Patent Application
20220361483
The present invention relates to a cryopreservation jig fixture suitable for use in thawing of frozen cells or tissues, and a freezing and thawing method that uses the fixture.
Excellent cell or tissue preservation techniques are desired in various industrial fields. For example, in the bovine embryo transfer technology, embryos are cryopreserved in advance and thawed and transferred according to the estrous cycle of a recipient cow. In the human fertility treatment, eggs or ovaries harvested from a woman's body are cryopreserved until appropriate timing for transplantation, and the cryopreserved eggs or ovaries are thawed for use in transplantation.
Generally, cells or tissues harvested from living bodies gradually become inactive and/or undergo transformation even in a culture medium. Thus, long-term culture of cells or tissues in vitro is undesirable. For this reason, techniques for long-term preservation of cells or tissues with their bioactivity maintained are essential. Excellent preservation techniques enable more accurate analysis of cells or tissues harvested. Such excellent preservation techniques also enable transplantation of cells or tissues with their bioactivity maintained at a higher level, thus likely improving the engraftment rate. The techniques also enable sequential production and preservation of artificial tissues for transplantation, such as skins cultured in vitro and what is called “cell sheets” formed in vitro, and storage thereof until needed. Therefore, such excellent preservation techniques are expected to bring great advantages not only in the medical science fields but also in the industrial fields.
One of known cell or tissue cryopreservation methods is a slow freezing method, for example. In this method, cells or tissues are immersed in a preservation solution prepared by adding a cryoprotectant to a physiological solution such as phosphate buffered saline, for example. Examples of the cryoprotectant include compounds such as glycerol, ethylene glycol, and dimethyl sulfoxide. The cells or tissues immersed in the preservation solution are cooled down to −30° C. to −35° C. at a relatively slow cooling rate (for example, 0.3° C. to 0.5° C./min), whereby the solution inside and outside the cells or tissues is sufficiently cooled and increases its viscosity. Further cooling down the cells or tissues in the preservation solution in such a state to the temperature of liquid nitrogen (−196° C.) allows a slight amount of the solution both inside and outside (surrounding) the cells or tissues to become a solid while the amorphous state thereof is maintained, that is, to vitrify. Vitrification solidifies the solution inside and outside the cells or tissues, which substantially immobilizes the molecules. Thus, the vitrified cells or tissues can be semipermanently preserved in the liquid nitrogen.
Another known cell or tissue cryopreservation method is a vitrification method. The vitrification method is a technique using a principle that addition of a large amount of a cryoprotectant, such as glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO), to a preservation solution decreases the freezing point of the preservation solution, which reduces or prevents ice crystal formation at sub-zero temperatures. When quickly cooled in liquid nitrogen, the preservation solution can solidify without forming ice crystals. This solidification is called vitrification. The preservation solution containing a large amount of a cryoprotectant is called a vitrification solution.
The slow freezing method described above requires cooling at a relatively slow cooling rate, which prolongs the operation of cryopreservation. Disadvantageously, this method also requires a device or jig for controlling the cooling rate. In addition, the slow freezing method cannot avoid ice crystal formation in the preservation solution outside the cells or tissues, which may cause physical damage to the cells or tissues. In contrast, the vitrification method described above is a process that shortens the operation time and requires no special devices or jigs. In addition, the vitrification method provides high viability because it prevents ice crystal formation.
There have been reported various examples of cell or tissue cryopreservation by the vitrification method using various methods and various cells or tissues. For example, according to Patent Literature 1, application of the vitrification method to animal or human reproductive or somatic cells is very useful in terms of viability after cryopreservation and thawing.
The vitrification method is a technique which has been developed mainly using human reproductive cells. More recently, its application to iPS or ES cells has also been widely examined. Non-Patent Literature 1 discloses the effectiveness of the vitrification method in preservation of Drosophila embryos. Patent Literature 2 discloses the effectiveness of the vitrification method in preservation of plant culture cells and tissues. As mentioned here, the vitrification method is known to be useful for preservation of various kinds of cells and tissues.
It is known that a higher freezing rate is better for suitable vitrification. Also in a thawing step after cryopreservation, it is known that a higher freezing rate is better for reduction or prevention of ice crystal reformation in cells or tissues.
The freezing rate and the thawing rate are important factors for suitable vitrification. Of these, the thawing rate is considered to be particularly important. For example, as described in Non-Patent Literature 2, it is known that rapidly frozen cells may have a low viability, if the thawing rate is slow. According to Patent Literature 3, the viability of human induced pluripotent stem (iPS) cell-derived neurons/precursor cells after thawing is improved by thawing frozen samples by the rapid thawing method.
As a general freezing method applicable to the vitrification method, Patent Literature 4 suggests a method in which mammalian embryos or eggs are attached to an inner surface of a cryopreservation container such as a cryostraw, cryovial, or cryotube, with a vitrification solution in a minimum amount sufficient to cover these embryos or eggs, and the container is brought into contact with liquid nitrogen for rapid cooling. In a thawing method that is performed after the freezing method, the cryopreservation container stored by the above-described method is taken out from the liquid nitrogen, and one end of the container is opened to inject a diluent at 33° C. to 39° C. into the container, whereby the frozen embryos or eggs are thawed and diluted. This method is considered to be capable of preserving, thawing, and diluting mammalian embryos or eggs with high viability, without risk of infection with viruses and/or bacteria. However, it is highly difficult to attach embryos or eggs to an inner surface of a cryopreservation container such as a cryostraw, cryovial, or cryotube, and it is also difficult to confirm that the embryos or eggs are deposited in the cryopreservation container.
