of the egg or embryo is removed by sucking from the lower part. , A method of cryopreserving with excellent survival rate has been proposed. As the storage liquid removing material, a film-like material made of a natural product such as wire mesh or paper or a synthetic resin having through holes is described. Further, as a cryopreservation jig capable of removing excess preservative solution and improving workability when placing cells, for example, a preservation solution absorber having a specific haze value is described in Patent Document 6. Further, in Patent Document 7, Patent Document 8 and the like, a vitrification cryopreservation jig having a porous sintered form or a porous structure formed of a material having a specific refractive index as a storage liquid absorber. Is described.

Japanese Patent No. 3044323 Japanese Unexamined Patent Publication No. 2008-5846 Japanese Unexamined Patent Publication No. 2002-315573 Japanese Unexamined Patent Publication No. 2006-271395 International Publication No. 2011/070973 Pamphlet Japanese Unexamined Patent Publication No. 2014-183757 Japanese Unexamined Patent Publication No. 2015-142523 International Publication No. 2015/064380 Pamphlet

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

Patent Documents 3 and 4 propose a method of cryopreserving an egg or embryo with a small amount of a storage solution by limiting the width of the film on which the egg or embryo is placed to obtain an excellent survival rate. There is. In this method, an egg or an embryo is placed on a film together with a small amount of a preservative solution by an 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 an egg or embryo with a smaller amount of preservation solution, once the egg or embryo is placed on a film together with the preservation solution, and then an extra preservation solution is added. A complicated operation such as suctioning and removing from the film may be performed. Further, for example, it is not suitable for application to cryopreservation of a sheet-shaped tissue having a large area such as a cell sheet.

Patent Document 5 proposes a method for cryopreserving these germ cells with an excellent survival rate by removing excess preservative solution adhering to the periphery of the egg or embryo. In Patent Documents 6 to 8, unlike the above-mentioned Patent Document 5 and the like, it is not necessary for the operator to remove the excess preservative solution adhering to the periphery of the egg or the embryo, and good workability can be obtained. However, in the cryopreservation jig having the function of absorbing the preservation liquids of Patent Documents 5 to 8, at least when the sample to be preserved and the preservation liquid are dropped and adhered (before the dropping and adhering), the preservation liquid is adhered. Due to the poor light transmission of the absorber itself, the work under a transmission microscope may be complicated, and further improvement has been desired.

The main object of the present invention is to provide a jig for cryopreservation of cells or tissues capable of easily and surely performing cryopreservation work of cells or tissues. More specifically, after immersing the cells or tissues in the preservation solution, the work of dropping and adhering the cells or tissues to the placement portion on the preservation solution absorber under observation with a transmission microscope should be performed with good visibility. It is an object of the present invention to provide a cryopreservation jig having excellent absorption performance capable of absorbing excess storage solution on the outer periphery of cells or tissues.

As a result of diligent studies to solve the above problems, the present inventor has referred to a jig for cryopreservation of cells or tissues having the following constitution (in the present specification, "jig for cryopreservation of cells or tissues"). It has been found that the above-mentioned problems can be solved by simply using a "jig for cryopreservation").

It has a glass porous structure having a pore diameter of 4 nm or more and 15 nm or less as a preservative solution absorber , the thickness of the glass porous structure is 0.5 mm or more, and further, the glass porous structure per 1 μL of the preservative liquid to be dropped. A jig for cryopreservation of cells or tissues having a body volume of 0.5 mm ³ or more .

According to the above invention, it is possible to perform the work of dropping and adhering cells or tissues to the placement portion on the preservation solution absorber with good visibility under the observation of a transmission type microscope, and the preservation solution. It is possible to provide a jig for cryopreservation having excellent absorbability.

It is an overall view which shows an example of the cryopreservation jig of this invention. It is an enlarged view of the preservation liquid absorber in FIG. It is a schematic diagram which shows an example of the case where a storage liquid absorber has a support. It is a schematic diagram which shows an example of the preservation liquid absorber used when a plurality of cells or tissues are cryopreserved by one cryopreservation jig. It is a schematic diagram which shows another example of the preservation liquid absorber used when a plurality of cells or tissues are cryopreserved by one cryopreservation jig.

