JP2021177725A - Fixture of freezing preservation jig - Google Patents

Abstract

To provide a fixture of a freezing preservation jig capable of firmly holding a freezing preservation jig, allowing a user to confirm that frozen cells or tissues are in a surely cooled state, and quickly transferring the cells or tissues into fusion liquid.SOLUTION: There is provided a fixture of a freezing preservation jig comprising, on a top face of a base material comprising the top face, side faces and a bottom face, a slit part whose one end is opened to the side face part of the base material, or on the top face of the base material, an insertion part for inserting the freezing preservation jig and a slit part whose one end is opened on the side face of the insertion part. The fixture has a specific shaped taper structure part for fixing the freezing preservation jig into the slit part.SELECTED DRAWING: Figure 1

Description

The present invention relates to a fixture for a cryopreservation jig that is suitably used when a frozen cell or tissue is thawed.

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

In general, cells or tissues collected from an in-vivo body gradually lose their activity or change their traits even in a culture medium, so that the cells or tissues in vitro are used for a long period of time. Culture is not preferred. Therefore, a technique for long-term storage while maintaining biological activity is important. Superior preservation techniques allow for more accurate analysis of harvested cells or tissues. In addition, excellent preservation technology makes it possible to use cells or tissues for transplantation while maintaining higher biological activity, and it is expected that the engraftment rate after transplantation will be improved. Furthermore, it is also possible to sequentially produce and store artificial tissues for transplantation, such as cultured skin cultured in vitro and so-called cell sheets constructed in vitro, and use them when necessary. Great benefits can be expected not only in the medical aspect but also in the industrial aspect.

As a method for cryopreserving cells or tissues, for example, a slow freezing method is known. In this method, first, cells or tissues are immersed in a preservation solution obtained by containing a freezing agent in a physiological solution such as phosphate buffered saline. As the antifreeze agent, compounds such as glycerol, ethylene glycol and dimethyl sulfoxide are used. After immersing the cells or tissues in the preservation solution, the cells or tissues are cooled to −30 to −35 ° C. at a relatively slow cooling rate (for example, a rate of 0.3 to 0.5 ° C./min) to inside or outside the cells or the tissues. The inner and outer solutions are sufficiently cooled and become more viscous. In such a state, when the cells or tissues in the preservation solution are further cooled to the temperature of liquid nitrogen (-196 ° C.), the minute solutions inside and around the cells or tissues are solidified in an amorphous state. Vitrification occurs, which is a phenomenon that occurs. When the inside and outside of the cell or the inside and outside of the tissue are solidified by vitrification, the movement of the molecule is substantially stopped. Therefore, it is considered that the vitrified cell or tissue can be stored semi-permanently by storing it in liquid nitrogen.

A vitrification freezing method is also known as a method for cryopreserving cells or tissues. The vitrification freezing method uses the principle that ice crystals are less likely to form even below freezing point due to the freezing point depression of a storage solution containing a large amount of antifreezing agent such as glycerol, ethylene glycol, and dimethylsulfoxide. When this preservation solution is rapidly cooled in liquid nitrogen, it can be solidified without forming ice crystals. This solidification is called vitrification freezing. A preservative solution containing a large amount of antifreeze is called a vitrification solution.

In the slow freezing method described above, it is necessary to cool at a relatively slow cooling rate, so that the operation for cryopreservation takes time. Further, there is a problem that a device or a jig for controlling the cooling rate is required. In addition, in the slow freezing method, ice crystals are formed in the storage solution outside the cells or tissues, so that the cells or tissues may be physically damaged by the ice crystals. On the other hand, in the above-mentioned vitrification freezing method, the operation time is short and the process does not require a special device or jig. In addition, the vitrification freezing method provides high survival rates by not producing ice crystals.

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

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

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

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

Generally, as a freezing method related to the vitrification freezing method, in Patent Document 4, a mammalian embryo or an egg is wrapped in an inner surface of a freezing storage 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 vitrifying solution sufficient to be used, and the container is brought into contact with liquid nitrogen to be rapidly cooled. In the thawing method performed after the freezing method, the cryopreservation container stored by the above method is taken out from liquid nitrogen, one end of the container is opened, and a diluted solution at 33 to 39 ° C. is injected into the container. It is used to thaw and dilute frozen embryos or eggs. According to this method, mammalian embryos or eggs can be preserved and thawed and diluted with a high survival rate without being infected with viruses or bacteria. However, the difficulty of the step of attaching the embryo or egg to the inner surface of the cryopreservation container such as a frozen straw, a frozen vial or a freezing tube is high, and the embryo or egg is surely placed in the cryopreservation container. It was difficult to confirm.