Patent Literature 5 discloses a cell cryopreservation tool including a cell holding member having a thermal conductive member and a tubular storage member. With this tool, the problems in the freezing and thawing method disclosed in Patent Literature 4 are solved to some degree. The cryopreservation tool disclosed in Patent Literature 5 is used in the following manner: eggs are attached to the cell holding member under a microscope; the cell holding member is housed in the tubular storage member; and the cryopreservation tool is then immersed in liquid nitrogen for vitrification. Subsequently, according to a storage method disclosed in Patent Literature 5, a lid is attached to an opening of the tubular storage member, and the tubular storage member is stored in a liquid nitrogen tank. According to Patent Literature 6, a cryopreservation jig including an egg-holding strip on which eggs are deposited with a small amount of a vitrification solution is entirely housed in a tubular storage container, and then immersed in liquid nitrogen for vitrification.
Patent Literature 7 and Patent Literature 8 each disclose a freezing method applicable to the vitrification method with fewer steps, which is called the Cryotop® method that has been used in the field of human fertility treatment. Freezing in these methods is performed using an egg cryopreservation tool including a flexible, clear, and colorless film strip as an egg-holding strip. Eggs or embryos are deposited with a very small amount of a preservation solution on the film under microscope observation, and the film with the eggs attached thereto is immersed in liquid nitrogen for vitrification. The egg-holding strip holding the frozen eggs or embryos is protected by a cap or the like, and then stored in a liquid nitrogen tank.
As a cryopreservation jig suitable for the Cryotop method, for example, Patent Literatures 9 to 11 each suggest a method of cryopreserving eggs or embryos with high viability by using a cryopreservation jig with absorbency to remove an excess preservation solution surrounding these reproductive cells.
Eggs or embryos frozen by these methods are thawed by immersing the egg-holding strip in a thawing solution kept warm, and the eggs or embryos deposited on the strip are recovered in the thawing solution.
Meanwhile, for example, Patent Literature 12 discloses a liquid nitrogen container intended to improve working efficiency in thawing in the cryopreservation method. The liquid nitrogen container includes a lid having a slit formed therein. A cryopreservation jig housed in a tubular storage member is temporarily leaned against the slit.
In this manner, the cryopreservation jig is temporarily held in a state where the cells or tissues frozen are below a liquid surface of the liquid nitrogen. Thus, for example, in the methods disclosed in Patent Literature 5 and Patent Literature 6, the lid above the liquid surface of the liquid nitrogen can be easily removed, which allows the cells or tissues to be rapidly transferred to the thawing solution, thus preventing the slowdown of the thawing rate, while in the methods disclosed in Patent Literatures 7, 8, and 9 to 11, the cells or tissues can be rapidly transferred to the thawing solution by pulling out the cryopreservation jig from the cap, thus preventing the slowdown of the thawing rate.
Patent Literature 1: JP 3044323 B
Patent Literature 2: JP 2008-5846 A
Patent Literature 3: JP 2017-104061 A
Patent Literature 4: JP 2000-189155 A
Patent Literature 5: JP 5798633 B
Patent Literature 6: WO 2019/004300
Patent Literature 7: JP 2002-315573 A
Patent Literature 8: JP 2006-271395 A
Patent Literature 9: JP 2014-183757 A
Patent Literature 10: JP 2015-142523 A
Patent Literature 11: WO 2015/064380
Patent Literature 12: U.S. Design Pat. No. D642,697
Non-Patent Literature 1: Steponkus et al., Nature 345:170-172 (1990)
Non-Patent Literature 2: “Seisaibo no toketsu ni yoru shogai to hogo no kiko” (Mechanism of damage to living cells by freezing and protection) written by Hiroshi Souzu, Kagaku to Seibutsu (Chemistry and Biology), vol. 18 (1980), no. 2. pp. 78-87, published by The Japan Society for Bioscience, Biotechnology, and Agrochemistry
However, in the method that uses the liquid nitrogen container according to Patent Literature 12, while it is possible to stably hold a cryopreservation jig when it is leaned against the slit formed in the lid, it is difficult to confirm that a deposition part of the cryopreservation jig is below the liquid surface of the liquid nitrogen and the frozen state of the cells or tissues is maintained. In addition, the length of the tubular storage member is required to be longer than the length from the bottom of the liquid nitrogen container to the lid. In such a case, taking out the body of the cell holding member from the inside of the tubular resin member is complicated, and improvement has been required in terms of rapid thawing.
There have been suggested methods of cryopreserving eggs or embryos with a small amount of a preservation solution to provide high viability, for example, by limiting the width of the film on which eggs or embryos are to be deposited (Patent Literatures 7 and 8), or by using the cryopreservation jig with absorbency to remove an excess preservation solution surrounding eggs or embryos (Patent Literatures 9 to 11). However, in these methods, air bubbles are attached to the film during thawing in which the embryos or eggs are thawed and recovered, making it difficult to distinguish the deposited embryos or eggs from the air bubbles under microscope observation. Further, the air bubbles attached to the film may be attached to the eggs or embryos, impairing their visibility, or the eggs or embryos may suddenly disappear from the microscopic field due to buoyancy of the air bubbles. These problems interfere with the recovery of the embryos or eggs.
