Cryopreservation jig mounting device

Abstract

To provide a mounting tool for a cryopreservation jig that has excellent operability when mounting the cryopreservation jig and that has excellent holding stability of the cryopreservation jig.SOLUTION: A mounting tool 1 for a cryopreservation jig has a base material 2 having a top surface, a side surface, and a bottom surface, and a slit portion 3 provided in the top surface, including a curved structure, one end of which is open to the side surface of the base material 2, the position of the bottom surface of a mounting portion 6 located to the far side of the curved structure when seen from a slit opening portion 4 of the slit portion 3 is lower than the position of the bottom surface of an insertion portion 5 located to the near side of the curved structure.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a mounting device for a cryopreservation jig that is preferably used when thawing frozen cells or tissues. [Background technology]

[0002] There is a demand for superior cell or tissue preservation technology in various industrial fields. For example, in the case of bovine embryo transfer technology, embryos are frozen and then thawed and transferred in accordance with the estrous 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.

[0003] In general, cells or tissues collected from a living body gradually lose activity or undergo changes in phenotype even in a culture solution, so that long-term culture of cells or tissues outside the body is not preferable. Therefore, technology for long-term preservation while maintaining bioactivity is important. Excellent preservation technology enables more accurate analysis of collected cells or tissues. In addition, it is possible to use cells or tissues for transplantation while maintaining higher bioactivity, and it is expected that the engraftment rate after transplantation will be improved. Furthermore, it is possible to sequentially produce and preserve 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 necessary, which is expected to be of great merit not only in medical terms but also in industrial terms.

[0004] As a method for cryopreserving cells or tissues, for example, the slow freezing method is known. 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 cryoprotectant, compounds such as glycerol, ethylene glycol, and dimethyl sulfoxide are used. After immersing the cells or tissues in the preservation solution, the solution inside and outside the cells or inside and outside the tissues is cooled sufficiently and the viscosity increases by cooling the solution to -30 to -35°C at a relatively slow cooling rate (for example, a rate of 0.3 to 0.5°C / min). 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 inside of the cells or tissues and the microsolutions around them both solidify while remaining amorphous. When the inside and outside of the cells or tissues solidify due to 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.

[0005] Vitrification is also known as a method for cryopreserving cells or tissues. Vitrification uses the principle that ice crystals are unlikely to form even below freezing due to the freezing point depression of a preservation solution containing a large amount of cryoprotectants such as glycerol, ethylene glycol, and dimethyl sulfoxide. If this preservation solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. This solidification is called vitrification. A preservation solution containing a large amount of cryoprotectants is also called a vitrification solution.

[0006] In the slow freezing method described above, since it is necessary to cool at a relatively slow cooling rate, the operation for cryopreservation takes time. In addition, there is a problem that a device or tool is required 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 that the cells or tissues may be physically damaged by the ice crystals. In contrast, in the vitrification freezing method, the operation is completed in a short time, and the process does not require a special device or tool. In addition, the vitrification freezing method can obtain a high survival rate by not generating ice crystals.

[0007] It is known that the faster the freezing rate, the more preferable it is to achieve proper vitrification freezing. Furthermore, it is known that the faster the thawing rate, the more preferable it is to suppress re-ice crystal formation in cells or tissues during the thawing process after cryopreservation. In addition, 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 1, it is known that even if a cell is rapidly frozen, the survival rate is low if the thawing rate is slow. In addition, Patent Document 1 describes that the survival rate of human iPS cell-derived neural stem cells / progenitor cells after thawing is improved by thawing a frozen sample by a rapid thawing method.

[0008] Generally, as a freezing method related to the vitrification freezing method, Patent Document 2 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. The thawing method performed after the freezing method involves removing the cryopreservation container stored by the above-mentioned method from liquid nitrogen, opening one end of the container, and injecting a diluent at 33 to 39°C into the container to thaw and dilute the frozen embryo or egg.

[0009] Patent Document 3 describes a cell cryopreservation tool having a cell retention member with a thermally conductive member and a cylindrical storage member, and the method of using the cryopreservation tool described in the document includes attaching eggs to the cell retention member under microscopic observation, storing the cell retention member in a cylindrical storage member, and then immersing the member in liquid nitrogen for vitrification and freezing. The document also describes a method of attaching a lid member to the opening of the cylindrical storage member and storing the member in a liquid nitrogen tank. Patent Document 3 also describes placing eggs on an egg attachment / retention strip together with a small amount of vitrification liquid, storing the entire cryopreservation tool in a cylindrical storage container, and then immersing the container in liquid nitrogen for vitrification and freezing.

[0010] 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 Document 4 and Patent Document 5, is known. In the freezing operation in these methods, an egg freezing storage tool equipped with a rectangular flexible and colorless transparent film is used as an egg attachment and retention strip, and eggs or embryos are placed on the film together with a very small amount of preservation solution under microscopic observation, and the film with the eggs attached is immersed in liquid nitrogen to perform vitrification freezing. The egg attachment and retention 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.

