JP7471836B2 - Fixture for cryopreservation jig - Google Patents

Description

The present invention relates to a fixing device for a cryopreservation jig that is suitable for use when thawing frozen cells or tissues.

Excellent cell or tissue preservation technology is required in various industrial fields. For example, in cattle embryo transfer technology, embryos are frozen and then thawed and transferred in accordance with the estrus cycle of the recipient cow. In human infertility treatment, eggs or ovaries are collected from the mother, then frozen and stored to match the timing of transfer, and then thawed at the time of transfer.

In general, cells or tissues collected from a living body gradually lose activity or undergo changes in phenotype, even in a culture medium, so long-term ex vivo culture of cells or tissues is not desirable. Therefore, technology for long-term preservation while maintaining bioactivity is important. Excellent preservation technology enables more accurate analysis of collected cells or tissues. Excellent preservation technology also enables cells or tissues to be used for transplantation while maintaining higher bioactivity, which is expected to improve the survival rate after transplantation. Furthermore, it will be possible to sequentially produce and preserve artificial tissues for transplantation, such as cultured skin cultured ex vivo and so-called cell sheets constructed ex vivo, and use them when necessary, which is expected to be of great merit not only in medical terms but also in industrial terms.

For example, slow freezing is known as a method for cryopreserving cells or tissues. In this method, cells or tissues are first immersed in a preservation solution obtained by adding a cryoprotectant to a physiological solution such as phosphate buffered saline. Compounds such as glycerol, ethylene glycol, and dimethyl sulfoxide are used as the cryoprotectant. After immersing the cells or tissues in the preservation solution, the solution is cooled to -30 to -35°C at a relatively slow cooling rate (for example, 0.3 to 0.5°C/min), so that the solution inside and outside the cells or tissues is sufficiently cooled and the viscosity increases. In this state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (-196°C), vitrification occurs, which is a phenomenon in which the microsolutions inside and outside the cells or tissues and the surrounding microsolutions outside the cells or tissues are both solidified while remaining amorphous. When the inside and outside of the cells or tissues or the inside and outside of the tissues are solidified by vitrification, the movement of molecules is substantially eliminated, so it is considered that the vitrified cells or tissues can be preserved semi-permanently by storing them in liquid nitrogen.

Vitrification is also known as a method for cryopreserving cells or tissues. Vitrification uses the principle that ice crystals are less likely to form even below freezing due to the freezing point depression of a preservation solution that contains a large amount of cryoprotectants such as glycerol, ethylene glycol, or dimethyl sulfoxide. If this preservation solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. This type of solidification is called vitrification. A preservation solution that contains a large amount of cryoprotectants is also called a vitrification solution.

The slow freezing method described above requires a relatively slow cooling rate, so the operation for cryopreservation takes time. There is also the problem of needing a device or tool to control the cooling rate. In addition, the slow freezing method forms ice crystals in the preservation solution outside the cells or tissues, which may physically damage the cells or tissues. In contrast, the vitrification freezing method described above requires a short operation time and is a process that does not require special devices or tools. In addition, the vitrification freezing method achieves a high survival rate by not generating ice crystals.

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

Vitrification is a technique that has been developed primarily using human germ cells, but recently its application to iPS cells and ES cells has also been widely considered. Non-Patent Document 1 shows that vitrification is effective for preserving Drosophila embryos. Furthermore, Patent Document 2 shows that vitrification is effective for preserving plant cultured cells and tissues. In this way, vitrification is known to be useful for preserving a wide variety of cell and tissue species.

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

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

In general, as a freezing method related to the vitrification freezing method, Patent Document 4 proposes a method in which a mammalian embryo or egg is attached to the inner surface of a cryopreservation container such as a freezing straw, a freezing vial, or a freezing tube with a minimum amount of vitrification liquid sufficient to encapsulate the embryo or egg, and the container is brought into contact with liquid nitrogen to rapidly cool it. The thawing method performed after the freezing method involves removing the cryopreservation container stored by the above method from liquid nitrogen, opening one end of the container, injecting a diluent at 33 to 39°C into the container, and thawing and diluting the frozen embryo or egg. This method is said to enable the preservation and thawing/dilution of mammalian embryos or eggs with a high survival rate without the risk of infection by viruses or bacteria. However, the task of attaching an embryo or egg to the inner surface of a cryopreservation container such as a freezing straw, a freezing vial, or a freezing tube is highly difficult, and it is difficult to confirm that the embryo or egg has been placed in the cryopreservation container.

Patent Document 5 describes a cell cryopreservation tool having a cell retention member with a thermally conductive member and a cylindrical storage member, and the problems with the freezing and thawing method of Patent Document 4 are resolved to some extent. As a method of using the cryopreservation tool described in Patent Document 5, eggs are attached to the cell retention member under a microscope, the cell retention member is stored in a cylindrical storage member, and then immersed in liquid nitrogen for vitrification and freezing. A method is then described in which a lid member is attached to the opening of the cylindrical member and stored in a liquid nitrogen tank. In Patent Document 6, eggs are placed on an egg attachment and retention strip together with a small amount of vitrification liquid, and the entire cryopreservation tool is stored in a cylindrical storage container and then immersed in liquid nitrogen to perform vitrification and freezing.

As a freezing method related to the vitrification freezing method with fewer processes, the so-called Cryotop (registered trademark) method, which is used in the field of human infertility treatment, as described in Patent Documents 7 and 8, has been disclosed. In these methods, the freezing operation uses an egg freezing storage device equipped with a rectangular flexible, colorless and transparent film as an egg attachment and retention strip, and the egg or embryo is placed on the film together with a very small amount of preservation solution under microscope observation, and the film with the egg attached is immersed in liquid nitrogen to perform vitrification freezing. The egg attachment and retention strip with the frozen egg or embryo placed on it is then protected with a cap or the like and stored in a liquid nitrogen tank.

The eggs and embryos frozen by these methods are thawed by immersing the egg attachment and retention strip in a warm thawing liquid, and the eggs and embryos placed on the strip in the thawing liquid are then collected.

On the other hand, in order to improve the workability of the thawing operation in the cryopreservation method, a liquid nitrogen container as described in Patent Document 9 is known. The liquid nitrogen container has a slit formed in the lid, and the cryopreservation jig stored in a cylindrical storage member is temporarily placed against the slit.