A main object of the present invention is to provide a fixture for achieving good working efficiency in thawing in cell or tissue vitrification. More specifically, the present invention aims to provide a cryopreservation jig fixture that can stably hold a cryopreservation jig; that allows, prior to transfer of frozen cells or tissues to a thawing solution, an operator to confirm that the cells or tissues are in a cooled state; and that enables rapid transfer of the frozen cells or tissues to the thawing solution kept warm.
The present invention also aims to provide a freezing and thawing method that facilitates recovery of cells or tissues when thawing the cells or tissues after freezing, without requiring complicated work.
As a result of extensive studies to solve the above problems, the present inventors found that a cryopreservation jig fixture configured as described below (hereinafter also referred to as “the fixture of the present invention”) can solve the above problems. The present inventors also found that a method of freezing and thawing a cell or tissue described below can solve the above problems.
(1) A cryopreservation jig fixture including: a base having an upper surface, side surfaces, and a bottom surface; and either a slit on the upper surface of the base having at least one end opened at the side surface of the base or an insertion portion for inserting a cryopreservation jig on the upper surface of the base and a slit having one end opened at a lateral portion of the insertion portion, wherein the slit has a fixing structure for fixing a cryopreservation jig, and the fixing structure is at least one of the following structures (I) to (III):
(T21−T22)/T11≤0.03 formula (1)
where T21 is a width of the tapered structure at a portion closest to the slit opening, T22 is a width of the tapered structure at a portion farthest from the slit opening, and T11 is a length of the tapered structure.
The present invention can provide a fixture for achieving good working efficiency in thawing in cell or tissue vitrification. More specifically, the present invention can provide a cryopreservation jig fixture that can stably hold a cryopreservation jig; that allows, prior to transfer of frozen cells or tissues to a thawing solution, an operator to confirm that the cells or tissues are in a cooled state; and that enables rapid transfer of the frozen cells or tissues to the thawing solution kept warm.
The present invention can also provide a freezing and thawing method that facilitates recovery of cells or tissues when thawing the cells or tissues after freezing, without requiring complicated work.
The cryopreservation jig fixture of the present invention (hereinafter described as “the fixture of the present invention”) is used to fix a cell or tissue cryopreservation jig. The fixture of the present invention is also suitably used for cell or tissue cryopreservation called “vitrification method”. The term “cell” herein encompasses not only a single cell but also a biological cell population composed of multiple cells. The cell population composed of multiple cells may be a cell population composed of a single kind of cells or may be a cell population composed of multiple kinds of cells. The tissue may be composed of a single kind of cells or may be composed of multiple kinds of cells, or may contain a non-cellular substance like an extracellular matrix in addition to the cells. The fixture of the present invention can be particularly suitably used in egg or embryo cryopreservation.
The fixture of the present invention can also be referred to as a “fixture 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”, a “fixture for a cell or tissue cryopreservation tool”, or a “fixture for a cell or tissue cryopreservation device”.
Hereinafter, the fixture of the present invention is described in detail.
The fixture of the present invention is a cryopreservation jig fixture, which includes a base having an upper surface (top surface), side surfaces, and a bottom surface, and a slit on the upper surface of the base for fixing a cryopreservation jig. Alternatively, the fixture includes, on the upper surface of the base, an insertion portion into which a cryopreservation jig is inserted and a slit whose one end is opened at a lateral portion of the insertion portion. In the present invention, the fixture only needs one slit on the upper surface of the base, but may include multiple slits.
In fixing a cryopreservation jig using the fixture of the present invention, a tip of a tubular storage member of the cryopreservation jig or a tip of a cap member of the cryopreservation jig is inserted into the slit of the fixture of the present invention, and the cryopreservation jig is moved along the slit to the back of the slit. The width of the slit of the fixture of the present invention is preferably 90 to 110% relative to the width of the tip of the tubular storage member or the width of the tip of the cap member in order to achieve both handleability when moving the cryopreservation jig and fixing stability of the cryopreservation jig. The tubular storage member may be one having one end blocked, which is capable of housing a cell holding member and is formed from a cold-resistant material, as disclosed in Patent Literature 5.
The slit of the fixture of the present invention has a fixing structure for securely fixing and holding the cryopreservation jig inserted thereinto, and the slit has at least one of the following structures (I) to (III) as the fixing structure:
The following describes the irregular structure formed on at least one inner side surface of the slit. The irregular structure formed on at least one inner side surface of the slit can reliably fix a cryopreservation jig to the fixture when the cryopreservation jig has an irregular structure on its tip (the tip of the tubular storage member or the tip of the cap member), because the irregular structure of the fixture and the irregular structure of the cryopreservation jig fit into each other. Preferably, the irregular structure on at least one inner side surface of the slit has a shape of a recess and/or protrusion extending in a direction parallel to the bottom surface of the fixture.