[0011] Furthermore, as a cryopreservation device suitable for the Cryotop method, for example, Patent Documents 6 to 8 propose a method for cryopreserving these reproductive cells with an excellent survival rate using a cryopreservation device having an absorbent strip that removes excess preservation solution adhering to the periphery of the egg or embryo.

[0012] The oocytes, embryos, etc. frozen by these methods are thawed by immersing the strip to which the oocytes, embryos, etc. are attached in a warmed thawing liquid, and the oocytes, embryos, etc. placed on the strip in the thawing liquid are then recovered.

[0013] On the other hand, in order to improve the workability of the thawing operation in the cryopreservation method, Patent Document 9 describes a freeze-thawing method in which cells or tissues are placed on a mounting part of a cryopreservation jig having at least a main body member of the cryopreservation jig and a cap member detachable from the main body member, together with a preservation liquid, and the mounting part is cooled with a cooling solvent while being sealed with the cap member, and before the start of the subsequent thawing process, the cells or tissues sealed with the cap member are located below the liquid level of the cooling solvent, and the joint part of the main body member and the cap member is held above the liquid level of the cooling solvent. In such a freeze-thawing method, it is described that a fixing tool having a slit part provided with a fixing part for fixing the cap member can be suitably used to hold the cells or tissues sealed with the cap member below the liquid level of the cooling solvent and the joint part of the main body member and the cap member above the liquid level of the cooling solvent. Patent Document 10 also describes a fixing tool for a cryopreservation jig having a slit part provided with a taper as a fixing structure for fixing the cryopreservation jig. Furthermore, Patent Document 11 describes a fixture for a cryopreservation jig having a slit section including a bent structure bent 90 degrees midway when viewed from above. In order to hold the cryopreservation jig in this fixture, it is necessary to push it all the way into the slit at the end of the 90-degree bend, but this operation is cumbersome for the operator, and there has been a demand for an auxiliary jig for the cryopreservation jig that is easier to operate and more stable in holding. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] JP 2017-104061 A [Patent Document 2] JP 2000-189155 A [Patent Document 3] Patent No. 5798633 [Patent Document 4] JP 2002-315573 A [Patent Document 5] JP 2006-271395 A [Patent Document 6] JP 2014-183757 A [Patent Document 7] JP 2015-142523 A [Patent Document 8] International Publication No. 2015 / 064380 Brochure [Patent Document 9] JP 2020-198845 A [Patent Document 10] International Publication No. 2020 / 250766 Brochure [Patent Document 11] Design Registration No. 1675557 [Non-patent literature]

[0015] [Non-Patent Document 1] 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 Summary of the Invention [Problem to be solved by the invention]

[0016] The main object of the present invention is to provide a mounting device that realizes favorable operability during the thawing operation for vitrification cryopreservation of cells or tissues. More specifically, the object of the present invention is to provide a mounting device for a cryopreservation jig that is excellent in operability when mounting the cryopreservation jig and in holding stability of the cryopreservation jig.

[0017] As a result of intensive research into solving the above problems, the present inventors have discovered that the above problems can be solved by a mounting device for a cryopreservation jig having the following configuration (hereinafter also referred to as the "mounting device of the present invention"). [Means for solving the problem]

[0018] A mounting device for a cryopreservation jig having a slit portion including a bending structure on the upper surface of a substrate having an upper surface, side surfaces and a bottom surface, one end of which opens to the side surface of the substrate, characterized in that the position of the bottom surface of the mounting portion, which is located further back than the bending structure when viewed from the slit opening of the slit portion, is lower than the position of the bottom surface of the insertion portion, which is located in front of the bending structure. Effect of the Invention

[0019] According to the present invention, it is possible to provide a mounting device for a cryopreservation device which is excellent in operability when mounting the cryopreservation device and in stability in holding the cryopreservation device. [Brief description of the drawings]

[0020] [Figure 1] FIG. 2 is a schematic top view showing an example of the mounting device of the present invention. [Diagram 2] FIG. 11 is a schematic top view showing another example of the mounting device of the present invention. [Diagram 3] FIG. 11 is a schematic top view showing still another example of the mounting device of the present invention. [Figure 4] 2 is a cross-sectional view taken along the line AA' of the mounting device shown in FIG. 1. [Diagram 5] FIG. 11 is a schematic top view showing still another example of the mounting device of the present invention. [Figure 6] FIG. 11 is a schematic top view showing still another example of the mounting device of the present invention. [Figure 7] FIG. 2 is a schematic diagram showing a state in which a cryopreservation jig is held using the mounting device of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention will be described in detail below.