Patent Document 5 describes a cell holding member having a heat conductive member and a cell cryopreservation tool having a tubular storage member, and the problem in the freeze-thaw method of Patent Document 4 is solved to some extent. In the method of using the cryopreservation tool described in Patent Document 5, an egg is attached to a cell holding member under a microscope observation, the cell holding member is stored in a tubular storage member, and then the egg is immersed in liquid nitrogen to glass. Freeze. After that, a method of attaching a lid member to the opening of the tubular member and storing it in a liquid nitrogen tank is described. Further, in Patent Document 6, an egg is placed on a strip for holding egg adhesion together with a small amount of vitrified liquid, and the entire cryopreservation jig is stored in a tubular storage container and then immersed in liquid nitrogen to glass. Freezing is carried out.

As a freezing method related to the vitrification freezing method with a smaller process, a method called the so-called cryotop (registered trademark) method used in the field of human infertility treatment as described in Patent Documents 7 and 8 is disclosed. Has been done. In these methods, the freezing operation uses an egg cryopreservation tool equipped with a strip-shaped flexible and colorless transparent film as a strip for holding the attachment of eggs, and ova with a very small amount of preservative solution on the film under microscopic observation. Alternatively, the embryo is placed and the film to which the egg is attached is immersed in liquid nitrogen to perform vitrification freezing. The egg attachment holding strip on which the frozen egg or embryo is placed is protected by a cap or the like and then stored in a liquid nitrogen tank.

Thawing of eggs, embryos, etc. frozen by these methods is performed by immersing the egg attachment holding strip in a warmed thaw solution, and the eggs, embryos, etc. placed on the strip in the thaw solution, etc. Will be recovered.

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

Once the cells or tissues frozen in this way are once held so as to be located below the liquid level of liquid nitrogen, for example, in the method described in Patent Document 5 or Patent Document 6, the liquid is used. The lid member located above the liquid surface of nitrogen can be easily removed, and the cells or tissues can be rapidly moved into the melting liquid to prevent a decrease in the melting rate. Further, in the method described in Patent Document 7 and Patent Document 8, by pulling out the cryopreservation jig from the cap, cells or tissues can be rapidly moved into the thaw solution, and a decrease in the thaw rate can be prevented. can.

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

Steponkus et al. , Nature 345: 170-172 (1990) Hiroshi Monk, "Mechanisms of Damage and Protection by Freezing of Living Cells" Chemistry and Biology Vol. 18 (1980) No. 2 P. 78-87 Published by Japan Society for Bioscience and Biotechnology

However, in the method using the liquid nitrogen container described in Patent Document 9 described above, even if it is possible to stably hold the cryopreservation jig when the cryopreservation jig is leaned against the slit portion formed in the lid portion, It was difficult to confirm whether the frozen state of the cells or tissues was surely maintained because the placement portion of the cryopreservation jig was located below the liquid level of liquid nitrogen. Further, the length of the tubular 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. In such a case, the cells are retained from the inside of the tubular resin member. The work of taking out the main body of the member is complicated, and improvement has been desired from the viewpoint of performing a quick melting operation.

The main object of the present invention is to provide a fixture for realizing suitable workability during a thaw operation of vitrified cryopreservation of cells or tissues. More specifically, it is possible to firmly hold the cryopreservation jig, and the cells or tissues are in a sufficiently cooled state prior to the operation of transferring the frozen cells or tissues to the thaw solution. It is an object of the present invention to provide a fixture of a cryopreservation jig capable of confirming that and rapidly transferring frozen cells or tissues into a warmed thaw solution.

As a result of diligent studies to solve the above problems, the present inventor solves the above problems with a fixture for a cryopreservation jig having the following configuration (hereinafter, also referred to as "fixing tool of the present invention"). I found out what I could do.

An insertion part and an insertion part for inserting a cryopreservation jig into the upper surface of the base material having a slit portion having at least one end opened in the side surface portion of the base material on the upper surface of the base material having an upper surface, a side surface and a bottom surface. A fixture for a cryopreservation jig having a slit portion with one end open on the side surface thereof, wherein the fixture has a tapered structure portion for fixing the cryopreservation jig inside the slit portion. A fixture for a cryopreservation jig, characterized in that the tapered structure part satisfies the following formula (1).
(T21-T22) /T11≤0.03 (1)
(In the formula, T21 represents the width of the tapered structure at the portion closest to the slit opening, T22 represents the width of the tapered structure at the portion farthest from the slit opening, and T11 represents the depth length of the tapered structure. Represents.)

