JP7341847B2 - Fixture for cryopreservation jig - Google Patents

Description

The present invention relates to a fixture for a cryopreservation jig that is suitably used when thawing frozen cells or tissues.

Excellent cell or tissue preservation techniques are required in various industrial fields. For example, in cow embryo transplantation technology, embryos are cryopreserved, thawed in accordance with the estrous cycle of the recipient cow, and then implanted. In addition, in human infertility treatment, eggs or ovaries are collected from the mother's body, then frozen and preserved in order to match the timing suitable for transplantation, and used after being thawed at the time of transplantation.

In general, cells or tissues collected from within a living body gradually lose activity or undergo changes in traits, even in culture, so cells or tissues collected from a living body can be stored for a long period of time in vitro. culture is not preferred. Therefore, techniques for long-term preservation while maintaining biological activity are important. Good preservation techniques allow for more accurate analysis of harvested cells or tissues. Furthermore, with excellent preservation techniques, cells or tissues can be used for transplantation while maintaining higher biological activity, and it is hoped that the survival rate after transplantation will improve. Furthermore, it has become possible to sequentially produce and store artificial tissues for transplantation, such as cultured skin cultured outside the body and so-called cell sheets constructed outside the body, and use them when needed. Great benefits can be expected not only in the medical field but also in the industrial field.

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. As the antifreeze agent, compounds such as glycerol, ethylene glycol, and dimethyl sulfoxide are used. After immersing 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, 0.3 to 0.5°C/min) to preserve the inside and outside of the cells or tissues. The inner and outer solutions are sufficiently cooled and become highly viscous. In this state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (-196°C), both the inside of the cells or tissues and the surrounding minute solution solidify in an amorphous state. Vitrification, a phenomenon that occurs when When the inside and outside of a cell or the outside of a tissue is solidified by vitrification, there is virtually no movement of molecules, so it is thought that vitrified cells or tissues can be preserved semi-permanently by storing them in liquid nitrogen.

Furthermore, a vitrification freezing method is also known as a method for cryopreservation of cells or tissues. The vitrification freezing method uses the principle that ice crystals are difficult to form even at subzero temperatures by lowering the freezing point of a preservation solution containing a large amount of antifreeze agents such as glycerol, ethylene glycol, and dimethyl sulfoxide. If this storage solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. This solidification is called vitrification freezing. Furthermore, a preservation solution containing a large amount of antifreeze agent is called a vitrification solution.

In the slow freezing method described above, since it is necessary to cool at a relatively slow cooling rate, the cryopreservation operation takes time. There is also the problem of requiring a device or jig to control the cooling rate. In addition, in the slow freezing method, ice crystals are formed in the preservation solution outside the cells or tissues, so there is a risk that cells or tissues may be physically damaged by the ice crystals. In contrast, in the vitrification freezing method described above, the operation time is short and the process does not require any special equipment or jigs. In addition, the vitrification freezing method provides a high survival rate by not producing ice crystals.

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

The vitrification freezing method is a technology that has been developed mainly using human germ cells, but recently, its application to iPS cells and ES cells has also been widely considered. Furthermore, Non-Patent Document 1 shows that the vitrification freezing method was effective for preserving 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 preserving cells and tissues of a wide variety of species.

It is known that in order to achieve appropriate vitrification freezing, the faster the freezing rate is, the more preferable it is. Furthermore, it is known that even during the thawing process after cryopreservation, the faster the thawing rate is, the more preferable it is from the viewpoint of suppressing the formation of ice crystals again in cells or tissues.

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

In general, as a freezing method related to the vitrification freezing method, Patent Document 4 discloses that mammalian embryos or eggs are wrapped in the inner surface of a cryopreservation container such as a freezing straw, a freezing vial, or a freezing tube. A method has been proposed in which the container is affixed with a minimum amount of vitrification liquid sufficient to achieve this, and the container is brought into contact with liquid nitrogen to rapidly cool the container. The thawing method performed after the freezing method involves removing the cryopreservation container stored in the above-described method from liquid nitrogen, opening one end of the container, and injecting a diluted solution at 33 to 39° C. into the container. This involves thawing and diluting frozen embryos or eggs. According to this method, it is said that mammalian embryos or eggs can be preserved and thawed and diluted with a high survival rate without fear of being infected by viruses or bacteria. However, the process of pasting embryos or eggs onto the inner surface of a cryopreservation container such as a freezing straw, cryovial, or cryotube is difficult, and it is difficult to ensure that the embryo or egg is placed in the cryopreservation container. It was difficult to confirm.

Patent Document 5 describes a cell cryopreservation device having a cell holding member having a thermally conductive member and a cylindrical storage member, and the problems in the freezing and thawing method of Patent Document 4 are solved to some extent. As a method of using the cryopreservation tool described in Patent Document 5, eggs are attached to a cell holding member under a microscope, the cell holding member is stored in a cylindrical storage member, and then immersed in liquid nitrogen to be vitrified. to freeze. After that, a method is described in which a lid member is attached to the opening of the cylindrical member and the cylindrical member is stored in a liquid nitrogen tank. Furthermore, in Patent Document 6, eggs are placed on a strip for retaining egg attachment together with a small amount of vitrification liquid, the entire cryopreservation jig is stored in a cylindrical storage container, and then immersed in liquid nitrogen. freezing is performed.