[0022] The mounting device of the present invention is used for mounting a device for cryopreservation of cells or tissues. The mounting device of the present invention is also preferably used when cryopreserving cells or tissues by the so-called vitrification cryopreservation method and then thawing the cells or tissues. 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. The 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. The 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 a non-cellular substance such as an extracellular matrix in addition to cells. The mounting device of the present invention is particularly preferably used in the thawing operation of a cryopreserved egg or embryo.

[0023] The mounting device of the present invention has a base material having an upper surface, a side surface, and a bottom surface, and the base material is preferably formed of a liquid nitrogen resistant material. As the liquid nitrogen resistant material, 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 can be suitably used. In particular, metals are preferable from the viewpoint of excellent durability, large weight, and good retention of the cryopreservation jig in the cooling solvent. Among them, aluminum is preferable from the viewpoint of processability. The base material of the mounting device of the present invention may be made of one type of material or may be made of multiple materials.

[0024] FIG. 1 is a top schematic diagram showing an example of the mounting device of the present invention. In FIG. 1, the mounting device 1 has a slit section 3 including a bent structure on the upper surface of a substrate 2, one end of which is opened to a side surface of the substrate. The slit section 3 has an insertion section 5 located on the front side of the bent structure (a point where the insertion section 5 and the mounting section 6 intersect) as viewed from the slit opening 4, and a mounting section 6 located on the back side of the bent structure as viewed from the opening 4. The mounting section 6 shown in FIG. 1 extends in a direction bent 90° to the right in the drawing with respect to the extension direction of the insertion section 5, but the bending direction may be bent to either the left or right with respect to the extension direction of the insertion section 5, and the bending angle α in this case is preferably 80 to 125°, and more preferably 85 to 95° from the viewpoint of operability.

[0025] The shape of the mounting device of the present invention (the general shape when it is assumed that the substrate does not have the slit portion and the liquid level gauge portion described later) may be a prism shape such as a triangular prism, a square prism, or a hexagonal prism, as well as a truncated pyramid shape such as a truncated triangular pyramid or a truncated square pyramid. A prism shape is preferable, and a square prism shape is more preferable. In addition to the above shapes, a cylindrical shape or a truncated cone shape may be exemplified.

[0026] When mounting a cryopreservation jig using the mounting tool of the present invention, the tip of a tubular storage member (a tubular storage member storing a cell retention member) of the cryopreservation jig described below or the tip of a cap member of the cryopreservation jig is inserted into the slit of the mounting tool of the present invention, and then the cryopreservation jig is moved to the back side of the slit along the wall of the slit. From the viewpoint of achieving both operability when moving the cryopreservation jig and fixation stability of the cryopreservation jig after mounting, the width of the slit of the mounting tool of the present invention is preferably 90 to 120% of the width of the tip of the tubular storage member or the width of the tip of the cap member.

[0027] In the present invention, the width W1 of the insertion portion 5 and the width W2 of the placement portion 6 of the slit portion 3 may be the same or different, but from the viewpoint of operability, it is preferable that they are the same, as shown in FIG. 1.

[0028] In FIG. 1, the slit portion 3 of the mounting tool 1 has an insertion portion 5 and a mounting portion 6 having a bottom surface lower than the position of the bottom surface of the insertion portion 5 (hereinafter, such a structure is also referred to as a stepped structure). The relationship between the depth of the insertion portion 5 (B11 in FIG. 4 described later) and the depth of the mounting portion 6 (B21 in FIG. 4 described later) is B11 < B21. At the same time, it is preferable to satisfy the following formula (1). Thereby, the operability when mounting the freezing storage jig is enhanced, and the held freezing storage jig can be easily taken out. 1.1 < (B21 / B11) < 1.3 (1)

[0029] FIG. 2 is a schematic top view showing another example of the mounting tool of the present invention. The slit portion 3 shown in FIG. 2 has a different boundary position between the insertion portion 5 and the mounting portion 6 compared to the slit portion 3 shown in FIG. 1. In FIG. 1 described above, the mounting portion 6 is given priority at the intersection of the insertion portion 5 and the mounting portion 6, and this portion is used as the mounting portion. However, in FIG. 2, the insertion portion 5 is given priority at the intersection. In the present invention, when the mounting portion 6 is given priority at the intersection of the insertion portion 5 and the mounting portion 6, a mounting tool with particularly excellent operability when mounting the freezing storage jig can be obtained, which is preferable. The mounting portion 6 shown in FIG. 2 extends in a direction bent 90° to the right in the drawing with respect to the extending direction of the insertion portion 5, similar to the mounting portion 6 shown in FIG. 1. However, the bending direction may be bent to the left or right with respect to the extending direction of the insertion portion 5. In that case, the bending angle α is preferably 80 to 125°, and more preferably 85 to 95° from the viewpoint of operability.