If the frozen cells or tissues are temporarily held below the liquid nitrogen level in this manner, then in the methods described in Patent Documents 5 and 6, for example, the lid member located above the liquid nitrogen level can be easily removed, and the cells or tissues can be quickly moved into the melting liquid, preventing a decrease in the melting rate. In the methods described in Patent Documents 7 and 8, the cryopreservation tool can be pulled out of the cap, allowing the cells or tissues to be quickly moved into the melting liquid, preventing a decrease in the melting rate.

Patent No. 3044323 JP 2008-5846 A JP 2017-104061 A JP 2000-189155 A Patent No. 5798633 International Publication No. 2019/004300 JP 2002-315573 A JP 2006-271395 A U.S. Design Registration No. 642,697

Steponkus et al., Nature 345:170-172 (1990) Hiroshi Sozu, "Mechanism of freezing-induced damage and protection of living cells," Chemistry and Biology, Vol. 18 (1980), No. 2, pp. 78-87, Published by the Agricultural Chemical Society of Japan

However, in the method using a liquid nitrogen container described in Patent Document 9, although it is possible to stably hold the cryopreservation jig when it is leaned against the slit formed in the lid, the mounting portion of the cryopreservation jig is located below the liquid nitrogen surface, making it difficult to confirm whether the cells or tissues are reliably maintained in a frozen state. In addition, the length of the cylindrical storage member must be longer than the length from the bottom of the liquid nitrogen container to the position where the lid is placed. In such cases, the task of removing the cell retention member main body from inside the cylindrical resin member is cumbersome, and improvements are desired from the perspective of performing a rapid thawing operation.

The main objective of the present invention is to provide a fixing device that realizes favorable operability during the thawing operation for vitrification cryopreservation of cells or tissues. More specifically, the objective of the present invention is to provide a fixing device for a cryopreservation jig that can stably hold a cryopreservation jig, can easily confirm that the frozen cells or tissues are in a reliably cooled state prior to the operation of transferring the frozen cells or tissues to a melting liquid, and can quickly transfer the frozen cells or tissues to a warm melting liquid.

As a result of intensive research into solving the above problems, the present inventors have found that the above problems can be solved by a fixing device for a cryopreservation jig having the following configuration (hereinafter also referred to as the "fixing device of the present invention").

A fixing device for a cryopreservation jig made of a base material having a top surface, sides, and a bottom surface, the fixing device has a slit portion on the top surface of the base material with at least one end opening into the side surface of the base material in order to fix the cryopreservation jig, or has an insertion portion on the top surface of the base material for inserting the cryopreservation jig and a slit portion on the side surface of the insertion portion with one end opening, and further has a liquid level gauge portion on the top surface or side surface of the base material that extends above the top surface of the base material.

According to the present invention, it is possible to provide a fixing device for a cryopreservation jig that can stably hold the cryopreservation jig and that can easily confirm that the frozen cells or tissues are in a completely cooled state prior to the operation of transferring the frozen cells or tissues to a melting liquid. Furthermore, the fixing device of the present invention makes it possible to quickly transfer the frozen cells or tissues to a warm melting liquid.

FIG. 2 is a schematic top view showing an example of a fixing device of the present invention. FIG. 1 is a schematic side view showing an example of a slit portion of a fixing device of the present invention. 13 is a schematic side view showing another example of a slit portion of the fixing device of the present invention. FIG. 13 is a schematic side view showing another example of a slit portion of the fixing device of the present invention. FIG. FIG. 11 is a schematic top view showing another example of the fixing device of the present invention. FIG. 11 is a schematic top view showing another example of the fixing device of the present invention. FIG. 11 is a schematic top view showing another example of the fixing device of the present invention. FIG. 11 is a schematic top view showing another example of the fixing device of the present invention. FIG. 11 is a schematic top view showing another example of the fixing device of the present invention. FIG. 11 is a schematic top view showing another example of the fixing device of the present invention. FIG. 11 is a schematic top view showing another example of the fixing device of the present invention. FIG. 11 is a schematic side view showing another example of the fixing device of the present invention. FIG. 11 is a schematic side view showing another example of the fixing device of the present invention. FIG. 11 is a schematic side view showing another example of the fixing device of the present invention. FIG. 11 is a schematic side view showing another example of the fixing device of the present invention. FIG. 11 is a schematic side view showing another example of the fixing device of the present invention. FIG. 11 is a schematic side view showing another example of the fixing device of the present invention. FIG. 2 is a schematic diagram showing an example of a state in which a cryopreservation jig is fixed using the fixing device of the present invention. FIG. 13 is a schematic diagram showing another example of a state in which a cryopreservation jig is fixed using the fixing device of the present invention. 1 is a schematic diagram showing a state in which the main body member and cap member of a cryopreservation jig are separated using the fixing device of the present invention and the mounting portion is immersed in a melting liquid. FIG.

The fixing device of the present invention is used to fix a jig for cryopreservation of cells or tissues. The fixing device of the present invention is also preferably used when cryopreserving cells or tissues by the so-called vitrification cryopreservation method. In the present invention, the term "cell" refers not only to a single cell but also to a cell population of an organism consisting of a plurality of cells. A cell population consisting of a plurality of cells may be a cell population consisting of a single type of cell, or may be a cell population consisting of a plurality of types of cells. Furthermore, the term "tissue" may be a tissue consisting of a single type of cell, or may be a tissue consisting of a plurality of types of cells, or may contain non-cellular substances such as extracellular matrix in addition to cells. The fixing device of the present invention is particularly preferably used in the cryopreservation of eggs or embryos.

The fixing device of the present invention can be rephrased as a fixing device for a device for cryopreservation of cells or tissue, a fixing device for a device for cryopreservation of cells or tissue, a fixing device for a device for cryopreservation of cells or tissue, a fixing device for a device for cryopreservation of cells or tissue, or a fixing device for a device for cryopreservation of cells or tissue.

The fixing device of the present invention is described in detail below.

The fixing device of the present invention is a fixing device for a cryopreservation jig made of a substrate having an upper surface (top surface), side surfaces, and a bottom surface, and has a slit portion on the upper surface of the substrate for fixing the cryopreservation jig, and at least a liquid level gauge portion extending above the upper surface of the substrate on the upper surface or side surface of the substrate. After inserting the tip end of the cylindrical storage member or the tip end of the cap of the cryopreservation jig into the slit portion of the fixing device of the present invention, the cryopreservation jig is fixed by moving the cryopreservation jig along the slit to the back side of the slit. Meanwhile, the liquid level of the cooling medium can be easily adjusted based on the liquid level gauge portion attached to the upper surface or side surface of the substrate, and according to the present invention, the cryopreservation jig can be fixed while reliably maintaining the frozen state of the cells or tissue.