The cryopreservation jig of the present invention is used for cryopreserving cells or tissues. In the present invention, a cell includes not only a single cell but also a cell population of an organism composed of a plurality of cells. The cell population composed of a plurality of cells may be a cell population composed of a single type of cells or a cell population composed of a plurality of types of cells. Further, the tissue may be a tissue composed of a single type of cells or a tissue composed of a plurality of types of cells, and may contain a non-cellular substance such as an extracellular matrix in addition to the cells. It may be a thing.

The cryopreservation work according to the present invention includes a freezing work in which cells or tissues are frozen using a cryogenic refrigerant, a storage task in which cells or tissues are stored in a cryogenic refrigerant, and cells or tissues are thawed in a thaw solution. It shall include a series of melting operations.

The jig for cryopreservation of the present invention is used for cryopreservation work of cells or tissues, and is preferably used for vitrification cryopreservation work. Specifically, the cryopreservation jig of the present invention is for immersing cells or tissues immersed in a preservation solution in a cooling solvent such as liquid nitrogen and freezing them. When the cells or tissues placed on the placement portion are thawed, the cells or tissues are taken out from the cooling medium together with a cryopreservation jig and immersed in a thaw solution to thaw. When the cryopreservation jig of the present invention is used, the preservation solution absorber has excellent light transmittance. Therefore, during the freezing operation, the cells or tissues are placed under observation using a transmission microscope. Becomes easier. Further, since the preservation solution absorber quickly absorbs the excess preservation solution, stable cell or tissue survival rate can be achieved even if the amount of the preservation solution that drops and adheres together with the cells or tissue is large. Further, as described above, the cells or tissues subjected to the freezing operation are covered with a very small amount of storage solution, and a rapid freezing rate can be achieved in an extremely low temperature refrigerant environment. In other words, rapid vitrification freezing is possible. In addition, due to the minimal amount of preservation solution that covers the cells or tissue, the preservation solution can be immediately diluted when the cryopreserved cells or tissue is thawed. The cryopreservation jig of the present invention can be paraphrased as a cell or tissue preservation tool, a cell or tissue cryopreservation instrument, or a cell or tissue preservation instrument.

The cryopreservation jig of the present invention is characterized in that the storage liquid absorber is a glass porous structure. The glass porous structure is what is called porous glass, and is formed by, for example, molding glass together with a spacer component and then removing the spacer by a chemical treatment called acid treatment. Further, the glass 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 cryopreservation jig of the present invention has the glass porous structure as a preservative liquid absorber, and has the above-mentioned porous structure itself as a preservative liquid absorber without having a support. You may be. Alternatively, the above-mentioned glass porous structure may be provided on the support. When the glass porous structure is laminated on the support, an adhesive layer can be provided between the support and the glass porous structure. Further, in addition to the glass porous structure and the adhesive layer, the support may have, for example, an undercoat layer or the like for the purpose of obtaining a uniform adhesive layer on the support.

The pore size of the glass porous structure of the cryopreservation jig of the present invention is 15 nm or less. When the pore size is 15 nm or less, excellent light transmittance can be obtained in addition to good absorption of the preservative solution, so that the work under transmission electron microscope observation can be performed with good visibility. A more preferable pore diameter is 10 nm or less. When the pore diameter exceeds 15 nm, scattering depending on the glass particles and pores / voids becomes remarkable, the light transmittance of the glass porous structure is lowered, and the workability is also lowered. The pore diameter of the porous body of the present invention is the diameter of the largest pore measured by the bubble point test described in JIS K 3832.

The glass porous structure of the cryopreservation jig of the present invention has a pore diameter of 4 nm or more . When the pore diameter is within the above range, the absorption of the preservative solution is good, and excellent workability can be obtained during the freezing operation.

The surface of the above-mentioned glass porous structure can be electropolished for the purpose of enhancing light transmission. By performing electrolytic polishing on the surface, it is possible to suppress the scattering of the surface and further enhance the light transmission.

The surface of the above-mentioned glass porous structure can be hydrophilized in order to enhance the absorption performance of the preservative solution. As a method for the hydrophilization treatment, a graft modification method, a coating method using a hydrophilic polymer compound or the like, a corona discharge, a plasma treatment, a general surface modification method using an excimer laser or the like can be used. ..