In the fixture of the present invention, when the slit includes the irregular structure on at least one inner side surface thereof, the irregular structure may be a recessed structure and/or a protruding structure of any shape according to the shape of the tip of the cryopreservation jig to be fixed, preferably, according to the shape of the tip of the cap member. Preferably, the irregular structure of the slit of the fixture has a cross-sectional shape that is the same as the cross-sectional shape of the irregular structure of the tip of the cryopreservation jig to be fixed.
In the fixture of the present invention, when the slit includes the irregular structure on at least one inner side surface thereof, it suffices as long as the slit includes at least one irregular structure on the inner side surface thereof. In order to increase the fixing stability of the cryopreservation jig, it is more preferred that the slit includes the irregular structures on both side surfaces facing each other inside the slit. Here, the expression “both side surfaces facing each other inside” refers to both side surfaces facing each other in a schematic top view when the fixture of the present invention is viewed from the top. The irregular structures on both side surfaces may be continuously connected to each other in the back of the slit.
When the slit of the fixture of the present invention has the tapered structure as the fixing structure, the slit is configured such that the distance between the inner side surfaces of the slit gradually decreases from the slit opening into which the cryopreservation jig is inserted toward the back of the slit. When the slit of the fixture of the present invention has such a tapered structure, advantageously, the cryopreservation jig inserted can be reliably fixed to the fixture because it is fitted into the tapered structure. The tapered structure may have any shape according to the shape of the tip of the cryopreservation jig to be fixed, specifically, the shape of the tip of the tubular storage member or the tip of the cap member. It is favored that the tapered structure satisfies the following formula (1), because the resulting fixture will be particularly excellent in terms of fixing stability of the cryopreservation jig.
(T21−T22)/T11≤0.03 formula (1)
In the formula, T21 is a width of the tapered structure at a portion closest to the slit opening, T22 is a width of the tapered structure at a portion farthest from the slit opening, and T11 is a length of the tapered structure.
When the slit of the fixture of the present invention has the bent structure as the fixing structure, the slit is configured such that the slit linearly extends for a certain distance from the slit opening into which a cryopreservation jig is inserted toward the inside of the base, and then the slit bends in a direction different from the linearly extending direction. When the slit has such a bent structure, the cryopreservation jig inserted from the slit opening is further linearly inserted in a direction different from the direction which linearly extends from the opening, whereby the cryopreservation jig is fixed through the bent structure. Advantageously, this can prevent sudden slipping of the cryopreservation jig toward the opening. Preferably, the certain distance that the slit linearly extends toward the inside of the base is at least three times the external length of the cap member to be fixed (the external length of the cap member in the insertion direction).
Of these fixing structures (I) to (III) described above, preferred is the tapered structure (II), which can reliably fix a cryopreservation jig and provides excellent handleability when fixing. The bent structure (III) capable of preventing sudden slipping of a cryopreservation jig toward the opening is also preferred. Further, the fixture in which the slit has the bent structure (III) in which the slit bends in a direction different from its linearly extending direction and the tapered structure (II) located after the bend is particularly preferred because such a fixture can prevent sudden slipping of a cryopreservation jig toward the opening and can also provide both excellent fixing stability of the cryopreservation jig and excellent handleability when fixing.
The shape of the fixture of the present invention (the approximate shape of the fixture, assuming that no slit is provided in the base or no insertion portion for inserting a cryopreservation jig is provided on the upper portion of the base, and no liquid surface gauge portion (described later) is attached) may be a prism such as a triangular prism, a quadrangular prism, or a hexagonal prism. The shape may also be a partial pyramid such as a triangular pyramid with a top surface or a quadrangular pyramid with a top surface. A prism is preferred, and a quadrangular prism is more preferred. The shape can also be a cylinder or a partial cone, for example.
In the present invention, preferably, the base is formed from a material resistant to liquid nitrogen. Preferred examples of such a material include various metals such as aluminum, iron, copper, and stainless steel alloy, ABS resin, acrylic resin, polypropylene resin, polystyrene resin, polyethylene resin, fluororesin, various engineering plastics, glass, and rubber materials. Metals are particularly preferred because they have excellent durability and they can suitably fix a cryopreservation jig owing to their own heavy weight. Of these, aluminum is preferred in terms of processability. The fixture of the present invention may be made of one or more types of materials.
Preferably, the fixture of the present invention includes a liquid surface gauge portion on the upper surface or one of the side surfaces of the base, the liquid surface gauge portion extending above the upper surface of the base. The liquid surface gauge portion of the fixture of the present invention is a structure used for controlling the liquid surface of a coolant. The liquid surface gauge portion makes it possible to easily adjust the liquid surface of the coolant. In other words, when a cryopreservation jig holding a cell or tissue is fixed to the fixture of the present invention, the liquid surface gauge portion makes it possible to easily confirm that the cell or tissue deposited on the cryopreservation jig is in a cooled state.
Preferably, the liquid surface gauge portion, which is preferably included in the fixture of the present invention, has a chimney shape (a prism shape such as a quadrangular prism or a cylindrical shape) extending from the bottom of the container toward the liquid surface of the coolant. As is the case with the base described above, preferably, the liquid surface gauge portion is formed from a material resistant to liquid nitrogen.
The liquid surface gauge portion of the fixture of the present invention may have any shape as long as the liquid surface gauge portion extends above the upper surface of the base and provides a reference for controlling the liquid surface of the coolant. Yet, preferably, the liquid surface gauge is out of the liquid surface of the coolant or is slightly hidden below the liquid surface, so that an operator can easily visually check the level of the liquid surface.