As a freezing method related to the vitrification freezing method that requires fewer processes, a method called the so-called cryotop (registered trademark) method, which is used in the field of human infertility treatment, as described in Patent Document 7 and Patent Document 8, has been disclosed. has been done. In these methods, the freezing operation is carried out using an egg cryopreservation device equipped with a strip of flexible, colorless and transparent film as a strip for retaining egg attachment. Alternatively, vitrification freezing is performed by placing the embryo and immersing the film to which the egg is attached in liquid nitrogen. The egg attachment retaining strip on which the frozen eggs or embryos are placed is protected with a cap or the like, and then stored in a liquid nitrogen tank.

The eggs, embryos, etc. frozen by these methods are thawed by immersing the egg attachment retaining strip in a warmed melting solution, and the eggs, embryos, etc. placed on the strip are thawed in the melting solution. will be collected.

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

Once the cells or tissues frozen in this way are held below the level of liquid nitrogen, the method described in Patent Document 5 and Patent Document 6, for example, The lid member located above the nitrogen liquid level can be easily removed, and cells or tissues can be quickly moved into the lysis solution, thereby preventing a decrease in the lysis rate. Furthermore, in the methods described in Patent Document 7 and Patent Document 8, by pulling out the cryopreservation jig from the cap, cells or tissues can be quickly moved into the thawing solution, and a decrease in the thawing rate can be prevented. can.

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

Steponkus et al. , Nature 345:170-172 (1990) Hiroshi Sozu, “Mechanism of damage and protection caused by freezing of living cells”, Chemistry and Biology, Vol. 18 (1980), No. 2, P. 78-87 Published by the Japanese Society of Agricultural Chemistry

However, in the method using the liquid nitrogen container described in Patent Document 9 mentioned above, although it is possible to stably hold the cryopreservation jig when it is placed against the slit formed in the lid, The mounting part of the cryopreservation jig is located below the level of liquid nitrogen, making it difficult to ensure that the frozen state of cells or tissues is maintained. In addition, the length of the cylindrical storage member needs to be longer than the length from the bottom of the liquid nitrogen container to the position where the lid is installed. The work of taking out the main body of the member is complicated, and an improvement has been desired from the viewpoint of performing a rapid melting operation.

The main object of the present invention is to provide a fixture for realizing suitable workability during the thawing operation of vitrified cryopreservation of cells or tissues. More specifically, it is possible to stably hold the cryopreservation jig and ensure that the frozen cells or tissues are cooled prior to the operation of transferring them to a thawing solution. It is an object of the present invention to provide a fixing device for a cryopreservation jig, which allows easy confirmation of frozen cells or tissues and allows rapid transfer of frozen cells or tissues into a heated thawing solution.

As a result of intensive studies to solve the above problems, the present inventors have solved the above problems by using a fixing device for a cryopreservation jig (hereinafter also referred to as "fixing device of the present invention") having the following configuration. I found a solution.

A fixing device for a cryopreservation jig consisting of a base material having a top surface, a side surface, and a bottom surface, the base material having a slit portion on the top surface of the base material with at least one end opening in the side surface portion of the base material, and having a slit portion inside the slit portion. It is characterized by having at least an uneven structure part on the side surface for fixing the cryopreservation jig , and the uneven structure part is the uneven structure part of the tip of the cylindrical storage member of the cryopreservation jig. , or a fixture for a cryopreservation jig, which is a structure into which the uneven structure of the tip of the cap member fits .

According to the present invention, it is possible to stably hold the cryopreservation jig, and the frozen cells or tissues are reliably cooled prior to the operation of transferring the cells or tissues to a thawing solution. It is possible to provide a fixing device for a cryopreservation jig that can be easily confirmed to be in place. Further, according to the fixture of the present invention, it becomes possible to rapidly transfer frozen cells or tissues into a warmed thawing solution.

FIG. 2 is a schematic top view showing an example of the fixture of the present invention. FIG. 2 is a schematic side view showing an example of an uneven structure portion of the fixture of the present invention. It is a side schematic diagram which shows another example of the uneven|corrugated structure part which the fixture of this invention has. It is a side schematic diagram which shows another example of the uneven|corrugated structure part which the fixture of this invention has. It is a side schematic diagram which shows another example of the uneven|corrugated structure part which the fixture of this invention has. It is a side schematic diagram which shows another example of the uneven|corrugated structure part which the fixture of this invention has. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 7 is a schematic top view showing another example of the fixture of the present invention. FIG. 3 is a schematic side view showing another example of the fixture 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. It is a schematic diagram which shows another example of the state which fixed the jig for cryopreservation using the fixing tool of this invention. FIG. 2 is a schematic diagram showing a state in which the main body member and cap member of the cryopreservation jig are separated using the fixing device of the present invention, and the mounting portion is immersed in a melting liquid.

The fixing device of the present invention is used for fixing a cell or tissue cryopreservation jig. Furthermore, the fixing device of the present invention is suitably used when cryopreserving cells or tissues in the so-called vitrification cryopreservation method. In the present invention, a cell includes not only a single cell but also a cell population of a living 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 a cell population consisting of multiple types of cells. Furthermore, a tissue may be a tissue composed of a single type of cell or a tissue composed of multiple types of cells, and may include non-cellular substances such as extracellular matrix in addition to cells. Anything is fine. The fixing device of the present invention can be particularly suitably used in the cryopreservation of eggs or embryos.