[0030] FIG. 3 is a schematic top view showing still another example of the mounting tool of the present invention. In FIG. 3, the mounting tool 1 has slit portions 3 and 3' opened on different side surfaces of the base material 2 on the upper surface of the base material 2. In FIG. 3, the slit portions 3 and 3' opened on adjacent side surfaces of the base material 2 are respectively provided. However, slit portions opened on opposite side surfaces of the base material 2 may be respectively provided. Alternatively, it is also possible to have a plurality of slit portions for one side surface.

[0031] Fig. 4 is a cross-sectional view of the mounting tool shown in Fig. 1 at the position AA'. In Fig. 4, the slit portion 3 of the mounting tool 1 has, from the opening 4 side, an insertion portion 5 and a mounting portion 6 whose bottom surface is lower than that of the insertion portion 5. In this case, in the adjacent insertion portions 5 and mounting portions 6, when the bottom surface position of the mounting portion 6 located on the back side of the bent portion is made lower than the bottom surface position of the insertion portion 5 located in front of the bent portion, it is possible to provide the mounting portion 6 whose bottom surface position is lower by a slope whose bottom surface position changes continuously, but it is preferable to provide the mounting portion 6 whose bottom surface position is lower by a stepped height difference as shown in Fig. 4, because it is possible to obtain a mounting tool that is particularly excellent in mounting stability after placing the cryopreservation jig thereon.

[0032] The depth B11 of the insertion part 5 shown in Fig. 4 is not particularly limited as long as the cells or tissues placed on the cryopreservation jig are not exposed to liquid nitrogen when the cryopreservation jig is inserted, but is preferably 10 to 60 mm, and more preferably 20 to 40 mm, from the top surface of the substrate 2. The depth B21 of the mounting part 6 is not particularly limited as long as it is deeper than the bottom surface of the insertion part and does not cause any problems in holding the cryopreservation jig, but is preferably 12 to 75 mm, and more preferably 24 to 50 mm, from the top surface of the substrate 2. Furthermore, the step between the bottom surface of the insertion part 5 and the mounting part 6 is preferably 4 to 10 mm.

[0033] Fig. 5 is a schematic top view showing another example of the mounting tool of the present invention. In Fig. 5, the slit section 3 of the mounting tool 1 has a structure for holding a cryopreservation jig, and in addition to the slit section 3 including a bent structure and the step structure between the insertion section 5 and the mounting section 6, the mounting section 6 has a tapered structure at the back side. The tapered structure is a structure in which the width of the mounting section (the distance between the side surfaces of the mounting section) gradually decreases over the length T11 in the depth direction of the mounting section 6. That is, the relationship between the width (W2) of the tapered structure part closest to the opening 4 of the mounting section 6 and the width (T22) of the tapered structure part farthest from the opening 4 of the mounting section 6 is W2>T22.

[0034] When using a mounting tool having the shape of Fig. 5, a cryopreservation jig (not shown) is inserted into the insertion part 5 from the opening 4, the cryopreservation jig is slid along the side of the insertion part 5, and inserted into the mounting part 6 whose bottom is lower than the bottom of the insertion part 5, and while pressing the tip of the cap member (or the tip of the cylindrical insertion member) of the cryopreservation jig against the side of the lowermost part of the bent part, the gripping part of the cryopreservation jig is tilted toward the tapered structure part at the same time as the cryopreservation jig falls. This is preferable because it enables the cryopreservation jig to be fixed more firmly.

[0035] Furthermore, the above-mentioned tapered structure portion has a shape that satisfies the following formula (2), thereby making it possible to fix the cryopreservation jig particularly firmly. 0.001≦(W2-T22) / T11≦0.03 (2) (In the formula, W2 represents the width of the tapered structure at the part closest to the opening of the slit, T22 represents the width of the tapered structure at the part farthest from the opening of the slit, and T11 represents the depth length of the tapered structure.)

[0036] The tapered structure of the mounting tool shown in Fig. 5 does not have a shape that satisfies the above-mentioned formula (2), but for the sake of explanation, a shape in which the value of (W2-T22) / T11 exceeds 0.03 is shown as an example. This also applies to Fig. 6 described later.

[0037] Fig. 6 is a schematic top view showing another example of the mounting device of the present invention. In Fig. 6, the mounting device 1 has an insertion section 5 as a structure for holding a cryopreservation jig, and a slit section 3 having mounting sections 6 and 6' branched and bent in two directions from the insertion section 5, and both ends of the mounting sections 6 and 6' each have a tapered structure. With the shape as shown in Fig. 6, even a single opening makes it possible to fix two cryopreservation devices at the same time.

[0038] In the mounting tool shown in FIG. 6, the shapes of the tapered structures provided in two locations and the values ​​of (W2-T22) / T11 and (W2-T22') / T11' may be the same or different, but are preferably the same.

[0039] T11 and T11' shown in Figures 5 and 6 are not particularly limited as long as they are long enough to fix the cryopreservation device after insertion, but when cells or tissues are frozen and thawed by the Cryotop method, they are preferably 3 to 30 mm, and more preferably 5 to 20 mm.