The fixing device of the present invention has a slit portion for fixing and holding the cryopreservation jig, and may have one slit portion on the upper surface of the substrate, or may have multiple slit portions.

The slit portion of the fixing device of the present invention preferably has a fixing structure for firmly fixing and holding the cryopreservation jig inserted therein. When the slit portion has a fixing structure, the cryopreservation jig inserted in the slit portion can be more firmly fixed and held by the fixing structure. Examples of the fixing structure include an uneven structure formed on the inner side of the slit portion, a tapered structure in which the distance between the inner side of the slit portion gradually narrows, and a bent structure in which the slit portion bends in a different direction after moving straight ahead a certain distance from the opening.

The case where the slit portion of the fixing device of the present invention has a concave-convex structure as a fixing structure will be described. When the cryopreservation jig has a concave-convex structure, the concave-convex structure formed on the inner side of the slit portion can reliably fix the cryopreservation jig to the fixing device by fitting the concave-convex structure of the fixing device and the concave-convex structure of the cryopreservation jig into each other. It is preferable that the concave-convex structure of the slit portion has a shape in which the concave and/or convex portions of the concave-convex structure extend in a direction parallel to the bottom surface of the fixing device on the inner side of the slit portion.

When the fixing device of the present invention has a concave-convex structure, it can have a concave structure and/or a convex structure of any shape to match the shape of the tip of the cryopreservation jig to be fixed, preferably the shape of the tip of the cap member. In addition, it is more preferable that the cross-sectional shape of the concave-convex structure of the fixing device is the same as the cross-sectional shape of the concave-convex structure of the tip of the cryopreservation jig to be fixed.

When the fixing device of the present invention has an uneven structure, it is sufficient that it has it in at least one location on the side surface inside the slit portion. From the viewpoint of increasing the fixing stability of the cryopreservation jig, it is more preferable that the uneven structure be present on both of the side surfaces facing each other inside the slit portion. Here, both of the side surfaces facing each other mean both of the side surfaces facing each other in a top view schematic diagram of the fixing device of the present invention when viewed from above. The uneven structure portions located on both of these side surfaces may be continuously connected at the back side of the slit.

When the slit portion of the fixing device of the present invention has a tapered structure as a fixing structure, the distance between the side surfaces inside the slit portion decreases as the cryopreservation jig is inserted from the opening of the slit portion through which the cryopreservation jig is inserted. When the slit portion of the fixing device of the present invention has a tapered structure, the inserted cryopreservation jig fits into the tapered structure, and the cryopreservation jig can be securely fixed to the fixing device, which is preferable.

When the slit portion of the fixing device of the present invention has a bent structure portion as a fixing structure portion, the slit portion has a structure in which it advances straight a certain distance from the opening of the slit portion through which the cryopreservation jig is inserted and then bent in a different direction. When the slit portion has a bent structure portion, the cryopreservation jig inserted from the opening of the slit portion is inserted further straight through the bent structure portion in a direction different from the straight direction from the opening and fixed, which is preferable as it can prevent the jig from accidentally falling off toward the opening side.

The shape of the fixture of the present invention (the general shape when it is assumed that the substrate does not have a slit portion, or that an insertion portion for inserting a cryopreservation jig is not provided above the substrate, and the liquid level gauge portion described above is not provided) can be a prismatic structure such as a triangular prism, a square prism, or a hexagonal prism, as well as a partial structure of a pyramid such as a triangular pyramid or a square pyramid with an upper surface. A prismatic structure is preferable, and a square prism structure is more preferable. In addition to the above, a cylindrical structure or a conical partial structure can also be exemplified.

In the present invention, the substrate is preferably formed of a liquid nitrogen resistant material. Suitable materials for use include, for example, various metals such as aluminum, iron, copper, and stainless steel alloys, ABS resin, acrylic resin, polypropylene resin, polystyrene resin, polyethylene resin, fluorine-based resin, various engineering plastics, and even glass and rubber materials. Metals are particularly preferred from the viewpoints of excellent durability, large weight, and ability to secure the cryopreservation jig well. Among these, aluminum is preferred from the viewpoint of workability. The fixture of the present invention may be made of one type of material or multiple materials.

The fixing device of the present invention has a liquid level gauge section on the upper or side surface of the substrate, which extends above the upper surface of the substrate. The liquid level gauge section of the fixing device of the present invention is a structure used to manage the liquid level of the cooling solvent. The presence of the liquid level gauge section makes it easy to adjust the liquid level of the cooling solvent. In other words, when a cryopreservation jig on which cells or tissues are placed is fixed to the fixing device of the present invention, it is easy to confirm that the cells or tissues placed on the cryopreservation jig are in a reliably cooled state.

The liquid level gauge portion of the fixture of the present invention is preferably a chimney-shaped (prism-shaped such as a square prism or cylinder-shaped) structure extending from the bottom of the container toward the liquid level of the cooling solvent.

The liquid level gauge portion of the fixture of the present invention can take any shape as long as it extends above the top surface of the base material and can serve as a reference for managing the liquid level of the cooling solvent. However, from the viewpoint of allowing an operator to easily visually confirm the position of the liquid level, it is preferable that it protrudes above the liquid level of the cooling solvent or is slightly hidden by the liquid level.

When the liquid level gauge portion of the fixture of the present invention has a height that protrudes from the liquid level of the cooling solvent, the liquid level gauge portion preferably has a marking portion. The shape of the marking portion is not particularly limited, but it is preferable that the marking portion is a line segment parallel to the liquid level of the cooling solvent. From the viewpoint of providing a reference for the height of the liquid level of the cooling solvent, it is sufficient that there is one marking portion in the height direction, and two or more marking portions may be provided.

Next, we will explain in detail the freezing and thawing method using the fixing device of the present invention.