The fixing device of the present invention includes a fixing device for a cell or tissue cryopreservation jig, a cell or tissue cryopreservation jig fixing device, a cell or tissue cryopreservation jig fixing device, and a cell or tissue cryopreservation jig fixing device. It can also be referred to as a fixture for a cryopreservation device or a fixture for a cell or tissue cryopreservation device.

The fixture of the present invention will be explained in detail below.

The fixing device of the present invention includes a base material having an upper surface (top surface), side surfaces, and a bottom surface, and the upper surface of the base material has a slit section with at least one end opening into the side surface of the base material. That is, the base material has a groove-like structure on its upper surface. From the opening position of the slit on the side surface of the base material, the uneven structure of the cryopreservation jig (for example, the uneven structure of the tip of the cylindrical storage member mentioned above, or the tip of the cap member) The cryopreservation jig is fixed by inserting the concavo-convex structure (having a concavo-convex structure) along the slit.

The slit portion for fixing and holding the cryopreservation jig, which the fixture of the present invention has, may be provided at one location on the upper surface of the base material, or may be provided at multiple locations.

The slit portion of the fixture of the present invention has an uneven structure on the inner side surface of the slit portion for fixing the cryopreservation jig. The uneven structure of the cryopreservation jig described above fits into the uneven structure, so that the cryopreservation jig can be reliably fixed. Preferably, the uneven structure of the slit has a shape in which the recesses and/or the protrusions of the uneven structure extend in a direction parallel to the bottom surface of the fixture on the side surface inside the slit.

The concavo-convex structure of the fixture of the present invention can have a concave structure and/or a convex shape of any shape, depending on the shape of the tip of the cryopreservation jig to be fixed, preferably the shape of the tip of the cap member. It can have a structure. Moreover, it is more preferable that the cross-sectional shape of the uneven structure of the fixture is the same as the cross-sectional shape of the uneven structure of the tip of the cryopreservation jig to be fixed.

The uneven structure portion of the fixture of the present invention may be provided at at least one location on the side surface inside the slit portion with at least one end open. From the viewpoint of increasing the fixation stability of the cryopreservation jig, it is more preferable that the slit has an uneven structure on both sides of the opposing sides. Here, both sides of the side faces facing each other mean both sides of the side faces facing each other in a schematic top view when the fixture of the present invention is viewed from above. Note that the slit portions located on both sides of these side surfaces may be connected continuously.

The shape of the fixture of the present invention (the approximate shape assuming that the base material is not provided with a slit) includes prismatic structures such as triangular prisms, square prisms, and hexagonal prisms, as well as triangular pyramids with upper surfaces, and Examples include partial structures of pyramids such as pyramids. Preferably, it has a prismatic structure, more preferably a quadrangular prismatic structure. Moreover, in addition to the above-mentioned structure, a cylindrical structure and a conical structure can also be exemplified.

In the present invention, the substrate is preferably formed of a liquid nitrogen resistant material. Examples of such materials include various metals such as aluminum, iron, copper, and stainless steel alloys, ABS resin, acrylic resin, polypropylene resin, polystyrene resin, polyethylene resin, fluorine resin, and various engineering plastics, as well as glass and rubber materials. etc. can be suitably used. In particular, metal is preferable from the viewpoint of not only having excellent durability but also having a large self-weight and being able to securely fix the cryopreservation jig. Among them, aluminum is preferred from the viewpoint of workability. The fixture of the present invention may be made of one type of material or may be made of a plurality of materials.

Next, a freeze-thaw method using the fixture of the present invention will be explained in detail.

The fixing device of the present invention is suitable for a series of processes from cryopreservation to thawing of cells or tissues, particularly for cold storage operations once removed from a liquid nitrogen tank and for thawing operations when thawing cells or tissues. Can be used. Generally, when cells or tissues are preserved by the so-called vitrification freezing method, cryopreservation is performed using a cryopreservation jig having a strip-shaped strip. Such a cryopreservation jig is disclosed in, for example, International Publication No. 2011/070973 pamphlet, International Publication No. 2015/064380 pamphlet, etc. in addition to the above-mentioned Patent Document 7 and Patent Document 8. As a series of processes from cryopreservation to thawing, first, equilibrated cells or tissues are dropped onto a strip of a cryopreservation jig together with a preservation solution, and then liquid nitrogen is used to freeze the cells or tissues. Cool and perform freezing operation. Second, a cold storage operation is performed to preserve the frozen cells or tissues for a long period of time while maintaining an extremely low temperature environment. Third, frozen cells or tissues are transferred from an extremely low temperature environment into a solution called a thawing solution, and a thawing operation is performed in which they are thawed and thawed in the thawing solution. The fixing device of the present invention can be suitably used for fixing and holding a cryopreservation jig in liquid nitrogen in the above-described cold storage operation and thawing operation.

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

In the closed-type cryopreservation-thaw process, cells or tissues are dripped onto the strip along with a preservation solution during the freezing operation, and then a cylindrical storage member with a completely closed end is used for cryopreservation. Encapsulate the jig and isolate it from the outside world. Alternatively, after cells or tissues are dripped onto the strip together with a preservation solution, the strip is covered with a cap member and isolated from the outside world. After such blocking, 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 through 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 fixture of the present invention, prior to the operation of transferring frozen cells or tissues to a thawing solution, the tip of the cylindrical storage member described above or The cryopreservation jig is fixed by fitting the uneven structure of the tip of the cap member into the uneven structure of the inner side surface of the slit of the fixture of the present invention. By fixing in this way, it is possible to fix the cells or tissues in a position below the liquid level of the cooling medium, and the cells or tissues do not come into contact with liquid nitrogen and are contaminated via the liquid nitrogen. Never.