[0040] 1, 2, 5 and 6 showing examples of the mounting device of the present invention, when looking at the depth of the slit section 3 from the opening 4, the depth of the slit section 3 is the sum of the centerline length T1A from the side surface of the mounting device 1 to where the slit section 3 bends and the centerline length T1B (or T1B') from the bending point to the innermost part of the slit section. The bending point is the position where the centerlines of the insertion section 5 and mounting section 6 intersect. In the present invention, when cells or tissues are frozen and thawed by the above-mentioned Cryotop method, the depth of the slit section is preferably 10 to 60 mm, and more preferably 15 to 50 mm.

[0041] Next, the freezing and thawing method using the mounting device of the present invention will be described in detail.

[0042] The mounting device of the present invention can be suitably used in a series of processes from freezing and thawing cells or tissues, particularly in a cooling operation after the cryopreservation jig on which the cells or tissues are mounted is removed from a tank containing a cooling medium, and in a thawing operation when the cells or tissues that have been kept cold are thawed. In general, when preserving cells or tissues by the so-called vitrification freezing method, cryopreservation is performed using a cryopreservation jig having a rectangular strip. Such a cryopreservation jig is disclosed, for example, in the pamphlet of International Publication No. 2011 / 070973, in addition to the above-mentioned Patent Documents 4 to 8. As a series of processes from freezing and thawing, first, the equilibrated cells or tissues are dropped onto the strip of the cryopreservation jig together with a preservation solution, and then the cells or tissues are cooled using a cooling medium, preferably liquid nitrogen, to perform the freezing operation. Secondly, a cooling operation is performed to preserve the cells or tissues that have been subjected to the freezing operation for a long period of time while maintaining an extremely low temperature environment. Thirdly, a thawing operation is performed in which the frozen cells or tissues are transferred from the cryogenic environment into a solution called a thawing liquid, and melted or thawed in the thawing liquid.

[0043] The mounting 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 a cooling medium in the series of processes from cryopreservation to thawing of the cells or tissues described above.

[0044] In the closed type cryopreservation-thawing process, during the freezing operation, cells or tissues are dripped onto the strip together with a preservation solution, and then the cryopreservation tool is stored in a cylindrical storage member with a completely closed end, and isolated from the outside world. Alternatively, cells or tissues are dripped onto the strip together with a preservation solution, and then the strip is encapsulated with a cap member and isolated from the outside world. After such storage or isolation, the cryopreservation tool is immersed in a cooling solvent to freeze the cells or tissues. During the closed type freezing operation and subsequent cold storage, the air and preservation solution around the cells or tissues are cooled by the cooling solvent via the cylindrical storage member or cap member, and the vitrified state is maintained.

[0045] In the thawing operation in the closed cryopreservation-thawing process using the mounting device of the present invention, prior to the operation of transferring frozen cells or tissues to a thawing liquid, one end of a cylindrical storage member housing a cryopreservation device on which cells are placed, or one end of a cap member enclosing a mounting part on which cells or tissues are placed, is temporarily held by a mounting device having a slit part including a bent structure. The bent structure of such a slit part is useful from the viewpoint of preventing the cylindrical storage member housing a cryopreservation device, or the cryopreservation device with a mounting part enclosing a cap member, from falling off into the cooling solvent, but on the other hand, the operation of inserting the cryopreservation device behind the bent part is cumbersome for those skilled in the art.

[0046] The present invention solves such problems by the step structure, and the mounting tool of the present invention remarkably improves the operability when moving the cryopreservation jig to the mounting part via the bent structure. In addition, the step structure prevents the cryopreservation jig once placed from falling off the insertion part, and good holding stability is obtained. Specifically, for example, in the mounting tool 1 shown in FIG. 1, the cryopreservation jig inserted from the opening 4 is moved to the bent part, and the tip part of the cap member (or the tip part of the cylindrical insert member) of the cryopreservation jig is pressed against the lowest part of the side of the bent part (at this time, almost at the same time as the tip part of the cap member of the cryopreservation jig or the tip part of the cylindrical insert member falls down the step structure), and the grip part of the cryopreservation jig (or the rear end part of the cylindrical insert member) is tilted toward the back side of the mounting part 6, so that the cryopreservation jig can be easily fixed. 2, the cryopreservation jig can be easily fixed by moving the cryopreservation jig inserted from the opening 4 to the bent portion, and then dropping the step between the insertion portion 5 and the mounting portion 6, while tilting the gripping portion of the cryopreservation jig (or the rear end portion of the tubular insertion member) toward the back side of the mounting portion 6, using the step portion as a fulcrum. Note that, as shown in the examples described later, the former mounting device is preferable because it allows the cryopreservation jig to be mounted with superior operability.