The fixing device of the present invention can be suitably used in a series of processes from freezing and thawing cells or tissues, particularly in the cooling operation after the frozen cells or tissues are removed from the liquid nitrogen tank, and in the thawing operation when thawing the cells or tissues. In general, when preserving cells or tissues by the so-called vitrification freezing method, the cells or tissues are frozen using a cryopreservation jig having a rectangular strip. Such cryopreservation jigs are disclosed in, for example, WO 2011/070973 and WO 2015/064380, in addition to the above-mentioned Patent Document 7 and Patent Document 8. The series of processes from freezing and thawing includes, first, dropping equilibrated cells or tissues together with a preservation solution onto the strip of the cryopreservation jig, and then cooling the cells or tissues using liquid nitrogen to perform the freezing operation. Secondly, the cells or tissues that have been frozen are preserved for a long period of time while maintaining an extremely low temperature environment inside the liquid nitrogen tank. Thirdly, the frozen cells or tissues are transferred from the cryogenic environment to a solution known as a melting liquid, and melted and thawed in the melting liquid (melting operation). The fixation device of the present invention can be suitably used to fix and hold the cryopreservation jig during the cooling operation and melting operation after the cells or tissues are removed from the liquid nitrogen tank described above.

The fixation device of the present invention can be preferably used in a so-called closed type cryopreservation-thawing process in which the cells or tissues do not come into contact with liquid nitrogen during the series of processes from cryopreservation to thawing of the cells or tissues.

In the closed-type cryopreservation-thawing process, during the freezing operation, the cells or tissues are dripped onto the strip together with the preservation solution, and then the cryopreservation jig is enclosed in a cylindrical storage member with a completely closed end, isolating it from the outside world. Alternatively, the cells or tissues are dripped onto the strip together with the preservation solution, and then the strip is encapsulated with a cap member, isolating it from the outside world. After such isolation, the cryopreservation jig is immersed in liquid nitrogen to cool and freeze the cells or tissues. During the closed-type freezing operation and storage described above, the air and preservation solution around the cells or tissues are cooled via the cylindrical storage member or cap member, and the vitrified state is maintained.

In the thawing operation in the closed cryopreservation-thawing process using the fixing device of the present invention, the cryopreservation jig stored in the liquid nitrogen tank is removed from the liquid nitrogen tank, and prior to the operation of transferring the cells or tissue to the thawing liquid, the liquid level (volume) of the liquid nitrogen is controlled using the liquid level gauge of the fixing device of the present invention when cooling in liquid nitrogen using the fixing device of the present invention. In other words, when the tip of the cylindrical storage member or the tip of the cap member is fitted into the slit of the fixing device of the present invention, the liquid level (volume) of the liquid nitrogen is controlled so that the cells or tissue is located below the liquid nitrogen level. By holding the cryopreservation jig in this state, it is easy to confirm that the cells or tissue placed on the cryopreservation jig is in a reliably cooled state.

Thereafter, by removing the lid fixed to the cylindrical storage member located above the liquid nitrogen surface, the cryopreservation jig on which the cells or tissues are placed can be quickly removed without touching the liquid nitrogen and immersed in the melting liquid. Alternatively, by pulling out the cryopreservation jig having the strip on which the cells or tissues are placed from the cap member fixed by the fixing device of the present invention, the cells or tissues held on the strip can be quickly removed without touching the liquid nitrogen and immersed in the melting liquid. The latter cryopreservation jig is preferred from the viewpoint of enabling faster operation.

The cells or tissues immersed in the melting liquid by the above-mentioned operation are preferably immersed in the melting liquid within 5 minutes, more preferably within 1 minute, after the lid member is removed from the cylindrical storage member. In addition, when the cryopreservation jig having the strip is pulled out from the cap member, it is preferably immersed in the melting liquid within 5 minutes, more preferably within 1 minute, after the fitting is loosened. If the so-called sealed state is opened for a long period of time, such as after the lid member is removed from the cylindrical storage member or after the fitting with the cap member is loosened when the cryopreservation jig is pulled out, gases such as nitrogen and oxygen in the air may enter the cylindrical storage member or the cap member, and then be cooled and liquefied. The liquefied gas may adhere to the placement part and be carried into the melting liquid, lowering the temperature of the melting liquid. If the temperature of the melting liquid drops, the melting speed will slow down, which is not preferable.

The fixing device of the present invention can also be used in the so-called open type cryopreservation/thawing process in which the cells or tissues come into contact with liquid nitrogen during the series of processes from cryopreservation to thawing of cells or tissues. Even in the open type process, it is preferable to fix and hold the cryopreservation jig using the fixing device of the present invention prior to the operation of transferring the frozen cells or tissues to the thawing liquid, since this allows the cells or tissues to be quickly transferred from liquid nitrogen to the thawing liquid.

The freeze-thaw method using the fixing device of the present invention has been described above. Next, the fixing device of the present invention will be described in more detail with reference to the drawings.

Figure 1 is a top schematic diagram showing an example of a fixing device of the present invention. In Figure 1, fixing device 1 has one slit section 2, which is a groove-like structure into which a cryopreservation jig is inserted, on the top surface of substrate 20. One side of slit section 2 has a slit opening 4 on the side surface of substrate 20, and the other side is closed at the center side of the top surface in the figure. Fixing device 1 also has a liquid level gauge section 16 in the shape of a rectangular prism attached to the top surface (top surface) of substrate 20. When inserting a cryopreservation jig (not shown) into slit section 2, the cryopreservation jig is inserted from the slit opening 4 side along slit section 2 toward the center of the top surface in the figure.

T1 in Figure 1 indicates the length (depth) of the slit portion 2 of the fixing device 1. There are no particular limitations on T1 as long as it is a length that can hold the cryopreservation jig after insertion, but when freezing and thawing cells or tissues using the above-mentioned Cryotop method, T1 is preferably 3 to 50 mm, and more preferably 5 to 40 mm. Note that all dimensions of the fixing device 1 described below are dimensions intended for freezing and thawing operations using the Cryotop method.

T2 in FIG. 1 indicates the length in the width direction of the slit portion 2. In the present invention, T2 is not particularly limited as long as it is a length that allows the cryopreservation jig to be inserted and fixed and held, but it is preferably 1 to 10 mm, and more preferably 2 to 5 mm.

Figure 2 is a schematic side view showing an example of a slit portion of the fixing device of the present invention (fixing device of Figure 1). In Figure 2, fixing device 1 has a slit portion 2 whose one end opens into the side portion of substrate 20 and into which a cryopreservation jig (not shown) is inserted.

T3 shown in FIG. 2 indicates the length in the depth direction of the slit portion 2 of the fixing device 1. In the present invention, T3 is not particularly limited as long as it is a length that can fix and hold a cryopreservation jig (not shown), but it is preferably 3 to 50 mm, and more preferably 5 to 40 mm.