Then, by removing the lid fixed to the cylindrical storage member located above the liquid nitrogen level, the cryopreservation jig containing the cells or tissues can be quickly taken out without touching the liquid nitrogen. , can be immersed in a melting liquid. Alternatively, by pulling out a cryopreservation jig having a strip on which cells or tissues are placed from the cap member fixed by the uneven structure, cells or tissues can be quickly removed without touching liquid nitrogen, and the thawed liquid can be removed. Can be immersed in. The latter method is preferred from the viewpoint of enabling faster operation.

The cells or tissues immersed in the molten liquid by the above operation are preferably immersed in the molten liquid within 5 minutes, more preferably within 1 minute, after the lid member is removed from the cylindrical storage member. . In addition, when pulling out the cryopreservation jig having the strip on which cells or tissues are placed from the cap member, it is preferable to immerse it in the thawing solution within 5 minutes after the fitting is loosened, and more preferably 1 Within minutes. If the so-called sealed state has been 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 pulling out the cryopreservation jig, air Gases such as nitrogen and oxygen may enter the cylindrical storage member or the cap member, and then be cooled and liquefied. If the above-mentioned time is long, the liquefied gas may adhere to the mounting portion and be carried into the melt during the melting operation, thereby lowering the temperature of the melt.

The fixation device of the present invention can also be used in a so-called open-type cryopreservation/thawing process in which cells or tissues come into contact with liquid nitrogen in a series of processes from cryopreservation to thawing of cells or tissues. Even in an open process, if the cryopreservation jig is fixed and held using the fixing device of the present invention prior to transferring frozen cells or tissues to a thawing solution, cells or tissues can be This is preferred because it can be quickly transferred from liquid nitrogen to a molten liquid.

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

FIG. 1 is a schematic top view showing an example of the fixture of the present invention. In FIG. 1, the fixture 1 has one slit portion 2, which is a groove-like structure, into which a cryopreservation jig is inserted on the upper surface of a base material 20. One side of the slit portion 2 is open as a slit opening 4 on the side surface of the base material 20, and the other side is closed on the center side of the upper surface. The slit part 2 has an uneven structure part 3 on its inner side surface in order to fix the cryopreservation jig. Note that FIG. 1 shows a convex concavo-convex structure portion 3. As shown in FIG. When inserting a cryopreservation jig (not shown) into the slit 2, the cryopreservation jig is inserted from the slit opening 4 side along the slit 2 toward the center of the upper surface of the base material.

T1 shown in FIG. 1 indicates the length (depth) of the slit portion 2 of the fixture 1. T1 is not particularly limited as long as it can hold the cryopreservation jig after insertion, but when freezing and thawing cells or tissues by the cryotop method described above, T1 is preferably 3 to 50 mm; More preferably, it is 5 to 40 mm. In addition, each dimension of the fixture 1 described in the following description is a dimension intended for freezing and thawing operations by the cryotop method.

T2 shown in FIG. 1 indicates the length of the slit portion 2 of the fixture 1 in the width direction. In the present invention, T2 is not particularly limited as long as it is long enough to insert, fix and hold a cryopreservation jig, but is preferably 1 to 10 mm, more preferably 2 to 5 mm.

T4 shown in FIG. 1 indicates the length in the width direction of the portion of the slit portion 2 of the fixture 1 narrowed by the uneven structure portion 3. In the present invention, T4 is not particularly limited as long as it has a length that can fix and hold a cryopreservation jig (not shown), but is preferably 0.5 to 5 mm, more preferably 1.5 to 4 mm.

FIG. 2 is a schematic side view showing an example of an uneven structure portion of the fixture of the present invention (the fixture of FIG. 1). In FIG. 2, the fixture 1 has a slit part 2 on the upper surface (top surface) of the base material 20 into which the cryopreservation jig is inserted. It has an uneven structure 3 on the inner side surface. The concavo-convex structure portion 3 of the fixture shown in FIG. 2 has a convex structure. Similarly to FIG. 1, the length in the width direction of the slit portion 2 is shown as T2, and the length in the width direction of the portion narrowed by the convex uneven structure 3 of the slit portion 2 is shown as T4.

T3 shown in FIG. 2 indicates the length of the slit portion 2 of the fixture 1 in the depth direction. In the present invention, T3 is not particularly limited as long as it can fix and hold a cryopreservation jig (not shown), but is preferably 3 to 50 mm, more preferably 5 to 20 mm.

FIG. 3 is a schematic side view showing another example of the uneven structure portion of the fixture of the present invention. In FIG. 3, the slit part 2 of the fixture 1 has an uneven structure part 3 on its inner side surface in order to fix the cryopreservation jig. The concavo-convex structure 3 of the fixture shown in FIG. 3 is concave.

T5 shown in FIG. 3 indicates the length in the width direction expanded by the concave and convex structure portion 3 of the slit portion 2 of the fixture 1. T5 is not particularly limited as long as it is long enough to fix and hold a cryopreservation jig (not shown), but in the present invention, T5 is preferably 2 to 11 mm, more preferably 2.5 to 5.5 mm. be. As shown in FIG. 3, when the concave and concave structure 3 is provided, the length T5 in the width direction expanded by the concave and convex structure 3 is larger than the length T2 of the slit portion 2 in the width direction.