According to the present invention, it is possible to firmly hold the cryopreservation jig, and the cells or tissues are surely cooled prior to the operation of transferring the frozen cells or tissues to the thaw solution. It is possible to provide a fixture for a cryopreservation jig that can be confirmed to be present and can rapidly transfer frozen cells or tissues into a warmed thaw.

It is a top view schematic showing an example of the fixture of this invention. It is a top view schematic showing another example of the fixture of this invention. It is a top schematic which shows another example of the fixture of this invention. It is a top schematic which shows another example of the fixture of this invention. It is a top schematic which shows another example of the fixture of this invention. It is a schematic diagram which shows an example of the state which fixed the cryopreservation jig by using the fixture of this invention.

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

The fixing tool of the present invention is a fixing tool for a cell or tissue cryopreservation jig, a fixing tool for a cell or tissue cryopreservation jig, a fixing device for a cell or tissue cryopreservation jig, or a cell or tissue. It can be paraphrased as a fixture of a cryopreservation tool or a fixture of a cell or tissue cryopreservation device.

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

The fixture of the present invention has 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 portion having at least one end open to the side surface portion of the base material. Alternatively, it has an insertion portion for inserting a cryopreservation jig on the upper surface of the base material and a slit portion with one end open on the side surface of the insertion portion. Then, in the fixture of the present invention, the cryopreservation jig is inserted into the slit through the slit opening to fix the cryopreservation jig.

The slit portion for fixing and holding the cryopreservation jig of the fixture of the present invention may be provided at one place on the upper surface of the base material or at a plurality of places. Alternatively, it may be present at one place on the side surface of the insertion portion provided on the upper surface of the base material, or may be held at a plurality of places.

When fixing the cryopreservation jig using the fixture of the present invention, the tip of the tubular storage member or the tip of the cap member of the cryopreservation jig is slit in the fixture of the present invention. After inserting into the portion, the cryopreservation jig is moved to the back side of the slit along the slit. The width of the slit portion of the fixture of the present invention is the tip of the tubular storage member from the viewpoint of achieving both operability when moving the cryopreservation jig and fixing stability of the cryopreservation jig. It is preferably 90 to 110% with respect to the width or the width of the tip portion of the cap member.

The shape of the fixture of the present invention (rough shape when it is considered that the base material is not provided with a slit portion) includes a triangular prism structure such as a triangular prism, a quadrangular prism, and a hexagonal prism, as well as a triangular pyramid having an upper surface and four. A partial structure of a pyramid such as a pyramid can be mentioned. It is preferably a prismatic structure, and more preferably a prismatic structure. In addition to the above, a cylindrical structure or 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 alloys, ABS resin, acrylic resin, polypropylene resin, polystyrene resin, polyethylene resin, fluororesin, various engineer plastics, and glass and rubber materials. Etc. can be preferably used. In particular, metal is preferable from the viewpoint of having excellent durability, having a large weight, and being able to satisfactorily fix the cryopreservation jig. Of these, aluminum is preferable from the viewpoint of workability. The fixture of the present invention may be composed of one kind of material or may be composed of a plurality of materials.

The slit portion of the fixture of the present invention is a taper structure portion in which the distance between the side surfaces inside the slit portion is gradually narrowed as a fixing structure portion for firmly fixing and holding the inserted cryopreservation jig. Have. Further, the tapered structure portion has a shape satisfying the above-mentioned formula (1), so that the cryopreservation jig can be firmly fixed.

Hereinafter, 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 slit portion 2 of the fixture 1 has a tapered structure portion as a fixing structure portion for fixing the cryopreservation jig.

The tapered structure portion of the fixture 1 of FIG. 1 extends from the slit opening 3 toward the center portion side of the base material 5 over the depth length T11 of the tapered structure portion, and the width of the slit portion 2 (side surface of the slit portion). It has a structure in which the distance between the two is gradually attenuated, that is, the width of the tapered structure portion closest to the slit opening (T21) and the width of the tapered structure portion farthest from the slit opening (T22). ) Is T21> T22. In the present invention, the tapered structure portion satisfies the above-mentioned formula (1), so that the cryopreservation jig can be firmly held and the frozen cells or tissues are kept warm in the thawed liquid. It will be possible to transfer quickly to. It is preferable that the value of (T21-T22) / T11 described above is 0.001 or more from the viewpoint of fixing stability of the cryopreservation jig.