[0047] It is particularly preferable to use the mounting device of the present invention to position the cells or tissue sealed by the cap member below the liquid level of the cooling solvent and temporarily hold the joint between the main body and the cap member above the liquid level of the cooling solvent, since this allows the frozen cells or tissue to be quickly moved into the melting liquid.

[0048] The cells or tissues to be immersed in the melting liquid are preferably immersed in the melting liquid within 5 minutes, more preferably within 1 minute, after the cover member is removed from the cylindrical storage member. Also, when removing the cryopreservation jig having the strip on which the cells or tissues are placed from the cap member, the cells or tissues are preferably immersed in the melting liquid within 5 minutes, more preferably within 1 minute, after the fitting is loosened once.

[0049] The mounting device of the present invention can also be used in so-called open-type cryopreservation and thawing processes in which cells or tissues come into contact with a cooling solvent such as liquid nitrogen. Even in open-type processes, it is preferable to use the mounting device of the present invention to hold or fix the cryopreservation jig prior to the operation of transferring the frozen cells or tissues to a thawing liquid, since this allows the cells or tissues to be quickly transferred from liquid nitrogen to the thawing liquid.

[0050] Fig. 7 is a schematic diagram showing an example of a state in which a cryopreservation jig is held using the mounting device of the present invention. In Fig. 7, the cryopreservation jig 7 has at least a main body 8 and a cap member 9 that is detachable from the main body 8. Cells 12 are mounted on a strip 10 of the cryopreservation jig 7 together with a very small amount of preservation solution 13.

[0051] The strip 10 on which the cells 12 are placed together with a very small amount of preservation solution 13 is sealed with a cap member 9, and the cells 12 placed on the strip 10 are cooled by being located below the liquid level 15 of the liquid nitrogen 14, and the frozen cells 12 are maintained in a frozen state. When the frozen cells 12 are melted, the joint between the cap member 9 and the main body 8 is located above the liquid level 15 of the liquid nitrogen 14, so that the main body 8 can be quickly pulled out and separated from the cap member 9, and the mounting part can be quickly immersed in the melting liquid, which is preferable. In addition, when the mounting tool 1 has a liquid level gauge part 16, the operator can easily grasp the position of the liquid level 15, which improves workability, and is therefore preferable. FIG. 7 illustrates an example in which the upper surface of the liquid level gauge part 16 attached to the upper surface (top surface) of the mounting tool 1 is used as the reference for the position of the liquid level 15. By using the liquid level gauge portion 16, it is easy to position the liquid level 15 above the cells 12 and below the joint between the cap member 9 and the main body portion 8.

[0052] Next, a cryopreservation jig that is held or fixed using the mounting device of the present invention will be described.

[0053] The strip 10 of the main body 8 is preferably rectangular in shape, which is preferable since it is easy to store the strip 10 in a cap member or a cylindrical storage member.

[0054] In the present invention, the strip 10 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, the resin film is preferably used from the viewpoint of handling. Specific examples of the resin film 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 10 is 80% or more, the cells or tissues mounted on the mounting portion can be easily confirmed using a transmission microscope, which is preferable.

[0055] In addition, a metal plate can be suitably used as the strip 10 from the viewpoint of 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, and stainless steel. The thickness of the above-mentioned various resin films, metal plates, glass plates, rubber plates, etc. 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 an electrical method such as corona discharge treatment or a chemical method, and can also be roughened.

[0056] A preservation solution absorbent may also be used as the strip 10. When the preservation solution absorbent is used for the strip 10, the excess preservation solution 13 can be effectively removed, and the freezing speed is improved. Examples of the preservation solution absorbent include a film-like material made of wire mesh, paper, or synthetic resin, which has through-holes. Other examples of the preservation solution absorbent include a porous structure formed using a material with a refractive index of 1.45 or less. The porous structure allows the preservation solution 13 present around the cells 12 to be efficiently removed. In addition, under observation with a transmission optical microscope, the operation of placing and freezing the cells 12 and the operation of thawing after freezing can be easily and reliably performed with good visibility.

[0057] The refractive index of the material of the porous structure 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 forming the porous structure and having 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.

[0058] The pore size of the preservation solution absorbent with 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 solution absorbent 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 size of the preservation solution absorbent 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 size is the average pore size measured from image observation of the surface and cross section of the porous structure.

[0059] The porosity of the storage liquid absorbent is preferably 30% or more, and more preferably 70% or more. The porosity is defined by the following formula. Here, the pore volume V is calculated by multiplying the cumulative pore volume (ml / g) of the pores with a radius of 3 nm to 400 nm in the storage liquid absorbent, measured and processed using a mercury porosimeter (measuring device name: Autopore II 9220, manufacturer: Micromeritics Instrument Corporation), by the dry solid content (g / m 2 ) to obtain the unit area (m 2 The thickness T of the storage liquid absorbing body can be obtained by photographing the cross section of the storage liquid absorbing body with an electron microscope and measuring the length. P = (V / T) x 100 (%) P: Porosity (%) V: void volume (ml / m 2 ) T: Thickness (μm)

[0060] The cap member 9 of the cryopreservation jig in the present invention 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 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 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 placement part inside the cap can be easily confirmed after the cap member is attached.