Figure 3 is a schematic side view showing another example of a slit portion of the fixing device of the present invention. In Figure 3, the slit portion 2 of the fixing device 1 has an uneven structure portion 3 on its inner side as a fixing structure portion for firmly fixing the cryopreservation jig. The uneven structure portion 3 of the fixing device in Figure 3 is a convex type.

Figure 4 is a schematic side view showing another example of a slit portion of the fixing device of the present invention. In Figure 4, the slit portion 2 of the fixing device 1 has an uneven structure portion 3 on its inner side as a fixing structure portion for firmly fixing the cryopreservation jig. The uneven structure portion 3 of the fixing device in Figure 4 is concave.

Figure 5 is a top schematic diagram showing another example of the fixing device of the present invention. In Figure 5, the slit portion 2 of the fixing device 1 has a tapered structure as a fixing structure for firmly fixing the cryopreservation jig. The tapered structure of the fixing device in Figure 5 is a structure in which the distance between the side surfaces of the slit portion 2 gradually decreases from the slit opening 4 toward the center of the substrate, that is, T21>T22. When the slit portion 2 has a tapered structure as in the fixing device shown in Figure 5, the cryopreservation jig inserted along the slit portion 2 from the slit opening 4 can be firmly fixed, which is preferable.

Figure 6 is a top schematic diagram showing another example of the fixing device of the present invention. In Figure 6, the slit portion 2 of the fixing device 1 has a bent structure portion as a fixing structure portion for firmly fixing the cryopreservation jig. The slit portion 2 of the fixing device of Figure 6 having a bent structure portion is shaped so that it advances in a straight line from the slit opening 4 toward the center of the substrate for a certain distance, and then bends 90 degrees midway. The use of a fixing device of this shape is preferable because it can prevent the fixed and held cryopreservation jig from accidentally falling off the slit opening 4.

Figure 7 is a top schematic diagram showing another example of the fixing device of the present invention. In Figure 7, the fixing device 1 has an insertion section 23 for inserting a cryopreservation jig on the upper surface (top surface) of the substrate 20, and a slit opening 4 on the side of the insertion section 23. The fixing device as shown in Figure 7 has no opening on the side of the substrate, so that the structure is such that the fixed and held cryopreservation jig is less likely to fall off unexpectedly.

Figure 8 is a top schematic diagram showing another example of the fixing device of the present invention. In Figure 8, the slit portion 2 of the fixing device 1 has both a bent structure portion and a tapered structure portion as a fixing structure portion for firmly fixing the cryopreservation jig. The slit portion 2 of the fixing device 1 in Figure 8 has a structure in which, after moving straight from the slit opening 4 toward the center side of the substrate for a certain distance, it is bent 90 degrees along the way and has a tapered structure after being bent 90 degrees. When using a fixing device of such a shape, it is preferable to insert the cryopreservation jig into the slit portion 2 from the slit opening 4, pass the 90-degree bent position, and further insert the cryopreservation jig to the closed surface side of the slit portion, because the tapered structure can fix and hold it more firmly.

Figure 9 is a top schematic diagram showing another example of the fixing device of the present invention. In Figure 9, the slit opening 4 of the fixing device 1 is wider than the width of the slit portion 2, which provides excellent operability when inserting a cryopreservation jig.

Figure 10 is a top schematic diagram showing another example of the fixing device of the present invention. In Figure 10, the fixing device 1 has two slit openings 4 for one slit portion 2. In addition, the fixing device 1 has a tapered structure in which the width of the groove of the slit portion 2 decreases from the slit openings 4 toward the center of the substrate. With the shape shown in Figure 10, a cryopreservation jig (not shown) can be inserted from either end of the slit portion 2, so a fixing device capable of simultaneously storing multiple cryopreservation jigs is obtained. In addition, the fixed cryopreservation jig is expected to be cooled more efficiently because a larger area of the jig comes into contact with the cooling solvent compared to when the slit portion 2 is closed.

Figure 11 is a top schematic diagram showing another example of the fixing device of the present invention. In Figure 11, the fixing device 1 has multiple independent slit sections 2 on the upper surface of the substrate 20. Each of the slit sections 2 is capable of fixing a cryopreservation jig. According to this embodiment, since more slit sections 2 are provided on the upper surface of the substrate 20, a large number of cryopreservation jigs can be fixed and held at one time.

Figure 12 is a schematic side view showing another example of the fixing device of the present invention. In Figure 12, the fixing device 1 has a hollow structure 5. Having a hollow structure 5 is preferable because it reduces the thermal capacity of the fixing device 1 and reduces the amount of liquid nitrogen that evaporates when the fixing device 1 is immersed in liquid nitrogen.

Figure 13 is a schematic side view showing another example of the fixture of the present invention. The fixture 1 in Figure 13 has a slit portion 2 and a liquid level gauge portion 16 that is attached to the side of the substrate 20 and extends above the upper surface (top surface) of the substrate.

Figure 14 is a schematic side view showing another example of the fixing device of the present invention. The fixing device 1 in Figure 14 has a slit portion 2 and a liquid level gauge portion 16 in the shape of a rectangular prism attached to the upper surface (top surface) of the substrate 20. The liquid level gauge portion 16 of the fixing device 1 in Figure 14 has one marking portion, and the marking portion shown in Figure 14 is an upper limit marking portion 17. The upper limit marking portion 17 is a line segment parallel to the liquid nitrogen surface (not shown). By using this position as a guide to manage the amount of liquid nitrogen, the operator can easily confirm that the frozen cells or tissues have been reliably cooled.

Figure 15 is a schematic side view showing another example of the fixture of the present invention. The fixture 1 in Figure 15 has a square prism-shaped liquid level gauge section 16 attached to the upper surface (top surface) of the substrate 20, together with the slit section 2. The liquid level gauge section 16 of the fixture 1 in Figure 15 has a total of two marking sections, an upper limit marking section 17 and a lower limit marking section 18. When the fixture 1 has the upper limit marking section 17 and the lower limit marking section 18, for example, the operator only needs to set and prepare the liquid nitrogen level between the upper limit marking section 17 and the lower limit marking section 18, making it even easier to manage the amount of liquid nitrogen.