The fixture 1 in FIG. 1 described above is also an example in which the slit portion 2 is composed of only a tapered structure portion. Therefore, in FIG. 1, the width (T2) of the slit opening is equal to the width (T21) of the tapered structure portion closest to the slit opening, and similarly, the depth (T1) of the slit opening is the tapered structure portion. Equal to the depth (T11).

The tapered structure portion of the fixture 1 shown in FIG. 1 does not have a shape satisfying the above-mentioned equation (1), but for convenience, the value of (T21-T22) / T11 is 0. It is an example of a shape exceeding 03. This also applies to FIGS. 2 to 5 described later.

FIG. 2 is a schematic top view showing another example of the fixture of the present invention. In FIG. 2, the slit portion 2 of the fixture 1 has a slit portion 2 having a bent structure and a tapered structure portion of the present invention on the back side of the slit portion as a fixing structure portion for fixing the cryopreservation jig. Have. The slit portion 2 of the fixture 1 in FIG. 2 has a shape in which the slit portion 2 goes straight from the slit opening 3 toward the central portion side of the base material 5 by a certain distance (T1A) and is bent 90 degrees to the right side in the figure on the way. It also has a tapered structure after being bent 90 degrees to the right. Further, in FIG. 2, the relationship between the width of the tapered structure portion closest to the slit opening (T21) and the width of the tapered structure portion farthest from the slit opening (T22) is T21> T22.

When the fixture having the shape shown in FIG. 2 is used, a cryopreservation jig is inserted into the slit portion 2 through the slit opening 3, passes through a position bent by 90 degrees, and is further cryopreserved in the direction of the closed surface of the slit portion. A jig can be inserted and fixed by a tapered structure. By having both a bent structure portion and a tapered structure portion as the fixed structure portion, the cryopreservation jig can be held by the bent structure portion even when the cryopreservation jig comes off from the tapered structure portion. It is possible and preferable.

FIG. 3 is a schematic top view showing another example of the fixture of the present invention. In FIG. 3, the slit portion 2 of the fixture 1 has both a bent structure portion and a tapered structure portion as a fixing structure portion for fixing the cryopreservation jig. The fixture shown in FIG. 3 has a guide portion having a taper and a tapered structure portion for fixing the cryopreservation jig. In such a case, the tapered structure satisfying the relationship (T21> T22) between the width (T21) of the tapered structure portion closest to the slit opening and the width (T22) of the tapered structure portion farthest from the slit opening. The portion is preferably located at the back side of the slit. In this case, the cryopreservation jig is firmly fixed and held, and the workability when inserting into the substantially fixed tapered structure portion is improved. Is possible and preferable.

FIG. 4 is a schematic top view showing another example of the fixture of the present invention. In FIG. 4, the fixture 1 has an insertion portion 4 for inserting a cryopreservation jig on the upper surface of the base material 5, and a slit portion 2 having an open end on the side surface of the insertion portion, and the slit portion 2 is cryopreserved. It has a tapered structure for fixing a jig. By using a fixture having such a shape, even if the fixed / held cryopreservation jig falls off from the slit opening, the cryopreservation jig can stay in the insertion portion 4. ,preferable.

FIG. 5 is a schematic top view showing another example of the fixture of the present invention. In FIG. 5, the fixture 1 has two slit openings (slit opening 3 and slit opening 3') for one slit portion 2. Further, it has a tapered structure portion in which the width of the groove of the slit portion 2 is attenuated from each slit opening 3 toward the central portion side of the base material 5. With the shape as shown in FIG. 5, since the cryopreservation jig (not shown) can be inserted from either end of the slit portion 2, a fixture capable of simultaneously storing a plurality of cryopreservation jigs can be obtained. Further, since the fixed cryopreservation jig comes into contact with the cooling solvent in a larger area than when the slit portion 2 is closed, more efficient cooling can be expected. ..

In the fixture shown in FIG. 5, the shapes of the slit openings provided at two locations (the value of (T21-T22) / T11 in the slit opening 3 and (T21'-T22') in the slit opening 3'). / T11'value) may be the same or different, but is preferably the same.

T11 shown in FIGS. 1 to 5 described above indicates the depth length of the tapered structure portion of the fixture 1. The length of T11 is not particularly limited as long as it can be held after inserting the cryopreservation jig, but when cells or tissues are frozen and thawed by the above-mentioned cryotop method, T11 is preferably 3 to 50 mm. More preferably, it is 5 to 40 mm.