When the liquid surface gauge portion of the fixture of the present invention has a height that protrudes from the liquid surface of the coolant, preferably, the liquid surface gauge portion has a marking. The marking may be in any form, but preferably, it is a line segment parallel to the liquid surface of the coolant. Since the marking provides a reference for height of the liquid surface of the coolant, the liquid surface gauge only needs to have one marking or may have two or more markings in the height direction.
In a series of processes from cell or tissue cryopreservation to thawing, the fixture of the present invention can be suitably used particularly in refrigeration of a frozen cell or tissue taken out from a tank containing a coolant (typically, liquid nitrogen), and in thawing of the cell or tissue. Generally, when a cell or tissue is preserved by the vitrification method, a cryopreservation jig including a strip is used for cryopreservation. Such cryopreservation jigs are disclosed in Patent Literatures 7 to 11 described above and in WO 2011/070973, for example.
In the series of processes from cryopreservation to thawing, first, freezing is performed in which an equilibrated cell or tissue is dropped with a preservation solution to the strip of the cryopreservation jig, and then a coolant (preferably, liquid nitrogen) is used to cool the cell or tissue. Second, the frozen cell or tissue is preserved inside a coolant tank for a long time while a cryogenic environment is maintained. Third, thawing is performed in which the frozen cell or tissue is transferred from the cryogenic environment to a solution called a thawing solution, and is thawed and defrosted in the thawing solution. The fixture of the present invention can be suitably used to fix and hold the cryopreservation jig in the refrigeration and thawing of the cell or tissue taken out from the coolant tank.
In thawing in the series of processes from cryopreservation to thawing using the fixture of the present invention, the cryopreservation jig maintained in the vitrified state in the coolant tank is taken out from the tank, and is refrigerated in a coolant using the fixture of the present invention prior to transfer of the cell or tissue to the thawing solution. Here, the cell or tissue can be rapidly transferred to the thawing solution when the cryopreservation jig is held by the fixture of the present invention such that the tip of the tubular storage member or the tip of the cap member of the cryopreservation jig (i.e., the frozen cell or tissue) is below a liquid surface of the coolant and a portion of the cryopreservation jig is above the liquid surface of the coolant. Thus, the present invention can prevent the slowdown of the thawing rate of the frozen cell or tissue.
In the series of processes from cell or tissue cryopreservation to thawing, the fixture of the present invention can be suitably used in what is called a “closed-type series of processes from cryopreservation to thawing” in which a cell or tissue does not contact a coolant.
In freezing in the closed-type series of processes from cryopreservation to thawing, after a cell or tissue is dropped with a preservation solution to the strip, the cryopreservation jig is covered by a tubular storage member whose ends are completely blocked and is thus shielded from the outside environment. Alternatively, after a cell or tissue is dropped with a preservation solution to the strip, the strip is covered by the cap member and is thus shielded from the outside environment. After the shielding, the cryopreservation jig is immersed in a coolant (preferably, liquid nitrogen), whereby the cell or tissue is cooled and frozen. In freezing, storing, and the like in the above-described closed-type series of processes, the air and the preservation solution surrounding the cell or tissue is cooled and the vitrification state is maintained via the tubular storage member or the cap member.
After the vitrification state is maintained, the lid fixed to the tubular storage member above the liquid surface of the liquid nitrogen is removed, whereby the cryopreservation jig holding the cell or tissue can be rapidly taken out without contact with the liquid nitrogen and immersed in the thawing solution. Alternatively, the cryopreservation jig including the strip holding the cell or tissue is pulled out from the cap member fixed to the fixture of the present invention, whereby the cell or tissue held on the strip can be rapidly taken out without contact with the liquid nitrogen and immersed in the thawing solution. The latter cryopreservation jig is preferred for a faster operation.
The cell or tissue to be immersed in the thawing solution by the above-described operation is immersed in the thawing solution preferably within five minutes, more preferably within one minute, after the lid is removed from the tubular storage member. When pulling out the cryopreservation jig including the strip from the cap member, the cryopreservation jig is immersed in the thawing solution preferably within five minutes, more preferably within one minute, after the fitting is loosened. When the sealed state is released for a long time (for example, when a long time has elapsed after the lid is removed from the tubular storage member or after the fitting with the cap member is loosened for pulling out the cryopreservation jig from the cap member), a gas such as nitrogen or oxygen in the air may enter the tubular storage member or the cap member, and then the gas may be cooled and liquefied. In some cases, the liquefied gas is attached to the deposition part and brought into the thawing solution, decreasing the temperature of the thawing solution. Disadvantageously, a decrease in the temperature of the thawing solution causes the slowdown of the thawing rate.
In the series of processes from cell or tissue cryopreservation to thawing, the fixture of the present invention can also be used in what is called an “open-type series of processes from cryopreservation to thawing” in which a cell or tissue contacts liquid nitrogen. Also in the open-type series of processes, when the cryopreservation jig is fixed and held using the fixture of the present invention prior to transfer of a frozen cell or tissue to the thawing solution, advantageously, the cell or tissue can be rapidly transferred from the liquid nitrogen to the thawing solution.