In the present invention, the area of the glass porous structure may be appropriately set according to the amount of the preservation solution dropped ^together with the cells or tissues, and is not particularly limited. It is preferably 2 to 400 mm ² , and more preferably 2 to 400 mm 2. Further, for the purpose of enhancing absorbability, the thickness is 0.5 mm or more . Further, the volume is 0.5 mm ³ or more per 1 μL of the stored solution to be dropped . In the same cryopreservation jig, when a plurality of glass porous structures of the present invention are provided as a storage liquid absorber on a non-absorbable support, one continuous glass porous structure is provided. Is preferably the above-mentioned area.

The cryopreservation jig of the present invention may have a support for the purpose of supporting the storage liquid absorber. As the support, a light-transmitting support is preferable, and a light-transmitting support having a total light transmittance of 80% or more is more preferable. Further, the haze value of the light transmissive support is preferably 10% or less. Examples of such a support include various resin films, glass, rubber and the like. Two or more kinds of supports may be used in combination as long as the effect of the present invention is obtained. Among them, the resin film is preferably used because of its excellent 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, polyarylate resins, and polychlorides. Examples thereof include a resin film made of a vinyl resin, a polysulfone resin, a polyether sulfone resin, a polyimide resin, a polyamide resin, a polyolefin resin, a cyclic polyolefin resin and the like, and the thickness thereof is preferably 10 to 1000 μm. Further, in order to increase the adhesive strength with the adhesive layer, the surface of the support can be easily adhered by a corona discharge treatment.

The storage liquid absorber in the present invention has been described above. The configuration of the cryopreservation jig using these will be described below. The cryopreservation jig of the present invention may be any as long as it has the preservation liquid absorber as described above, and the preservation liquid absorber may be connected to the grip portion. Having a grip portion is preferable because it has good workability during freezing / thawing.

FIG. 1 is an overall view showing an example of the cryopreservation jig of the present invention. In FIG. 1, the cryopreservation jig 5 is composed of a grip portion 1 and a storage liquid absorber 2. The grip portion 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, polyellene resin, fluororesin, various engineer plastics, and glass can be preferably used. Further, the storage liquid absorber 2 is preferably in the shape of a sheet or a strip in terms of handling.

FIG. 2 is an enlarged view of the storage liquid absorber of FIG. The storage liquid absorber 2a in FIG. 2 is an example of a form in which the glass porous structure 3 itself has no support and the storage liquid absorber is used as the storage liquid absorber. Further, FIG. 3 is a schematic view showing an example of a case where the storage liquid absorber has a support, and the storage liquid absorber 2b has a glass porous structure 3 on the support 4. Further, the storage liquid absorber 2b is an example of a form having a glass porous structure on the entire surface of the storage liquid absorber.

A method of joining the grip portion 1 and the storage liquid absorber 2 in FIG. 1 will be described. When the grip portion 1 is made of resin, for example, the storage liquid absorber 2 can be joined to the grip portion 1 by insert molding during molding. Further, a storage liquid absorber insertion portion (not shown) can be formed in the grip portion 1 and the storage liquid absorber 2 can be joined with an adhesive. Various adhesives can be used, but silicon-based or fluorine-based adhesives that are resistant to low temperatures can be preferably used.

When cells or tissues are cryopreserved for a long period of time using the cell or tissue cryopreservation jig of the present invention, the storage solution absorber portion of the cryopreservation jig shown in FIG. 1 is to be shielded from the outside world for safety. It is also possible to put a cap on the. Furthermore, liquid nitrogen is not normally sterilized, and when it is frozen in direct contact with liquid nitrogen, it may not be possible to guarantee a sterile state even if the cryopreservation jig is sterilized. Therefore, before freezing, the preservative solution absorber to which cells or tissues are attached may be capped and frozen without being in direct contact with liquid nitrogen. The cap is preferably made of various metals, various resins, glass, ceramics, etc., which are materials resistant to liquid nitrogen. The shape may be any shape such as a pencil cap or a cylindrical straw cap as long as it does not come into contact with the storage solution absorber and can block the outside world.