Further, the width of the tapered structure portion closest to the slit opening shown in FIGS. 1 to 5 (the width of the tapered structure portion that substantially fixes the cryopreservation jig and is closest to the slit opening). The length of T21 is not particularly limited as long as it can be fixed and held by inserting a cryopreservation jig, but is preferably 1 to 10 mm, more preferably 2 to 5 mm.

When the fixture 1 has a bent structure portion as described above with reference to FIGS. 2 and 3, the sum of the length T1A until the slit portion 2 bends and the length T1B after bending is the slit. The depth T1 of the portion. At this time, the length T1A until the slit portion 2 is bent is the length from the surface of the base material 5 to the center position of the slit portion 2 at the bending point, and the length T1B after bending is the length at the bending point. It is the length from the center position of the slit portion 2 to the innermost part of the tapered structure portion. In the present invention, the depth T1 of the slit portion is preferably 3 to 150 mm, more preferably 5 to 100 mm when the cells or tissues are frozen and thawed by the cryotop method described above.

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

The fixture of the present invention is suitable for a series of processes from cryopreservation to thawing of cells or tissues, particularly in a cold storage operation once removed from a liquid nitrogen tank, and a thawing operation when thawing cells or tissues. Can be used. Generally, when cells or tissues are stored 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 Documents 7 and 8. As a series of processes from cryopreservation to thawing, first, equilibrated cells or tissues are dropped and adhered to a strip of a cryopreservation jig together with a preservation solution, and then cells or tissues are adhered using liquid nitrogen. Cool and freeze. Secondly, a cold insulation operation is performed in which the cells or tissues subjected to the freezing operation are stored for a long period of time while maintaining the cryogenic environment. Third, the frozen cells or tissues are transferred from an extremely low temperature environment into a so-called thaw solution, and a thaw operation is performed in which the cells or tissues are thawed and thawed in the thaw solution. The fixture of the present invention can be suitably used for fixing and holding the cryopreservation jig in liquid nitrogen in the above-mentioned cold insulation operation and thawing operation.

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

In the closed cryopreservation-thaw process, cells or tissues are dropped and adhered to the strip together with the preservation solution during the freezing operation, and then cryopreserved by a tubular storage member whose ends are completely closed. Enclose the jig and block it from the outside world. Alternatively, after the cells or tissues are dropped and adhered to the strip together with the preservation solution, the strip is covered with a cap member to block it 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 the preservative solution around the cells or tissues are cooled through the tubular storage member or cap member, and the vitrified state is maintained.

In the thaw operation in the closed cryopreservation-thaw process using the fixture of the present invention, the tip of the tubular storage member or the tip of the tubular storage member described above is used prior to the operation of transferring the frozen cells or tissues to the thaw solution. The cryopreservation jig is fixed by fitting the tip of the cap member into the tapered structure of the fixture of the present invention. By fixing in this way, the cells or tissues can be fixed at a position below the liquid level of the cooling solvent, and the cells or tissues do not come into contact with liquid nitrogen and are contaminated through liquid nitrogen. There is no such thing.

After that, by removing the lid fixed to the tubular storage member located on the liquid surface of liquid nitrogen, the cryopreservation jig on which the cells or tissues are placed can be quickly taken out without touching the liquid nitrogen. , Can be immersed in a melt. Alternatively, by pulling out a cryopreservation jig having a strip on which the cells or tissues are placed from the cap member fixed by the tapered structure, the cells or tissues can be quickly taken out without touching liquid nitrogen, and the thaw liquid can be taken out. Can be immersed in. The latter method is preferable from the viewpoint of enabling quicker operation, and in particular, the cells or tissues sealed by the cap member are positioned below the liquid level of the cooling solvent, and the joint portion between the main body and the cap member is located. It is preferable to temporarily hold the cooling solvent in a state where it is located above the liquid surface because the main body portion and the cap member can be quickly separated and the mounting portion can be quickly immersed in the molten liquid.

The cells or tissues immersed in the melt by the above operation are preferably immersed in the melt within 5 minutes after the lid member is removed from the tubular storage member, more preferably within 1 minute. .. Further, when pulling out the cryopreservation jig having the strip on which the cells or tissues are placed from the cap member, it is preferable to immerse it in the thaw solution within 5 minutes after the fitting is loosened, and more preferably 1. Within minutes. If the so-called sealed state is opened for a long period of time, such as after removing the lid member from the tubular storage member or after loosening the fitting with the cap member when pulling out the cryopreservation jig, air Gases such as nitrogen and oxygen inside 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 melting liquid during the melting operation to lower the temperature of the melting liquid.