In the above-described method in which the cryopreservation jig is entirely housed in the tubular storage container, a cutting tool and/or an extraction tool may be required in the thawing step, or it may be complicated to take out the housed cryopreservation jig.
Because of the above reasons, a cryopreservation jig at least including a body including a deposition part and a cap member freely attachable to and detachable from the body is preferred; and a method of freezing and thawing a cell or tissue is preferred which at least includes the following steps: a sealing step of depositing a cell or tissue with a preservation solution on the deposition part of the cryopreservation jig, and sealing the deposition part with the cap member; a freezing step of cooling the sealed deposition part by a coolant to freeze the cell or tissue; a refrigerating step of maintaining the frozen state of the cell or tissue frozen in the freezing step;
and a thawing step of thawing the cell or tissue maintained in the frozen state, wherein prior to the thawing step, the cell or tissue sealed with the cap member is fixed to the fixing structure of the cryopreservation jig fixture according to any one of (1) to (5) above, and the cryopreservation jig is temporarily held in a state where the cell or tissue is below a liquid surface of the coolant and a joint portion between the body and the cap member is above the liquid surface of the coolant, and in the thawing step, the body and the cap member are separated from each other, and the deposition part of the body is immersed in a thawing solution to thaw the cell or tissue.
When the deposition part includes a preservation solution absorber, advantageously, an excess preservation solution can be effectively removed, resulting in high handleability and less preservation solution surrounding the cell or tissue. This achieves a high freezing rate and a high thawing rate.
In the freezing and thawing method of the present invention, first, a cell or tissue is deposited with a preservation solution on the deposition part of the above-described cryopreservation jig. Preferably, the operation is performed under transmission microscope observation (hereinafter also simply described as “under microscope observation”). Here, when the deposition part of the cryopreservation jig includes a preservation solution absorber, advantageously, an excess preservation solution can be effectively removed, resulting in high handleability and less preservation solution surrounding the cell or tissue. This achieves a high freezing rate and a high thawing rate.
Second, the deposition part of the body is inserted into the cap member so that the cap member is fitted and fixed. The deposition part may be inserted into the cap member under microscope observation. The deposition part inserted into the cap member is completely covered by the cap member, and the cap member is fitted and fixed to a fitting structural member of the body to complete sealing.
Then, the cap member is immersed in a coolant such as liquid nitrogen, and the cell or tissue deposited on the deposition part is cooled and frozen. Here, the deposition part covered by the cap member and completely sealed, and the cell or tissue on the deposition part are cooled without contact with a coolant.
Preferably, it takes not more than one minute from when the cell or tissue is deposited with the preservation solution on the deposition part and the deposition part is sealed to when the body sealed with the cap member in the sealing step is immersed in the coolant. More preferably, it takes not more than 30 seconds.
The cooled cell or tissue is refrigerated in a cold storage container maintained at a cryogenic temperature by a coolant or the like, while the vitrification state of the cell or tissue is maintained.
In the freezing and thawing method of the present invention, prior to the thawing step, the cryopreservation jig is temporarily held in a state in which the cell or tissue sealed with the cap member is below a liquid surface of the coolant and a joint portion of the cap member of the cryopreservation jig is above the liquid surface of the coolant. Here, preferably, the cryopreservation jig is held using a fixture having a function to fix the cap member in order to improve handleability.
In the freezing and thawing method of the present invention, prior to the thawing step, the cryopreservation jig is temporarily held in a state in which the cell or tissue sealed with the cap member of the cryopreservation jig is inside the coolant while a joint portion between the body and the cap member is outside the coolant. Subsequently, the fitting and fixing between the cap member and the body is loosened, and the cap member is then separated from the body. Then, the deposition part is immersed in a thawing solution (thawing step). These operations prior to the thawing step allow the deposition part of the body to be rapidly immersed in the thawing solution without contact with a coolant such as liquid nitrogen. Preferably, it takes not more than five minutes from when the fitting and fixing between the body and the cap member is loosened to when the deposition part is immersed in the thawing solution. More preferably, it takes not more than one minute. When the fitting and fixing is loosened and the sealed state is released for a long time, a gas such as nitrogen or oxygen may enter the cap member, and then the gas may be cooled and liquefied. Disadvantageously, the liquefied gas which is attached to the deposition part and brought into the thawing solution may decrease the temperature of the thawing solution.
The freezing and thawing method of the present invention has been described so far. Next, the cryopreservation jig used in the present invention and the fixture of the present invention are described in further detail with reference to the drawings.
In the present invention, preferably, the handle has a prism shape in order to improve grippability and handleability. Preferably, the handle is a member formed from a material resistant to coolants such as liquid nitrogen. Preferred examples of such a material include various metals such as aluminum, iron, copper, and stainless steel, ABS resin, acrylic resin, polypropylene resin, polyethylene resin, fluororesin, various engineering plastics, and glass.
The cap member 3 may not necessarily include the fixing portion 9 when the fixture of the present invention has the following (II) or (III) as the fixing structure: (II) the tapered structure in which a distance between inner side surfaces of the slit gradually decreases toward a back of the slit; or (III) the bent structure in which the slit extends linearly from a slit opening toward an inside of the base and then bends in a direction different from the linearly extending direction.