4 and 5 show an example of a preservative solution absorber used when a plurality of cells or tissues are cryopreserved with one cryopreservation jig. In the vitrification cryopreservation jig for cells or tissues shown in FIG. 1, the preservation solution absorber may be the one shown in FIGS. 4 and 5. In FIGS. 4 and 5, a plurality of glass porous structures 3 are discontinuously arranged on the support 4.

When the glass porous structure 3 has a continuous shape as shown in FIGS. 2 and 3 described above, when a plurality of cells or tissues are to be attached to the glass porous structure 3, the preservation solution has a glass porous structure. Since it spreads in the lateral direction and the thickness direction of the body, for example, when the second and subsequent cells or tissues are attached to the glass porous structure 3, the absorbability of the preservation solution may decrease. However, if a plurality of glass porous structures 3 are discontinuously provided on the support 4 as shown in FIGS. 4 and 5, there is no such concern, and the cells or tissues are made glass porous together with the preservation solution. It can be reliably attached to the structure 3 one by one. In FIGS. 4 and 5, as an example, a plurality of square-shaped porous glass structures 3 are arranged. The preservation solution absorber 2c and the preservation solution absorber 2d shown in FIGS. 4 and 5 are also examples of the preservation solution absorber having a glass porous structure in a part on the support.

The method for cryopreserving cells or tissues using the cryopreservation jig of the present invention is not particularly limited, and for example, first, the cells or tissues soaked in the preservation solution are dropped onto the placement portion together with the preservation solution. This automatically removes excess preservatives. Then, the cells or tissues can be frozen by immersing the cryopreservation jig in liquid nitrogen or the like while holding the cells or tissues in the placement portion. At this time, a cap capable of blocking the cells or tissues on the mounting portion from the outside world may be attached to the mounting portion and immersed in liquid nitrogen or the like. As the storage solution, those usually used for freezing cells such as eggs and embryos can be used. For example, a freezing agent (glycerol, ethylene glycol, etc.) is added to a physiological solution such as the above-mentioned phosphate buffered saline. Preservation solution containing a large amount of various antifreeze agents such as glycerol, ethylene glycol, DMSO (dimethyl sulfoxide) (at least 10% by mass or more, more preferably 20% by mass or more based on the total mass of the preservation solution). Liquid can be used. During the thawing operation, the cryopreservation jig is taken out from a cooling solvent such as liquid nitrogen, and the placing portion on which the frozen cells or tissues are placed is immersed in the thawing solution, and then the cells or tissues are immersed. to recover.

Examples of cells that can be cryopreserved using the cryopreservation jig of the present invention include eggs, embryos, and sperms of mammals (for example, humans, cows, pigs, horses, rabbits, rats, mice, etc.). Such as germ cells; pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) can be mentioned. In addition, cultured cells such as primary cultured cells, subcultured cells, and cell line cells can be mentioned. Further, in one or more embodiments, the cells include cancer-derived cells such as fibroblasts, pancreatic cancer / hepatic cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, cartilage cells, and tissue stem cells. And adhesive cells such as immune cells. Further, examples of tissues that can be cryopreserved include tissues composed of cells of the same type or different species, such as tissues such as ovary, skin, corneal epithelium, periodontal ligament, and myocardium. The present invention is particularly suitable for cryopreservation of tissues having a sheet-like structure (for example, cell sheets, skin tissues, etc.). The cryopreservation jig of the present invention is not limited to tissues directly collected from a living body, for example, cultured skin cultured and grown in vitro, a so-called cell sheet constructed in vitro, Japanese Patent Application Laid-Open No. 2012-205516. It can also be suitably used for cryopreservation of artificial tissues such as the proposed tissue model having a three-dimensional structure. The cryopreservation jig of the present invention is suitably used as a cryopreservation jig for cells or tissues as described above.

Hereinafter, the present invention will be described in more detail with reference to Examples, and the present invention will be specifically described, but the present invention is not limited to the following Examples. In the examples, the pore diameters are all values measured by the bubble point test described in JIS K 3832.

(Example 1)
Porous glass (thickness 1 mm) with a pore diameter of 4 nm, which is a glass porous structure manufactured by Akakawa Hard Glass Industry Co., Ltd., is used as a preservative liquid absorber, and the preservative liquid absorber 400 mm ² (20 mm × 20 mm) is made of ABS resin. By joining to the grip portion, the cryopreservation jig of Example 1 was produced in the form shown in FIG.