FIG. 4 is a schematic side view showing another example of the uneven structure portion of the fixture of the present invention. In FIG. 4, the slit portion 2 of the fixture 1 has a plurality of convex structures as uneven structure portions 3 on the inner side surface of the slit portion 2 in order to fix the cryopreservation jig. By using a fixture having such a structure, the cryopreservation jig can be fixed and held more reliably. The length T4 in the width direction of the portion narrowed by the convex concave-convex structure 3 of the slit portion 2 shown in FIG. It's okay.

FIG. 5 is a schematic side view showing another example of the uneven structure portion of the fixture of the present invention. In FIG. 5, the slit portion 2 of the fixture 1 has an uneven structure portion 3a having a convex structure and an uneven structure portion 3b having a concave structure on its inner side surface in order to fix the cryopreservation jig.

FIG. 6 is a schematic side view showing another example of the uneven structure part of the fixture of the present invention. In FIG. 6, the slit portion 2 of the fixture 1 has one uneven structure portion 3 having a convex structure only on one side of the inside of the slit portion 2 in order to fix the cryopreservation jig. When a fixture having such a shape is used, it is easy to insert the cryopreservation jig along the slit portion 2 of the fixture. In addition, in FIGS. 2 to 6, only the shape of the side surface of the base material is shown, and the closed surface etc. that are visible inside the slit are omitted.

FIG. 7 is a schematic top view showing another example of the fixture of the present invention. In FIG. 7, the fixture 1 has two slit portions 2 on the upper surface of a base material 20, each having a slit opening 4 on a different side surface. Each slit portion 2 has an uneven structure portion 3 for fixing a cryopreservation jig. In the fixture of FIG. 7, a plurality of cryopreservation jigs (not shown) are inserted along the slit part 2 through the slit opening 4 of each slit part 2, and the plurality of cryopreservation jigs are fixed and held. be able to.

FIG. 8 is a schematic top view showing another example of the fixture of the present invention. In FIG. 8, the fixture 1 has two independent slit portions 2 on the upper surface of a base material 20, each having a slit opening 4 on the same side surface. Each slit section 2 has an uneven structure section 3 for fixing a cryopreservation jig. A cryopreservation jig (not shown) is inserted along the slit part 2 from the slit opening 4 of each slit part 2, and a plurality of cryopreservation jig can be fixed and held. In this embodiment, since the plurality of slit parts 2 are on the same surface of the base material 20, good workability can be obtained, which is preferable.

FIG. 9 is a schematic top view showing another example of the fixture of the present invention. In FIG. 9 , the fixture 1 has a plurality of independent slits 2 on the upper surface of a base material 20 . Each of the slit parts 2 has an uneven structure part 3 for fixing a cryopreservation jig. According to this embodiment, since more slits 2 are provided on the upper surface of the base material 20, a large number of cryopreservation jigs can be fixed and held at once.

FIG. 10 is a schematic top view showing another example of the fixture of the present invention. In FIG. 10, the fixture 1 has one slit part 2 on the upper surface of the base material 20 into which the cryopreservation jig is inserted, and the slit part 2 has an uneven surface for fixing the cryopreservation jig. It has a structure part 3. In FIG. 10, the uneven structure 3 has the uneven structure 3 not only on both sides of the side faces facing each other inside the slit 2 but also on the inner surface of the slit 2. For this reason, since the cryopreservation jig (not shown) is held from three directions, it is possible to more firmly fix and hold the cryopreservation jig, which is preferable.

FIG. 11 is a schematic top view showing another example of the fixture of the present invention. In FIG. 11, the fixture 1 has a slit portion 2 bent at 90 degrees in the middle. When using a fixture shaped like this, the cryopreservation jig is inserted into the slit part 2 through the slit opening 4, and the cryopreservation jig is pushed in until it bends 90 degrees, allowing the frozen material to be fixed and held. This is preferable because it can prevent the storage jig from accidentally falling off from the slit opening 4.

FIG. 12 is a schematic top view showing another example of the fixture of the present invention. In FIG. 12, the slit opening 4 of the fixing device 1 is wider than the width of the slit portion 2, so that the workability when inserting the cryopreservation jig is excellent.

FIG. 13 is a schematic top view showing another example of the fixture of the present invention. In FIG. 13, the length T2 of the slit portion 2 of the fixture 1 in the width direction gradually decreases as it advances from the slit opening 4 in the insertion direction. On the other hand, the width T4 narrowed by the convex concavo-convex structures 3 formed on both inner side surfaces of the slit section 2 is constant. When the fixture 1 shown in FIG. 13 is used, the cryopreservation jig inserted along the uneven structure 3 is not only affected by the uneven structure 3 but also by the wall surface of the slit 2 whose width becomes narrower as it is inserted deeper. Since it is fixed, stronger fixation and retention is possible.

FIG. 14 is a schematic top view showing another example of the fixture of the present invention. In FIG. 14, the length T2 in the width direction of the slit portion 2 of the fixture 1 is designed to gradually attenuate as it advances from the slit opening 4 in the insertion direction. Further, similarly to T2, the width T4, which is narrowed by the convex concave-convex structure 3 formed on both sides of the inside of the slit 2 of the fixture 1 shown in FIG. , the structure gradually narrows. When the fixture 1 shown in FIG. 14 is used, it becomes possible to more firmly fix and hold the cryopreservation jig inserted along the uneven structure 3.