Figure 16 is a schematic side view showing another example of the fixture of the present invention. The fixture 1 in Figure 16 has a slit portion 2 and a rectangular prism-shaped liquid level gauge portion 16 attached to the upper surface (top surface) of the substrate 20. The liquid level gauge portion 16 of the fixture 1 in Figure 16 has a convex upper limit marking portion 17 and a convex lower limit marking portion 18, and the operator can set and prepare the liquid nitrogen level between the convex upper limit marking portion 17 and the convex lower limit marking portion 18.

Figure 17 is a schematic side view showing another example of the fixture of the present invention. The fixture 1 in Figure 17 has a slit portion 2 and a three-dimensional liquid level gauge portion 16 with a stepped step attached to the upper surface (top surface) of the substrate 20. The liquid level gauge portion 16 of the fixture 1 in Figure 17 differs from the form shown in Figure 16 above in that it has an upper limit marking portion 17 and a lower limit marking portion 18 with stepped steps. As with the marking portions described above, the operator can set and prepare the liquid nitrogen level between the upper limit marking portion 17 and the lower limit marking portion 18.

Next, we will explain the method for freezing and thawing cells or tissues using the fixation device of the present invention.

In the present invention, the cells or tissue is placed together with a preservation liquid on the placement part of a cryopreservation jig having at least a main body member with a placement part and a cap member detachable from the main body member, the placement part is sealed with the cap member, and the sealed placement part is cooled with a cooling solvent to maintain the frozen state of the frozen cells or tissue. When the cells or tissue are subsequently thawed, it is preferable to temporarily hold the cells or tissue sealed with the cap member below the liquid level of the cooling solvent and the joint between the main body member and the cap member above the liquid level of the cooling solvent, because this allows the main body member and the cap member to be quickly separated and the placement part to be quickly immersed in the thawing liquid.

In addition, instead of the above-mentioned cryopreservation jig having at least a main body member with a mounting portion and a cap member that can be attached and detached to the main body member, a cryopreservation jig having a cell retention member to which eggs, embryos, etc. can be attached and the cell retention member stored in a cylindrical storage member is used, and when the eggs, embryos, etc. are temporarily held in a state where they are positioned below the liquid level of the cooling solvent and one end of the cylindrical storage member is positioned above the liquid level of the cooling solvent, the cell retention member can be removed from the cylindrical storage member by opening the end of the cylindrical storage member that is positioned above the liquid level of the cooling solvent, for example by cutting, and this can be quickly immersed in the melting liquid, which is preferable.

Figure 18 is a schematic diagram showing an example of a state in which a cryopreservation jig is fixed using the fixing device of the present invention. In Figure 18, the cryopreservation jig 6 has at least a main body member 7 and a cap member 8 that can be attached and detached to the main body member 7. Cells 10 are placed on a strip 9 of the cryopreservation jig 6 together with a very small amount of preservation liquid 11.

The strip 9 on which the cells 10 are placed together with a very small amount of preservation liquid 11 is sealed with a cap member 8, and the cells 10 placed on the strip 9 are cooled by being positioned below the liquid surface 14 of the liquid nitrogen 13, and the frozen cells 10 are maintained in a frozen state. When the frozen cells 10 are melted, the joint 12 between the cap member 8 and the main body member 7 is positioned above the liquid surface 14 of the liquid nitrogen 13, so that the main body member 7 can be quickly pulled out and separated from the cap member 8, and the placement portion can be quickly immersed in the melting liquid, which is preferable. In addition, by using the liquid level gauge portion 16 of the present invention, the operator can easily grasp the position of the liquid surface 14, improving workability. Figure 18 illustrates an example in which the upper surface of the liquid level gauge portion 16 attached to the upper surface (top surface) of the fixture 1 is used as the reference for the position of the liquid surface 14. By using the liquid level gauge portion 16, it is easy to position the liquid surface 14 above the cells 10 and below the joint portion 12.

Figure 19 is a schematic diagram showing another example of the state in which a cryopreservation jig is fixed using the fixing tool of the present invention. In Figure 19, the fixing tool 1 has a liquid level gauge section 16, which has an upper limit marking section 17. The liquid level 14 of the liquid nitrogen 13 is located between the upper limit marking section 17 and the upper surface (top surface) of the fixing tool 1. By managing the liquid level 14 using the liquid level gauge section 16 of the fixing tool 1 in this way, it is possible to more reliably maintain the frozen state of the cells 10 when the cryopreservation jig is fixed to the fixing tool, and even when the main body member 7 and the cap member 8 are disengaged, the cells 10 can be thawed quickly without coming into contact with liquid nitrogen.

Next, we will explain the cryopreservation jig that is fixed using the fixing device of the present invention.

The strip 9 of the main body member 7 is preferably rectangular. This is because rectangular shapes make it easier to store the strip 9 in a cap member or a cylindrical storage member.

In the present invention, the strip 9 of the cryopreservation jig may be, for example, various resin films, metal plates, glass plates, rubber plates, etc. The mounting portion may be made of one type of material, or may be made of two or more types of materials. Among them, resin films are preferably used from the viewpoint of handling. Specific examples of resin films include resin films made of polyester resins such as polyethylene terephthalate and polyethylene naphthalate, acrylic resins, epoxy resins, silicone resins, polycarbonate resins, diacetate resins, triacetate resins, polyacrylate resins, polyvinyl chloride resins, polysulfone resins, polyethersulfone resins, polyimide resins, polyamide resins, polyolefin resins, cyclic polyolefin resins, etc. In addition, if the total light transmittance of the strip 9 is 80% or more, it is preferable because the cells or tissues mounted on the mounting portion can be easily confirmed using a transmission microscope.

Metal plates can also be used as the strips 9 because they have excellent thermal conductivity and allow for rapid freezing. Specific examples of metal plates include copper, copper alloys, aluminum, aluminum alloys, gold, gold alloys, silver, silver alloys, iron, and stainless steel. The thickness of the various resin films, metal plates, glass plates, rubber plates, etc. described above is preferably 10 μm to 10 mm. Depending on the purpose, the surfaces of the various resin films, metal plates, glass plates, rubber plates, etc. can be made hydrophilic by electrical methods such as corona discharge treatment or chemical methods, and can also be roughened.