The fixture of the present invention can also be used in a so-called open 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 the open process, if the cryopreservation jig is fixed and held using the fixture of the present invention prior to the operation of transferring the frozen cells or tissues to the thaw solution, the cells or tissues can be obtained. It is preferable because it can be quickly transferred from liquid nitrogen to a melt.

FIG. 6 is a schematic view showing an example of a state in which the cryopreservation jig is fixed using the fixture of the present invention. In FIG. 6, the cryopreservation jig 6 has at least a main body 7 and a detachable cap member 8 on the main body 7. The cells 11 are placed on the strip 9 of the cryopreservation jig 6 together with a very small amount of the preservation solution 12.

The strip 9 on which the cells 11 are placed together with a very small amount of the storage solution 12 is sealed with a cap member 8, and the cells 11 placed on the strip 9 are located below the liquid level 14 of the liquid nitrogen 13. By doing so, the frozen cells 11 are kept in a frozen state. When the frozen cells 11 are thawed, the joint portion between the cap member 8 and the main body portion 7 is located above the liquid level 14 of the liquid nitrogen 13, so that the main body portion 7 is quickly pulled out from the cap member 8. It is preferable because it can be separated and the mounting portion can be quickly immersed in the melting liquid. Further, when the fixture 1 has the liquid level gauge portion 15, the operator can easily grasp the position of the liquid level 14, and the workability can be improved, which is preferable. FIG. 6 shows an example in which the upper surface of the liquid level gauge portion 15 attached to the upper surface (top surface) of the fixture 1 is used as a reference for the position of the liquid level 14. When the liquid level gauge portion 15 is used, it is easy to position the liquid level 14 above the cells 11 and to position the liquid level 14 below the joint portion between the cap member 8 and the main body portion 7.

Next, a cryopreservation jig fixed by using the fixture of the present invention will be described.

The strip 9 included in the main body 7 is preferably strip-shaped. The strip shape is preferable because it is easy to store the strip 9 in the cap member or the tubular storage member.

Examples of the strip 9 included in the cryopreservation jig in the present invention include various resin films, metal plates, glass plates, rubber plates and the like. The mounting portion may be made of one kind of material or may be made of two or more kinds of materials. Among them, the resin film is preferably used from the viewpoint of handling. Specific examples of the resin film include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, acrylic resins, epoxy resins, silicon resins, polycarbonate resins, diacetates resins, triacetate resins, polyacrylate resins, polyvinyl chloride resins, and polysulphon resins. , A resin film made of polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, cyclic polyolefin resin and the like. Further, when the total light transmittance of the strip 9 is 80% or more, the cells or tissues placed on the placement portion can be easily confirmed using a transmission microscope, which is preferable.

Further, as the strip 9, a metal plate can be preferably used 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, stainless steel and the like. The thickness of the various resin films, metal plates, glass plates, rubber plates and the like described above is preferably 10 μm to 10 mm. Further, depending on the purpose, the surfaces of various resin films, metal plates, glass plates, rubber plates and the like can be hydrophilized by an electric method such as corona discharge treatment or a chemical method, and further. Can also be roughened.

Further, as the strip 9 described above, a storage solution absorber can also be used. When a preservative solution absorber is used for the strip 9, the excess preservative solution 12 can be effectively removed, so that the freezing rate is improved. Examples of the storage liquid absorber include wire mesh, paper, and the like, which are film-like materials made of synthetic resin and have through holes. As another storage liquid absorber, a porous structure formed by using a material having a refractive index of 1.45 or less is exemplified. The porous structure can efficiently remove the preservation solution 12 existing around the cells 11. Further, under the observation of a transmission type optical microscope, the operation of placing and freezing the cells 11 and the operation of thawing after freezing can be easily and surely performed with good visibility.

The refractive index of the material of the above-mentioned porous structure 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 having a refractive index of 1.45 or less that form a porous structure include fluororesins such as polytetrafluoroethylene resin, polyvinylidene difluorolide resin, and polychlorotrifluoroethylene resin, and plastic resins such as silicon resin. Materials include metal oxide materials such as silicon dioxide, inorganic materials such as sodium fluoride, magnesium fluoride and calcium fluoride.

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

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

In the present invention, the cap member 8 or the above-mentioned tubular storage member included in the cryopreservation jig can be formed by using, for example, a material resistant to a cooling solvent such as liquid nitrogen such as various resins and metals. .. The cap member may be made of one kind of material or may be made of two or more kinds of materials. Above all, resin is preferable because a tapered structure or a thread cutting structure can be easily formed by a process such as injection molding. Specific examples of the resin include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, acrylic resins, epoxy resins, silicon resins, polycarbonate resins, diacetate resins, triacetate resins, polyacrylate resins, polyvinyl chloride resins, and polysulphon resins. Examples thereof include resins made of 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 cap member is 80% or more because the state of the mounting portion inside the cap can be easily confirmed after the cap members are assembled.