In FIG. 15, similarly to FIG. 10 shown earlier, a cryopreservation jig (not shown) can be held from three directions on the inner side surface of the slit portion 2, but the uneven structure portion 3 is held on the side surfaces facing each other. In addition to both sides, the inner surface of the slit 2 is connected by the semicircular concave-convex structure 3, so it is particularly strong when the fixed part of the cryopreservation jig (not shown) has a circular cross-sectional shape. This is preferable because it can be fixed and held.

FIG. 16 is a schematic top view showing another example of the fixture of the present invention. In FIG. 16, the fixture 1 has two slit openings 4 for one slit 2 having an uneven structure 3. With the shape shown in FIG. 16, a cryopreservation jig (not shown) can be inserted from either end of the slit portion 2, so that a fixing device with excellent workability can be obtained.

FIG. 17 is a schematic side view showing another example of the fixture of the present invention. In FIG. 17, the fixture 1 has an uneven structure 3 having a convex structure on the inner side surface of the slit portion 2, and also has a hollow structure 5. It is preferable that the fixture 1 has the hollow structure 5 because the thermal capacity of the fixture 1 is suppressed and the amount of volatilization of liquid nitrogen can be suppressed when the fixture 1 is immersed in liquid nitrogen.

Next, a method for freezing and thawing cells or tissues using the fixing device of the present invention will be described.

In the present invention, cells or tissues are placed together with a preservation solution on the mounting section of a cryopreservation jig, which has at least a main body member having a mounting section and a cap member that is detachably attached to the main body member, and After sealing with a cap member, the sealed mounting part is cooled with a cooling solvent to maintain the frozen state of the frozen cells or tissues, and then, when the cells or tissues are thawed, the sealed mounting part is sealed with the cap member. The cells or tissues that have been treated are placed below the liquid level of the cooling solvent, and once held in a state where the joint between the main body member and the cap member is located above the liquid level of the cooling solvent, the main body member This is preferable because the cap member and the cap member can be quickly separated and the mounting portion can be quickly immersed in the melt.

In addition, instead of the cryopreservation jig having at least a main body member having a mounting portion and a cap member removably attached to the main body member described above, a cell holding member to which eggs, embryos, etc. can be attached is provided, and the cell holding member is provided. Using a cryopreservation jig in which a holding member is housed in a cylindrical storage member, eggs, embryos, etc. are positioned below the liquid level of the cooling solvent, and one end of the cylindrical storage member is placed below the liquid level of the cooling solvent. Once the main body member is held in a state where it is located above the liquid level, by removing the lid member attached to the cylindrical storage member located above the liquid level of the cooling solvent, the mounting portion of the main body member can be placed in the melted liquid. This is preferable because it can be quickly immersed in water.

FIG. 18 is a schematic diagram showing an example of the former state in which the cryopreservation jig is fixed using the fixture of the present invention. In FIG. 18, the cryopreservation jig 6 has a main body member 7 and a strip 9 of the cryopreservation jig 6, which has at least a cap member 8 that is detachably attached to the main body member 7. It is placed.

The strip 9 on which the cells 10 are placed together with a very small amount of preservation solution 11 is sealed with a cap member 8, and the cells 10 placed on the strip 9 are located below the liquid level 14 of the liquid nitrogen 13. As a result, the frozen cells 10 are kept in a frozen state. When thawing frozen cells 10, since the joint 12 between the cap member 8 and the main body member 7 is located above the liquid level 14 of the liquid nitrogen 13, the main body member 7 can be quickly pulled out from the cap member 8. , which is preferable because it can be separated and the mounting part can be quickly immersed in the molten liquid. Further, by using the main body member 6 of the present invention, the operator can easily grasp the position of the liquid level 14, improving work efficiency.

FIG. 19 is a schematic diagram showing another example of a cryopreservation jig fixed using the fixing device of the present invention. In FIG. 19, the fixture 1 has a liquid level gauge 16, and the gauge 16 has an upper limit marking part 17 and a lower limit marking part 18. A liquid level 14 of liquid nitrogen 13 is located between the upper limit marking section 17 and the lower limit marking section 18 . It is preferable to control the liquid level 14 using the liquid level gauge 16 of the fixture 1 in this manner, since it is possible to prevent frozen cells or tissues from unexpectedly thawing.

Next, a cryopreservation jig fixed using the fixing device of the present invention will be explained.

It is preferable that the strip 9 of the main body member 7 has a rectangular shape. A rectangular shape is preferable because it is easy to store the strip 9 in a cap member or a cylindrical storage member.

In the present invention, the strip 9 included in the cryopreservation jig includes, for example, various resin films, metal plates, glass plates, rubber plates, and the like. 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 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, and polysulfone resins. , polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, cyclic polyolefin resin, and the like. Further, it is preferable that the total light transmittance of the strip 9 is 80% or more because the cells or tissues placed on the placement part can be easily confirmed using a transmission microscope.

Further, as the strip 9, a metal plate can also be suitably used from the viewpoint of having excellent thermal conductivity and enabling rapid freezing. Specific examples of the metal plate include copper, copper alloy, aluminum, aluminum alloy, gold, gold alloy, silver, silver alloy, iron, stainless steel, and the like. The thickness of the various resin films, metal plates, glass plates, rubber plates, etc. described above is preferably 10 μm to 10 mm. Furthermore, depending on the purpose, the surfaces of 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. It is also possible to roughen the surface.