The above-mentioned strip 9 can also be a preservation liquid absorbent. When the strip 9 is made of a preservation liquid absorbent, the excess preservation liquid 11 can be effectively removed, improving the freezing speed. Examples of the preservation liquid absorbent include a film-like material made of wire mesh, paper, or synthetic resin with through holes. Other examples of the preservation liquid absorbent include a porous structure made of a material with a refractive index of 1.45 or less. The porous structure can efficiently remove the preservation liquid 11 present around the cells 10. In addition, under observation with a transmission optical microscope, the operation of placing and freezing the cells 10 and the operation of thawing after freezing can be easily and reliably performed with good visibility.

The refractive index of the material of the porous structure described above can be measured, for example, using an Abbe refractometer (Na light source, wavelength: 589 nm) in accordance with JIS K 0062:1992 and JIS K 7142:2014. Examples of materials that form the porous structure and have a refractive index of 1.45 or less include plastic resin materials such as fluororesins such as polytetrafluoroethylene resin, polyvinylidene difluoride resin, and polychlorotrifluoroethylene resin, and silicone resin, metal oxide materials such as silicon dioxide, and inorganic materials such as sodium fluoride, magnesium fluoride, and calcium fluoride.

The pore diameter of the preservation liquid absorber made of a porous structure is preferably 5.5 μm or less, more preferably 1.0 μm or less, and even more preferably 0.75 μm or less. This can improve the visibility of cells or tissues under optical microscope observation. The thickness of the preservation liquid absorber is preferably 10 to 500 μm, more preferably 25 to 150 μm. In the case of a porous structure made of a plastic resin material, the pore diameter of the preservation liquid absorber is the diameter of the largest pore measured by a bubble point test. In the case of a porous structure made of a metal oxide or inorganic material, the pore diameter is the average pore diameter measured from image observation of the surface and cross section of the porous structure.

The porosity of the storage liquid absorbent is preferably 30% or more, more preferably 70% or more. The porosity is defined by the following formula. Here, the void volume V can be obtained as a value per unit area (m2) by multiplying the cumulative pore volume (ml/g) of the pore radius of 3 nm to 400 nm in the storage liquid absorbent measured and processed using a mercury porosimeter (measuring instrument name Autopore II 9220, manufacturer Micromeritics Instrument Corporation) by the dry solid content (g/m2) of the storage liquid absorbent. The thickness T of the storage liquid absorbent can be obtained by photographing and measuring the cross section of the storage liquid absorbent with an electron microscope.
P = (V/T) x 100 (%)
P: Porosity (%)
V: void volume (ml/m ² )
T: thickness (μm)

In the present invention, the cap member 8 of the cryopreservation jig or the above-mentioned cylindrical storage member can be formed using materials that are resistant to cooling solvents such as liquid nitrogen, such as various resins and metals. The cap member may be made of one type of material, or two or more types of materials. Among them, resin is preferable because it can easily form a tapered structure or a threaded structure by a process such as injection molding. Specific examples of resin include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, acrylic resins, epoxy resins, silicone resins, polycarbonate resins, diacetate resins, triacetate resins, polyacrylate resins, polyvinyl chloride resins, polysulfone resins, polyethersulfone resins, polyimide resins, polyamide resins, polyolefin resins, and cyclic polyolefin resins. In addition, if the total light transmittance of the cap member is 80% or more, it is preferable because the state of the mounting part inside the cap can be easily confirmed after the cap member is attached.

In the present invention, the main body member 7 of the cryopreservation jig preferably has a gripping portion from the viewpoint of workability, and in this case, the gripping portion is preferably prismatic in order to improve ease of gripping and operability. The gripping portion is preferably a member formed from a material that is resistant to cooling solvents such as liquid nitrogen. Suitable examples of such materials include various metals such as aluminum, iron, copper, and stainless steel, ABS resin, acrylic resin, polypropylene resin, polyethylene resin, fluororesin, various engineering plastics, and even glass.

When thawing cells or tissues using the fixation device of the present invention, the preservation liquid may be one that is normally used for freezing cells such as eggs and embryos. For example, a preservation liquid containing a cryoprotectant (glycerol, ethylene glycol, etc.) in a physiological solution such as the above-mentioned phosphate buffered saline, or a preservation liquid containing a large amount (at least 10% by mass or more, more preferably 20% by mass or more, based on the total mass of the preservation liquid) of various cryoprotectants such as glycerol, ethylene glycol, and DMSO (dimethyl sulfoxide) may be used. As for the melting liquid, one that is normally used for melting cells such as eggs and embryos may be used. For example, a melting liquid containing 1 M sucrose in a physiological solution such as the above-mentioned phosphate buffered saline for adjusting the osmotic pressure may be used.

Examples of cells that can be cryopreserved and thawed in the present invention include germ cells such as ova, embryos, and sperm of mammals (e.g., humans, cows, pigs, horses, rabbits, rats, mice, etc.); and pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). Examples of cultured cells include primary cultured cells, subcultured cells, and cell line cells. In one or more embodiments, the cells include fibroblasts, cancer-derived cells such as pancreatic cancer and hepatic cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, and adhesive cells such as immune cells. Examples of tissues that can be cryopreserved and thawed include tissues consisting of homogeneous or heterogeneous cells, such as ovaries, skin, corneal epithelium, periodontal ligament, and cardiac muscle.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

Example 1
A fixture according to Example 1 having one slit portion 2 and a rectangular column-shaped liquid level gauge portion 16 attached to the top surface, as shown in Figures 1 and 2, was produced by metal cutting using aluminum. The size of the base material 20 is 50 mm x 50 mm x 70 mm in length x width x height, the width T2 of the slit portion 2 is 3.2 mm, the length T1 of the slit portion 2 is 30 mm from the side surface of the base material 20, and the depth T3 of the slit portion 2 is 30 mm. The liquid level gauge portion 16 is attached to the top surface (top surface) of the base material 20, and the size of the liquid level gauge portion 16 is 15 mm x 15 mm x 15 mm.

Example 2
A fastener of Example 2 was produced by metal cutting using aluminum, having a convex structure as the uneven structure portion 3 on both side surfaces of the slit portion 2 shown in Fig. 3 over a length T1. The uneven structure portion 3 has a convex structure of 0.25 mm from each of the inner side surfaces of the slit portion 2, and the length in the width direction of the portion narrowed by the convex uneven structure portion 3 is 2.7 mm. The fastener of Example 2 was produced in the same manner as Example 1, except that it has the above-mentioned uneven structure portion.