In the present invention, the main body 7 of the cryopreservation jig preferably has a grip 10 from the viewpoint of workability, in which case the grip 10 is intended for ease of grip and improvement of operability. , Preferably prismatic. The grip portion 10 is preferably a member made of a material resistant to a cooling solvent such as liquid nitrogen. As such a material, 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 glass can be preferably used. ..

When thawing cells or tissues using the fixture of the present invention, the preservation solution can be one usually used for freezing cells such as eggs and embryos, for example, the above-mentioned phosphate buffered saline. A large amount of a preservation solution containing a freezing agent (glycerol, ethylene glycol, etc.) in a physiological solution such as glycerol, or various antifreezing agents such as glycerol, ethylene glycol, DMSO (dimethylsulfoxide) (at least for the total mass of the preservation solution). A preservative solution containing 10% by mass or more, more preferably 20% by mass or more) can be used. As the thawing solution, one usually used for thawing cells such as eggs and embryos can be used. For example, 1 M of a physiological solution such as the above-mentioned phosphate buffered saline is used for osmotic pressure adjustment. A melt containing sucrose can be used.

In the present invention, the cells to be cryopreserved and thawed include, for example, germ cells such as eggs, embryos, and sperms of mammals (for example, humans, cows, pigs, horses, rabbits, rats, mice, etc.); Examples thereof include pluripotent stem cells such as pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). In addition, cultured cells such as primary cultured cells, subcultured cells, and cell line cells can be mentioned. Further, in one or more embodiments, the cells include cancer-derived cells such as fibroblasts, pancreatic cancer / hepatic cancer cells, epithelial cells, vascular endothelial cells, lymphatic endothelial cells, nerve cells, cartilage cells, and tissue stem cells. And adhesive cells such as immune cells. Further, examples of tissues that can be cryopreserved and thawed include tissues composed of cells of the same species or different species, such as tissues such as ovary, skin, corneal epithelium, periodontal ligament, and myocardium.

Hereinafter, the present invention will be described in more detail with reference to Examples, and the present invention will be specifically described, but the present invention is not limited to the following Examples.

(Comparative Example 1)
The fixture of Example 1 having a tapered structure in the slit portion 2 shown in FIG. 1 was produced by metal cutting using aluminum. The size of the base material 5 is length × width × height 50 mm × 50 mm × 70 mm, and the width T2 of the slit opening 3 of the slit portion 2 is the slit portion at the position closest to the surface of the substrate in the tapered structure portion. The width is 4 mm, which is the same as the width T21, and the width T22 on the closed side is 3 mm. The length T1 of the slit portion 2 is the same as the depth T11 of the tapered structure portion, 30 mm from the side surface portion of the base material 5, and the depth of the slit portion 2 is 30 mm. The value of (T21-T22) / T11 is 0.033.

(Example 1)
Similar to Example 1, the tapered structure is the same as in Example 1 except that the width T21 of the slit portion 2 at the position closest to the surface of the base material is 3.8 mm and the width T22 on the closed side is 3 mm. The fixture of Example 2 having a portion was produced. The value of (T21-T22) / T11 is 0.027.

(Example 2)
By metal cutting using aluminum, the fixture of Example 2 having a tapered structure and a bent structure in the slit 2 shown in FIG. 2 was produced. The size of the base material 5 is length × width × height 50 mm × 50 mm × 70 mm, the width T2 of the slit portion 2 is 3.2 mm, and the depth of the slit portion 2 is 30 mm. The length of the slit portion 2 was set to go straight 30 mm from the insertion direction, then turn 90 degrees to the right and go straight 14 mm. In the tapered structure portion, the width T21 of the slit portion at the position closest to the surface of the base material is 3.2 mm, the width T22 on the closed side is 3 mm, and the depth T11 of the tapered structure portion is 10 mm. The value of (T21-T22) / T11 is 0.02.

Using the following cryopreservation jig, the cells or tissues were placed, sealed, frozen, cooled, and thawed according to the following procedure.