The tubular storage member and the cap member described above can be formed from materials, such as various resins and metals, which are resistant to coolants such as liquid nitrogen. The cap member may be made of one type or two or more types of materials. Particularly preferred is a resin because a tapered structure or a threaded 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 resin, epoxy resin, silicone resin, polycarbonate resin, diacetate resin, triacetate resin, polyacrylate resin, polyvinyl chloride resin, polysulfone resin, polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, and cyclic polyolefin resin. When the cap member has a total light transmittance of 80% or more, advantageously, the state of the deposition part inside the cap member can be easily checked after the cap member is fitted.
Preferably, the deposition part 4 of the body 2 described above has a strip shape. The deposition part having a strip shape can be easily covered and protected by the cap member. The shape of the deposition part 4 of the body 2 can be a flat planar sheet, a curved sheet, or a V-shaped sheet, for example. The flat planar sheet is preferred because it provides high working efficiency in depositing a cell or tissue on the deposition part.
In the present invention, examples of the deposition part of the cryopreservation jig include various resin films, metal plates, glass plates, and rubber plates. The deposition part may be made of one type or two or more types of materials. Particularly preferred is a resin film in terms of handling. Specific examples of the resin film include resin films made of polyester resins such as polyethylene terephthalate and polyethylene naphthalate, acrylic resin, epoxy resin, silicone resin, polycarbonate resin, diacetate resin, triacetate resin, polyacrylate resin, polyvinyl chloride resin, polysulfone resin, polyether sulfone resin, polyimide resin, polyamide resin, polyolefin resin, and cyclic polyolefin resin. When the deposition part has a total light transmittance of 80% or more, advantageously, the cell or tissue deposited on the deposition part can be easily checked under a transmission microscope.
In the present invention, the deposition part of the cryopreservation jig may preferably be a metal plate because it has excellent thermal conductivity and enables 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. Preferably, the various resin films, metal plates, glass plates, rubber plates, and the like described above each have a thickness of 10 μm to 10 mm. Depending on the purpose, it is possible to hydrophilize surfaces of these various resin films, metal plates, glass plates, rubber plates, and the like by an electrical method such as corona discharge treatment or a chemical method, and further to roughen these surfaces.
In the present invention, the deposition part of the cryopreservation jig can be a preservation solution absorber. When the deposition part is a preservation solution absorber, the preservation solution absorber can effectively remove an excess preservation solution, which improves handleability during deposition and freezing of a cell or tissue.
Examples of the preservation solution absorber described above include films made of wire mesh, paper, synthetic resin, or the like and having through holes. Another example of the preservation solution absorber may be a porous structure formed from a material having a refractive index of 1.45 or less. Owing to the porous structure, the preservation solution surrounding the cell or tissue can be removed. The porous structure also makes it possible to deposit and freeze a cell or tissue and thaw the cell or tissue after freezing with good visibility in an easy and reliable manner under transmission optical microscope observation.
The refractive index of the material of the porous structure described above can be measured using, for example, an Abbe's refractometer (Na light source; wavelength: 589 nm) in accordance with JIS K 0062:1992 and JIS K 7142:2014. Examples of the material having a refractive index of 1.45 or less to form the porous structure include plastic resin materials such as silicone resin and fluororesin (e.g., polytetrafluoroethylene resin, polyvinylidene difluoride resin, and polychlorotrifluoroethylene resin), metal oxide materials such as silicon dioxide, and inorganic materials such as sodium fluoride, magnesium fluoride, and calcium fluoride.
The pore size of the preservation solution absorber made of 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 can improve visibility of the cell or tissue under optical microscope observation. The thickness of the preservation solution absorber is preferably 10 to 500 μm, more preferably 25 to 150 μm. When the preservation solution absorber is a porous structure made of a plastic resin material, the pore size of the preservation solution absorber is the diameter of the largest pore measured by the bubble point test. When the preservation solution absorber is a porous structure made of a metal oxide or an inorganic material, the pore size is the average pore diameter determined by image observation of the surface and cross section of the porous structure.
The porosity of the preservation solution absorber is preferably 30% or more, more preferably 70% or more. The porosity is defined by a formula shown below. To determine the void volume V, a mercury porosimeter (name: Autopore II 9220, Micromeritics Instrument Corporation) is used to measure and process the cumulative pore volume (mL/g) of pores having a pore radius of 3 nm to 400 nm in the preservation solution absorber, and the cumulative pore volume (mL/g) is multiplied by the dry solids content (g/m2) of the preservation solution absorber, whereby the void volume V can be determined as the value per unit area (m2). The thickness T of the preservation solution absorber can be measured on a photograph of the cross section of the preservation solution absorber taken with an electron microscope.
P=(V/T)×100 (%)
P: porosity (%)
V: void volume (ml/m2)
T: thickness (μm)
T1 in
T2 in
T1 in
T2 in
T4 in
T3 in
T5 in
In the present invention, the slit 14 may include multiple irregular structures illustrated in
In
In
In
The fixture 13 in
In
When the fixture having the shape illustrated in
The length T1 of the slit 14 is the total of the length T11 of the tapered structure including the slit opening 16 and the length T11′ of the tapered structure including the slit opening 16′.
In the fixture illustrated in
The following describes in detail a method of freezing and thawing a cell or tissue using the fixture of the present invention.