[0061] In the present invention, the main body 8 of the cryopreservation jig preferably has a gripping part 11 from the viewpoint of workability, and in this case, the gripping part 11 is preferably prismatic in order to improve ease of gripping and operability. The gripping part 11 is preferably a member formed of a material that is resistant to a cooling medium 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.

[0062] When cells or tissues are thawed using the mounting 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, DMSO (dimethyl sulfoxide), etc. The thawing liquid may also be one that is normally used for thawing cells such as eggs and embryos, for example, a thawing liquid containing 1M sucrose in a physiological solution such as the above-mentioned phosphate buffered saline for adjusting the osmotic pressure.

[0063] In the present invention, examples of cells to be cryopreserved and thawed 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 the cells include cultured cells such as primary cultured cells, subcultured cells, and cell line cells. In one or more embodiments, examples of 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.

[0064] The mounting device of the present invention can be rephrased as a mounting device for a cell or tissue cryopreservation device, a mounting device for a cell or tissue cryopreservation device, a mounting instrument for a cell or tissue cryopreservation device, a mounting device for a cell or tissue cryopreservation device, or a mounting instrument for a cell or tissue cryopreservation device. EXAMPLES

[0065] 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.

[0066] Example 1 A mounting tool of Example 1 having a bent structure and a step structure in the slit portion 3 shown in Figs. 1 and 4 was produced by metal cutting using aluminum. In this mounting tool, the mounting portion 6 is prioritized at the intersection of the insertion portion 5 and the mounting portion 6. The size of the mounting tool 1 is 50 mm x 50 mm x 70 mm in length x width x height, and the width W1 of the opening 4 of the slit portion is 3.54 mm. This width W1 of the opening 4 corresponds to 114% of the width (3.1 mm) of the tip portion of the cap member 9 of the cryopreservation jig 7 described later. In addition, the depth B11 of the insertion portion 5 is 25 mm, and the depth B21 of the mounting portion 6 is 30 mm, so that B21 / B11 is 1.2. In addition, a step of 5 mm is generated between the insertion portion 5 and the mounting portion 6, and the bending angle α is 90°.

[0067] Example 2 The mounting tool of Example 2 was produced in the same manner as in Example 1, except that the slit portion 3 shown in FIG. 2 has a bent structure and a step structure by metal cutting using aluminum. In this mounting tool, the insertion portion 5 is prioritized at the intersection of the insertion portion 5 and the mounting portion 6. The size of the mounting tool 1 is 50 mm x 50 mm x 70 mm in length x width x height, similar to the mounting tool of Example 1 described above, and the width W1 of the opening 4 of the slit portion is 3.54 mm. This width W1 of the opening 4 corresponds to 114% of the width (3.1 mm) of the tip portion of the cap member 9 of the cryopreservation jig 7 described later. In addition, the depth B11 of the insertion portion 5 is 25 mm, and the depth B21 of the mounting portion 6 is 30 mm, so that B21 / B11 is 1.2, and a step of 5 mm is generated between the insertion portion 5 and the mounting portion 6, and the bending angle α is 90°.

[0068] (Comparative Example) A comparative mounting tool was produced in the same manner as in Example 1, except that the depth B11 of the insertion portion 5 was 30 mm and the depth B21 of the mounting portion 6 was also 30 mm. B21 / B11 in this mounting tool was 1.0.

[0069] Next, the cryopreservation jig described below was prepared, and using this cryopreservation jig, cells or tissues were frozen, cooled, and thawed according to the procedures described below.

[0070] <Preparation of cryopreservation jig> A cryopreservation jig 7 having the configuration shown in Fig. 7 was produced. A rectangular (1.5 mm wide, 20 mm long, 250 µm thick) polyethylene terephthalate resin film was used for the strip 10 of the main body 8 of the cryopreservation jig 7, and the resin film was attached to a gripping part 11 made of ABS resin. The cap member 9 was made of PP resin, and the main body 8 into which the cap member 9 was fitted had a tapered structure. The opening of the cap member 9 was circular and had a diameter of 2 mm. The outer shape of the cap member 9 was a square prism with one side measuring 3.1 mm.

[0071] <From placing cells or tissues to sealing, freezing, and cooling processes> The equilibrated mouse 8-cell stage embryos were dropped onto the strip 10 of the cryopreservation jig 7 together with the preservation solution using a pipette, and after removing excess preservation solution, the strip 10 was inserted into the cap member 9 under observation with a transmission microscope, and fitted and fixed (sealing step). Thereafter, the cap member 9 side of the cryopreservation jig 7 was immersed in liquid nitrogen 14 (freezing step). The preservation solution used was a commercially available Medium 199 medium (Life Technologies) containing L-glutamine, phenol red, and 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) as a base solution, containing 15% by volume of ethylene glycol, 15% by volume of DMSO (dimethyl sulfoxide), 0.5 M sucrose, and 50 mg / L of gentamicin.