(Example 2)
Cryopreservation of Example 2 in the same manner as in Example 1 except that a porous glass (thickness 1 mm) having a pore diameter of 15 nm, which is a glass porous structure manufactured by Akakawa Hard Glass Industry Co., Ltd., was used as a preservative solution absorber. I made a jig for it.

(Comparative Example 1)
Instead of the glass porous structure, a cellulose acetate membrane (pore size 0.5 μm, thickness 125 μm) manufactured by Advantech Toyo Co., Ltd. made of cellulose acetate was used as a storage solution absorber. Since the membrane is thin and the self-supporting property is not sufficient, the membrane was attached onto a PET film having a thickness of 250 μm using a urethane adhesive, and a jig for cryopreservation of Comparative Example 1 was prepared in the same manner as in Example 1. ..

(Comparative Example 2)
The cryopreservation jig of Comparative Example 2 was used in the same manner as in Example 1 except that a transparent PET film (thickness 250 μm), which is a colorless and transparent film having no pores, was used as it was as a storage solution absorber. Made.

(Comparative Example 3)
Comparative Example 3 was cryopreserved in the same manner as in Example 1 except that a porous glass (thickness 1 mm) having a pore diameter of 70 nm, which is a glass porous structure manufactured by Akakawa Hard Glass Industry Co., Ltd., was used as a preservative solution absorber. I made a jig for it.

<Evaluation of visibility>
Place the tip of the pipette on the storage solution absorber of each cryopreservation jig of Examples 1, 2 and Comparative Examples 1 to 3 and place it on a transmission type inverted optical microscope (SZH-121, manufactured by Olympus Corporation). The visibility of the tip of the pipette was evaluated according to the following criteria. These results are shown in the item "Evaluation of visibility" in Table 1.

⊚: The tip of the pipette can be observed well.
◯: The tip of the pipette can be observed.
X: The tip of the pipette is difficult to observe or cannot be observed.

<Evaluation of absorption of preservative solution>
0.4 μL of a storage solution containing a plurality of glass beads (diameter 100 μm) as pseudo cells was dropped and adhered onto the storage solution absorbers of the cryopreservation jigs of Examples 1, 2 and Comparative Examples 1 to 3. rice field. As the preservation solution, a modified TCM199 medium manufactured by Sigma-Aldrich Co., Ltd. containing 15% by volume DMSO, 15% by volume ethylene glycol, and 17% by mass sucrose was used. After dripping and adhering, observe how the preservative solution around the pseudo-cells placed on the preservative solution absorber is absorbed with an epi-illumination type upright optical microscope (VC4500-S1 manufactured by OMRON Corporation). Evaluated according to the criteria of. These results are shown in the item of "Evaluation of absorbability of preservative solution" in Table 1.

◯: Excess preservative solution was absorbed within 20 seconds after adhering under the preservative droplets.
X: After adhering under the preservation droplet, the absorption of the preservation solution was not sufficient, and it was confirmed that the preservation solution remained around the pseudo-cells.

From the above results, the cryopreservation jig of the present invention has good visibility in that cells or tissues are dropped and adhered to the placement portion on the preservation solution absorber under the observation of a transmission type microscope during the freezing operation. It can be seen that it is possible to do so and has excellent absorption performance so that excess storage solution on the outer periphery of cells or tissues can be absorbed.

The present invention relates to tests collected from living organisms including iPS cells, ES cells, commonly used cultured cells, embryos or eggs, as well as embryo transplantation and artificial insemination of domestic animals such as cattle and animals, and artificial insemination to humans. It can be used for cryopreservation of cells or tissues for use or transplantation, cells or tissues cultured in vitro.

1 Grips 2, 2a, 2b, 2c, 2d Preservative liquid absorber 3 Glass porous structure 4 Support 5 Freezing storage jig

Claims (1)

It has a glass porous structure having a pore diameter of 4 nm or more and 15 nm or less as a preservative solution absorber , the thickness of the glass porous structure is 0.5 mm or more, and further, the glass porous structure per 1 μL of the preservative liquid to be dropped. A jig for cryopreservation of cells or tissues having a body volume of 0.5 mm ³ or more .

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