<Making a jig for cryopreservation>
The main body 7 and the cap member 8 of the cryopreservation jig 6 having the form shown in FIG. 6 were manufactured. The strip 9 included in the main body 7 was made of a strip-shaped (width 1.5 mm, length 20 mm, thickness 250 μm) polyethylene terephthalate resin film and attached to the grip portion 10 made of ABS resin. The cap member 8 is made of ABS resin, and the main body 7 to which the cap member 8 is fitted has a tapered structure. The opening of the cap member 8 is circular and has a diameter of 2 mm. The outer shape of the cap member 8 is a 3.1 mm quadrangular prism.

<From cell or tissue placement to sealing process, freezing process, cold insulation process>
The equilibrated mouse 8-cell stage embryo was dropped and adhered to the strip 9 of the cryopreservation jig 6 together with the preservation solution using a pipette, and after removing the excess preservation solution, under a permeation-type microscopic observation. , The strip 9 was inserted into the cap member 8 and fitted / fixed. Then, the cap member 8 side of the cryopreservation jig 1 was immersed in liquid nitrogen 13 (freezing step). The preservative solution is ethylene glycol using a commercially available Medium 199 medium (Life Technologies) containing L-glutamine, phenol red, and 25 mM HEPES (4- (2-hydraxyethyl) -1-piperazinethalphonic acid) as a basal solution. Those containing 15% by volume of volume and DMSO (dimethyl sulfoxide), 0.5 M of sucrose, and 50 mg / L of gentamicin were used. The time from the time when the mouse 8-cell stage embryo was transferred from the equilibrium solution to the preservation solution until the immersion in liquid nitrogen was 1 minute. The cryopreservation jig subjected to the freezing step was stored under liquid nitrogen until the freezing step was carried out (cold insulation step).

<Melting process>
The cryopreservation jig 6 having been subjected to each of the above steps was taken out from the liquid nitrogen, and the cryopreservation jig 6 was fixed to the slit portion 2 of the fixtures of Examples 1 and 2 and Comparative Example 1. The joint portion between the main body 7 and the cap member 8 of the cryopreservation jig was temporarily held in a state of being located above the liquid level 14 of the liquid nitrogen 13. At that time, it was easily confirmed that the cells or tissues were reliably cooled. Next, the fitting between the cap member 8 and the main body 7 was loosened, the main body 7 was separated from the cap member 8, and the strip 9 of the main body 7 was immersed in a melting liquid kept at 37 ° C. Then, the embryo recovery operation from the strip 9 was performed. It should be noted that the work from loosening the fitting of the cap member 8 to separating the main body 7 from the cap member 8 is within 10 seconds, and after separating the main body 7 from the cap member 8, the strip 9 is immersed in the melting liquid. It was possible to operate within 1 second. As the melting solution, the medium 199 medium was used as the basal solution, and a solution containing 1 M of sucrose was used.

<Evaluation of the fixing condition of the cryopreservation jig>
With respect to the fixtures of Examples 1 and 2 and Comparative Example 1, the state when the cryopreservation jig was fixed and held was evaluated according to the following criteria. These results are shown in Table 1.

◎ ◎: It was particularly excellent because it could be fixed firmly.
⊚: It was firmly fixed and had a sense of stability.
◯: Stable fixing was possible.
Δ: It could be fixed, but it was a little unstable.

From the above results, the fixture of the present invention can be used to firmly fix and hold the cryopreservation jig prior to the operation of transferring cells or tissues to the thaw solution. Further, when the cryopreservation jig is fixed and held using the fixture of the present invention, it can be easily confirmed that the cells or tissues are in a cooled state, and further, a rapid thawing operation can be performed. It turns out that it is possible.

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

1 Fixture 2 Slit part 3, 3'Slit opening 4 Insertion part 5 Base material 6 Freezing storage jig 7 Main body part 8 Cap member 9 Strip 10 Grip part 11 Cell 12 Preservation liquid 13 Liquid nitrogen 14 Liquid level 15 Liquid level Gauge part

Claims (1)

An insertion part and an insertion part for inserting a cryopreservation jig into the upper surface of the base material having a slit portion having at least one end opened in the side surface portion of the base material on the upper surface of the base material having an upper surface, a side surface and a bottom surface. A fixture for a cryopreservation jig having a slit portion with one end open on the side surface thereof, wherein the fixture has a tapered structure portion for fixing the cryopreservation jig inside the slit portion. A fixture for a cryopreservation jig, characterized in that the tapered structure part satisfies the following formula (1).
(T21-T22) /T11≤0.03 (1)
(In the formula, T21 represents the width of the tapered structure at the portion closest to the slit opening, T22 represents the width of the tapered structure at the portion farthest from the slit opening, and T11 represents the depth length of the tapered structure. Represents.)

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