Example 3
A fixture of Example 3 having a tapered structure in the slit portion 2 shown in Fig. 5 was produced by metal cutting using aluminum. The fixture of Example 3 was produced in the same manner as Example 1, except that the width T21 of the opening 4 of the slit portion 2 was 4 mm, and the width T22 on the closed side was 3 mm.

Example 4
The fixture of Example 4 was produced in the same manner as in Example 3 except that 3D printer processing was performed using polyamide resin.

Example 5
Using polyamide resin, a 3D printer was used to fabricate a fixture of Example 5 having an insertion section 23 for inserting a cryopreservation jig into the upper surface (top surface) of the substrate 20, a slit opening 4 on the side of the insertion section 23, and a liquid level gauge section 16, in the shape shown in the top schematic diagram of FIG. 7. The size of the substrate 20 is 50 mm x 50 mm x 70 mm in length x width x height, the size of the insertion section 23 is 20 mm x 20 mm x 30 mm in length x width x depth, the width T2 of the slit section 2 is 3.2 mm, the length of the slit section 2 is 20 mm from the slit opening 4, and the depth T3 of the slit section 2 is 30 mm. The slit section 2 has a structure having a convex structure as the concave-convex structure section 3 on both sides thereof for a length of 20 mm. The concave-convex structure section 3 of the fixture of Example 5 has a convex structure of 0.25 mm from the side inside the slit section 2, and the width direction length of the part narrowed by the convex concave-convex structure section 3 is 2.7 mm. Moreover, the fixture of Example 5 has a liquid level gauge portion 16 attached to the upper surface (top surface) of the base material 20, and the size of the liquid level gauge portion 16 is 15 mm×15 mm×15 mm.

<Evaluation of fixation of cryopreservation jig>
In order to evaluate the fixation of the cryopreservation jig for the fixing devices of Examples 1 to 5, a cryopreservation jig 6 as shown in FIG. 18 was produced. The cryopreservation jig 6 was produced by forming a strip 9 of a rectangular polyethylene terephthalate resin film (width 1.5 mm, length 20 mm, thickness 190 μm) into a strip 9, which was attached to a gripping part of ABS resin to produce a main body member 7. The cap member 8 was produced from ABS resin. The outer shape of the cap member 8 was a square column of 3.1 mm, and it had a structure having a recess of 0.3 mm deep near the tip. Using the above cryopreservation jig, an equilibrated mouse 8-cell stage embryo was dropped and attached under a transmission microscope together with 0.1 μL of preservation solution. The preservation solution used was a composition containing 15% by volume DMSO, 15% by volume ethylene glycol, and 17% by mass sucrose in Medium 199 medium manufactured by Sigma-Aldrich. Next, under a transmission microscope, the strip including the placement portion was inserted into a cap member, and then immersed in liquid nitrogen to perform a freezing operation. Then, under liquid nitrogen, the cap member side of the cryopreservation jig was fixed to the slit portion 2. The liquid level 14 of the liquid nitrogen 13 was based on the liquid level gauge portion 16 of the fixture of Examples 1 to 5, and the preparation was performed in a state in which the liquid level gauge portion 16 was located slightly below the liquid level of the liquid nitrogen in the form shown in FIG. 18. The cap member side of the cryopreservation jig was inserted into the slit portion 2 of the fixture of Examples 1 to 5, and the state of fixation and retention in the form shown in FIG. 18 was evaluated according to the following criteria. These results are shown in the section "Evaluation of fixation during thawing operation" in Table 1.

○: Stable fixation was achieved.
△: Fixation was achieved, but was somewhat unstable.

<Evaluation of workability during melting operation>
In the same manner as in the above "Evaluation of the Fixation of the Cryopreservation Jig", the cryopreservation jigs were fixed and held using the fixtures of Examples 1 to 5 in the form shown in FIG. 18. Then, as shown in FIG. 20, the workability when separating the main body member and the cap member of the cryopreservation jig was evaluated. It was shown that all of the fixtures of Examples 1 to 5 were capable of rapid thawing operation and had high workability. Furthermore, in the thawing operation using the fixtures of Examples 1 to 5, when the cryopreservation jig was fixed and held in the fixture prior to the thawing operation, it was easy to confirm that the cells or tissues were located in liquid nitrogen, and it was easy to reliably maintain the frozen state of the cells 10. Furthermore, during the thawing operation, the cells 10 could be thawed without coming into contact with the liquid nitrogen 13.

The above results show that by using the fixing device of the present invention, it is possible to stably fix and hold the cryopreservation jig prior to the operation of transferring the cells or tissue to the melting liquid. When the cryopreservation jig is fixed and held using the fixing device of the present invention, it is easy to confirm that the cells or tissue placed on the strip of the cryopreservation jig are reliably located below the liquid nitrogen surface when the cryopreservation jig is fixed and held prior to the operation of transferring the cells or tissue to the melting liquid, and it is also possible to perform a rapid melting operation.

The present invention can be used for embryo transfer and artificial insemination of livestock such as cattle and other animals, artificial insemination of humans, etc., as well as for the freezing and thawing of iPS cells, ES cells, commonly used cultured cells, cells or tissues for testing or transplantation collected from living organisms, including embryos or eggs, and cells or tissues cultured in vitro.

Reference Signs List 1 Fixing tool 2 Slit section 3 Concave-convex structure section 4 Slit opening 5 Hollow structure section 6 Cryopreservation jig 7 Main body member 8 Cap member 9 Strip 10 Cell 11 Preservation liquid 12 Joint section 13 Liquid nitrogen 14 Liquid level 15 Freezing container 16 Liquid level gauge section 17 Upper limit marking section 18 Lower limit marking section 19 Melting liquid 20 Substrate 23 Insertion section

Claims (2)

A fixing device for a cryopreservation device consisting of a substrate having a top surface, sides and a bottom surface, the fixing device having a slit portion on the top surface of the substrate with at least one end opening into the side surface of the substrate in order to fix the cryopreservation device, the slit portion having a fixing structure portion , or having an insertion portion on the top surface of the substrate for inserting the cryopreservation device and a slit portion having one end opening into the side surface of the insertion portion, and further having a liquid level gauge portion on the top surface or side of the substrate that extends above the top surface of the substrate. 2. A fixing device for a cryopreservation jig as described in claim 1, wherein the fixing structure is an uneven structure formed on the inner side of the slit portion, a tapered structure in which the distance between the side surfaces inside the slit portion gradually narrows, or a bent structure in which the slit portion bends in a different direction after traveling straight ahead a certain distance from the opening.