[0072] The time from when the mouse 8-cell stage embryos were transferred from the equilibration solution to the preservation solution until they were immersed in liquid nitrogen was 1 minute. The cryopreservation jig used in the freezing process was stored under liquid nitrogen until the thawing process was performed (cooling process).

[0073] <Melting process> The cryopreservation jig 7 after the above steps was removed from the liquid nitrogen, and the cryopreservation jig 7 was fixed to the slit portion 3 of the fixture of Examples 1, 2, and Comparative Example, and the joint between the main body portion 8 and the cap member 9 of the cryopreservation jig was temporarily held above the liquid level 15 of the liquid nitrogen 14. At that time, it was confirmed that the cells or tissues were reliably cooled in all of the fixtures of Examples 1, 2, and Comparative Example. Next, the fitting between the cap member 9 and the main body portion 8 was loosened, the main body portion 8 was separated from the cap member 9, and the strip 10 of the main body portion 8 was immersed in a melting liquid kept at 37°C. Then, the embryo was recovered from the strip 10. Note that the operation from loosening the fitting of the cap member 9 to separating the main body portion 8 from the cap member 9 was possible within 10 seconds, and the operation from separating the main body portion 8 from the cap member 9 to immersing the strip 10 in the melting liquid was possible within 1 second. The thawing solution used was a solution containing 1M sucrose using the above-mentioned Medium 199 medium as a base solution.

[0074] <Evaluation of operability when placing cryopreservation equipment> In the fixing tool of Example 1, the cryopreservation jig 7 inserted from the opening 4 was moved to the bent portion, and then the cap member 9 was pressed against the bottom of the side of the bent portion while the gripping portion 11 of the cryopreservation jig 7 was tilted toward the back side of the mounting portion 6, so that the cryopreservation jig could be easily fixed (represented by ◎ in Table 1 described later). In the fixing tool of Example 2, the insertion portion 5 is prioritized at the intersection of the insertion portion 5 and the mounting portion 6. The cryopreservation jig 7 inserted from the opening 4 was moved to the bent portion, and then the step between the insertion portion 5 and the mounting portion 6 was dropped, and the gripping portion of the cryopreservation jig was tilted toward the back side of the mounting portion 6 with the step portion as a fulcrum, so that the cryopreservation jig 7 could be fixed, but the operability was slightly reduced compared to when the fixing tool of Example 1 described above was used (represented by ◯ in Table 1 described later). On the other hand, in the fixing device of the comparative example, the cryopreservation jig 7 was inserted from the opening 4 and moved to the bent portion, and then the cap member 9 was pressed against the bottommost side of the bent portion 4 while the gripping portion 11 of the cryopreservation jig 7 was tilted toward the rear side of the mounting portion 6; however, the operability at this time was even worse than that of the above-mentioned Example 2 (marked as △ in Table 1 described below).

[0075] <Evaluation of the retention stability of cryopreservation devices> The retention stability of the cryopreservation jigs 7 placed on the placement devices of Examples 1 and 2 and the Comparative Example as described above was evaluated according to the following criteria.

[0076] ◯: The cryopreservation jig 7 was stably held in the placement portion 6. In addition, the jig was stably held even when the liquid nitrogen surface moved. △: The cryopreservation jig 7 in the placement portion 6 shook with the movement of the liquid nitrogen surface, and lacked stability.

[0077] [Table 1]

[0078] From the above results, it is evident that the present invention provides a mounting device for a cryopreservation device that is excellent in operability when mounting the cryopreservation device and in stability in holding the cryopreservation device. [Industrial Applicability]

[0079] The present invention can be used in embryo transfer and artificial insemination of livestock such as cattle and other animals, artificial insemination in humans, and the like using the cryopreservation device, as well as in thawing procedures for 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. [Explanation of symbols]

[0080] 1 Mounting device 2 Base material 3 Slit section 4 Openings 5 Insertion section 6 Placement section 7. Cryopreservation fixture 8 Main body 9 Cap member 10. Strip 11 Gripping part 12 cells 13 Preservation solution 14 Liquid Nitrogen 15 Liquid level 16 Liquid level gauge section

Claims

[Claim 1] A mounting device for a cryopreservation jig having a slit portion including a bending structure on the upper surface of a substrate having an upper surface, side surfaces and a bottom surface, one end of which opens to the side surface of the substrate, characterized in that the position of the bottom surface of the mounting portion, which is located further back than the bending structure when viewed from the slit opening of the slit portion, is lower than the position of the bottom surface of the insertion portion, which is located in front of the bending structure.

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