Further, as the strip 9 described above, a storage liquid absorber can also be used. When a storage liquid absorber is used in the strip 9, excess storage liquid 11 can be effectively removed, thereby improving the freezing speed. As the storage liquid absorber, for example, a film-like material made of wire mesh, paper, or synthetic resin and having through holes is exemplified. Another example of the storage liquid absorber is a porous structure formed using a material having a refractive index of 1.45 or less. The porous structure allows the storage solution 11 present around the cells 10 to be efficiently removed. Further, under observation using a transmission type optical microscope, the operation of placing and freezing the cells 10 and the operation of thawing the cells 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) according to JIS K 0062:1992 and JIS K 7142:2014. Materials with a refractive index of 1.45 or less that form the porous structure include fluororesins such as polytetrafluoroethylene resin, polyvinylidene difluoride resin, and polychlorotrifluoroethylene resin, and plastic resins such as silicone resin. materials, metal oxide materials such as silicon dioxide, inorganic materials such as sodium fluoride, magnesium fluoride, calcium fluoride.

The pore diameter of the storage liquid absorber made of a porous structure is preferably 5.5 μm or less, more preferably 1.0 μm or less, and still more preferably 0.75 μm or less. This can improve the visibility of cells or tissues under optical microscopic observation. The thickness of the storage liquid absorber is preferably 10 to 500 μm, more preferably 25 to 150 μm. Note that, in the case of a porous structure made of a plastic resin material, the pore diameter of the storage 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 an inorganic material, it is the average pore diameter measured from image observation of the surface and cross section of the porous structure.

The storage liquid absorber preferably has a porosity of 30% or more, more preferably 70% or more. The porosity is defined by the following formula. Here, the pore volume V is the cumulative pore volume (ml/g) with a pore radius of 3 nm to 400 nm in the storage liquid absorber, which is measured and processed using a mercury porosimeter (meter name: Autopore II 9220, manufacturer: micromeritics instrument corporation). ) by the dry solid content (g/square meter) of the storage liquid absorber, it can be determined as a value per unit area (square meter). Further, the thickness T of the storage liquid absorber can be obtained by photographing a cross section of the storage liquid absorber with an electron microscope and measuring its length.
P=(V/T)×100(%)
P: Porosity (%)
V: void volume (ml/m ² )
T: Thickness (μm)

In the present invention, the cap member 8 or the above-mentioned cylindrical storage member included in the cryopreservation jig 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 may be made of 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 resins 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, Examples include resins such as polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, and cyclic polyolefin resin. Further, it is preferable that the total light transmittance of the cap member is 80% or more because it is possible to easily check the state of the mounting portion inside the cap after the cap members are assembled.

In the present invention, it is preferable that the main body member 7 of the cryopreservation jig has a gripping part from the viewpoint of workability. It is preferably columnar. The gripping portion is preferably a member made of a material that is resistant to cooling solvents such as liquid nitrogen. As such materials, for example, 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 can be suitably used. .

When cells or tissues are thawed using the fixation device of the present invention, the preservation solution that is normally used for freezing cells such as eggs and embryos can be used, such as the above-mentioned phosphate buffered saline. Preservation solutions containing cryoprotectants (glycerol, ethylene glycol, etc.) in physiological solutions such as (10% by mass or more, more preferably 20% by mass or more) can be used. As for the thawing solution, those normally used for thawing cells such as eggs and embryos can be used. For example, 1M solution is added to the above-mentioned phosphate buffered saline to adjust the osmotic pressure. A melt containing sucrose can be used.

Cells to be cryopreserved and thawed in the present invention include, for example, reproductive cells such as eggs, embryos, and sperm of mammals (e.g., humans, cows, pigs, horses, rabbits, rats, mice, etc.); Examples include pluripotent stem cells such as potent stem cells (iPS cells) and embryonic stem cells (ES cells). Also included are cultured cells such as primary cultured cells, subcultured cells, and cell line cells. In one or more embodiments, the cells include fibroblasts, cancer-derived cells such as pancreatic cancer and liver cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, chondrocytes, tissue stem cells, and adherent cells such as immune cells. Further, examples of tissues that can be cryopreserved and thawed include tissues made of cells of the same or different types, such as ovary, skin, corneal epithelium, periodontal ligament, and cardiac muscle.

EXAMPLES The present invention will be illustrated in more detail with reference to examples below, and the present invention will be specifically explained, but the present invention is not limited to the following examples.

(Example 1)
A fixture of Example 1 having a convex concavo-convex structure 3 in one slit 2 as shown in FIGS. 1 and 2 was produced by metal cutting using aluminum. Note that the width T2 of the slit portion is 3.2 mm, the length T1 of the slit portion 2 is 10 mm from the side surface of the base material 20, and the depth T3 of the slit portion is 10 mm. The concavo-convex structure portions each have a convex structure extending 0.25 mm from the inner side surface of the slit portion, and the length T4 in the width direction of the portion narrowed by the convex-convex concave-convex structure portion 3 is 2.7 mm.

(Example 2)
As shown in FIG. 13, one slit part 2 has a convex structure as an uneven structure part 3 by metal cutting using aluminum, and the width T2 of the slit part 2 increases as it advances from the slit opening in the insertion direction. A fixture of Example 2 having a gradually narrowed structure was produced. Note that the length T1 of the slit portion 2 is 10 mm from the side surface of the base material 20, and the depth T3 of the slit portion 2 is 10 mm. The width T2 of the slit portion was 4 mm at the slit opening 4, and the structure was such that the width T2 steadily decreases from the slit opening 4 toward the insertion direction to 3 mm at the closed portion of the slit portion 2 on the center side of the upper surface of the base material. The width T4 of the uneven structure was kept constant at 2.7 mm.

(Example 3)
A fixture of Example 3 having a convex concavo-convex structure only on one side of the inside of the slit, as shown in FIG. 6, was fabricated by metal cutting using aluminum. Note that the width T2 of the slit portion is 3.2 mm, the length T1 of the slit portion is 10 mm from the side surface of the base material 20, and the depth T3 of the slit portion is 10 mm. The concavo-convex structure is a convex structure extending 0.25 mm from the inner side surface of the slit, and the length T6 of the portion narrowed by the convex-convex concavo-convex structure 3 at one location is 2.95 mm.

(Example 4)
A fixture of Example 4 was produced in the same manner as Example 1 except that ABS resin was used and the processing was performed using a laminated 3D printer.

(Example 5)
A fixing device of Example 5 having a convex concavo-convex structure 3 on the inner side surface of the slit portion in the form shown in FIG. 11 was manufactured by processing with a laminated 3D printer using ABS resin. Note that the width T2 of the slit portion 2 is 3.2 mm, and the depth T3 of the slit portion is 10 mm. The length of the slit portion 2 was such that it went straight 20 mm from the insertion direction, turned 90 degrees to the right, and went straight 10 mm. The uneven structure portion 3 has a convex structure of 0.25 mm from the inner side surface of the slit portion 2, and the width T4 of the uneven structure portion is 2.7 mm.

<Evaluation of fixation of cryopreservation jig>
Regarding the fixtures of Examples 1 to 5, in order to evaluate the fixation of the cryopreservation jig, a cryopreservation jig as shown in FIG. 18 was prepared. For the cryopreservation jig, a strip 9 of a polyethylene terephthalate resin film in the form of a rectangle (width 1.5 mm, length 20 mm, thickness 190 μm) was bonded to a gripping portion of ABS resin to produce a main body member 7. The cap member 8 was made of ABS resin. The outer shape of the cap member 8 is a 3.1 mm rectangular prism, and has a structure having a recessed portion with a depth of 0.3 mm near the tip. Using the cryopreservation jig described above, an equilibrated mouse 8-cell stage embryo was dropped and attached together with 0.1 μL of preservation solution under a transmission microscope. The storage solution used was Medium 199 medium manufactured by Sigma-Aldrich, which had a composition containing 15% by volume of DMSO, 15% by volume of ethylene glycol, and 17% by mass of sucrose. Next, under a transmission microscope, the strip including the mounting part was inserted into the cap member, and then immersed in liquid nitrogen to perform a freezing operation. After the cold storage operation, the cap member side of the cryopreservation jig was prepared in Example 1 under liquid nitrogen in a state where the recessed portion of the tip of the cap member was fitted into the uneven structure of the cryopreservation jig. It was inserted into the slit part of the fixing tool No. 5 to 5, and the state when it was fixed and held in the form shown in FIG. 18 was evaluated based on the following criteria. These results are shown in Table 1.

◎: Firmly fixed and had a sense of stability.
○: Stable fixation was achieved.
△: Fixation was achieved, but it was somewhat unstable.

<Evaluation of workability during melting operation>
The cryopreservation jigs were each fixed and held using the fixing devices of Examples 1 to 5 in the form shown in FIG. 18 by the same method as in the above-mentioned "Evaluation of fixation of cryopreservation jigs." Thereafter, the workability of separating the main body member and cap member of the cryopreservation jig as shown in FIG. It was shown that it is possible and has high workability. Furthermore, in the thawing operation using the fixtures of Examples 1 to 5, it was easily confirmed that the cells or tissues were located in liquid nitrogen.

From the above results, when the fixing device of the present invention is used, it is possible to stably fix and hold the cryopreservation jig prior to the operation of transferring cells or tissues to a thawing solution. When a cryopreservation jig is fixed and held using the fixing device of the present invention, the cryopreservation jig can be fixed and held prior to the operation of transferring cells or tissues to a thawing solution. It can be seen that in addition to being able to easily confirm that the cells or tissues placed on the strip of the device are reliably located below the liquid nitrogen level, it is possible to perform a rapid thawing operation.

The present invention is applicable 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 livestock such as cattle and animals, artificial insemination of humans, etc. It can be used for cryopreservation and thawing of cells or tissues for use or transplantation, cells or tissues cultured in vitro, and the like.

1 Fixing device 2 Slit portion 3 Uneven structure portion 4 Slit opening portion 5 Cavity structure portion 6 Cryopreservation jig 7 Main body member 8 Cap member 9 Strip 10 Cell 11 Preservation solution 12 Joint portion 13 Liquid nitrogen 14 Liquid level 15 Freezing container 16 Liquid level gauge 17 Upper limit marking section 18 Lower limit marking section 19 Melt liquid 20 Base material

Claims (1)

A fixing device for a cryopreservation jig consisting of a base material having a top surface, a side surface, and a bottom surface, the base material having a slit portion on the top surface of the base material with at least one end opening in the side surface portion of the base material, and having a slit portion inside the slit portion. It is characterized by having at least an uneven structure part on the side surface for fixing the cryopreservation jig , and the uneven structure part is the uneven structure part of the tip of the cylindrical storage member of the cryopreservation jig. , or a fixture for a cryopreservation jig, which is a structure into which the uneven structure of the tip of the cap member fits .