In the present invention, a cell or tissue is deposited with a preservation solution on a deposition part of a cryopreservation jig at least including a body including the deposition part and a cap member freely attachable to and detachable from the body. The deposition part is sealed with the cap member. The sealed deposition part is then cooled by a coolant, and the frozen state of the frozen cell or tissue is maintained. Subsequently, the cell or tissue is thawed. For thawing of the cell or tissue, when the cryopreservation jig is temporarily held in a state in which the cell or tissue sealed with the cap member is below a liquid surface of the coolant and a joint portion between the body and the cap member is above the liquid surface of the coolant, advantageously, the body and the cap member can be quickly separated from each other, and the deposition part thus can be rapidly immersed in a thawing solution.
The deposition part 4 holding the cell 11 with a very small amount of the preservation solution 12 is sealed with the cap member 3. The cell 11 deposited on the deposition part 4 is cooled because it is below a liquid surface 19 of liquid nitrogen 18, and the frozen cell 11 is maintained in a frozen state. When thawing the frozen cell 11, the joint portion 10 between the cap member 3 and the body 2 is above the liquid surface 19 of the liquid nitrogen 18. Thus, even when the fitting and fixing between the body 2 and the cap member 3 is loosened and the fitting is released, advantageously, the body 2 can be quickly pulled out and separated from the cap member 3 while the surrounding coolant (liquid nitrogen) is prevented from directly entering the cap member 3, and the deposition part can be rapidly immersed in a thawing solution. When the fixture 13 includes the liquid surface gauge portion 21, advantageously, an operator can easily know the position of the liquid surface 19, which can improve working efficiency.
When thawing the cell or tissue using the fixture of the present invention, the preservation solution may be one commonly used for freezing cells, such as eggs and embryos. For example, the preservation solution may be the above-described preservation solution prepared by adding a cryoprotectant (e.g., glycerol or ethylene glycol) to a physiological solution such as a phosphate buffered saline, or a preservation solution containing a large amount (at least 10 mass % or more, more preferably 20 mass % or more relative to the total mass of the preservation solution) of a cryoprotectant such as glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO). The thawing solution may be one that is commonly used to thaw cells such as eggs and embryos. For example, a thawing solution prepared by adding 1 M sucrose for osmoregulation to the above-described physiological solution such as phosphate buffered saline may be used.
Examples of the cell that can be cryopreserved and thawed in the present invention include reproductive cells such as eggs, embryos, and sperms from mammals (for example, human, bovine, swine, equine, leporine, rat, and mouse); and pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells). Examples also include culture cells such as primary culture cells, subculture cells, and cell lines. In one or more embodiments, examples of the cell include adhesive cells such as fibroblasts, cancer-derived cells (e.g., pancreatic cancer cells and hepatoma cells), epithelial cells, vascular endothelial cells, lymphatic endothelial cells, neuronal cells, chondrocytes, tissue stem cells, and immune cells. Examples of the tissue that can be cryopreserved and thawed include tissues formed of homologous cells and tissues formed of heterologous cells, such as tissues of ovary, skin, corneal epithelium, periodontal ligament, and myocardium.
The present invention is specifically described in more detail below with reference to examples, but the present invention is not limited to the examples below.
In the following, deposition of a cell or tissue, sealing, freezing, refrigerating, and thawing steps were performed by the following procedure, using a cryopreservation jig.
<Production of Cryopreservation jig>
The body 2 and the cap member 3 in the forms illustrated in
An equilibrated mouse 8-cell stage embryo was dropped with a preservation solution to the deposition part 4 of the cryopreservation jig using a pipette. After an excess preservation solution was removed, the deposition part 4 was inserted into the cap member 3 under transmission microscope observation, whereby the cap member 3 was fitted and fixed (sealing step: the state illustrated in
The cryopreservation jig after the above steps was taken out, fixed in the slit of the fixture 13 made of aluminum in the form illustrated in
The series of steps of the freezing and thawing method was performed on a frozen mouse 8-cell stage embryo as in Example 1, except for applying a polyurethane adhesive to both ends (2 mm, each) of a polyethylene terephthalate resin film (width: 1.5 mm; length 20: mm; thickness: 180 μm) in the length direction and then bonding a porous resin sheet (pore size: 0.2 μm; porosity: 71%; thickness: 30 μm) made of hydrophilized polytetrafluoroethylene resin to the polyethylene terephthalate resin film to obtain the deposition part 4 of the cryopreservation jig. In the freezing and thawing method of Example 2, an excess preservation solution that was present when the embryo was dropped was automatically removed during deposition of the cell or tissue. Thus, there was no need to remove the excess preservation solution using a pipette.
A freezing and thawing method of Comparative Example 1 was performed as in Example 1, except for directly immersing the deposition part 4 in the liquid nitrogen without the cap member 3 thereon after the mouse 8-cell stage embryo was dropped to the deposition part 4 and then covering and protecting the deposition part 4 by the cap member 3 in the liquid nitrogen for freezing.
A freezing and thawing method of Comparative Example 2 was performed as in Example 2, except for directly immersing the deposition part 4 in the liquid nitrogen without the cap member 3 thereon after the mouse 8-cell stage embryo was dropped to the deposition part 4 and then covering and protecting the deposition part 4 by the cap member 3 in the liquid nitrogen for freezing.
In the thawing step of Example 1, while the joint portion 10 of the cap member 3 of the cryopreservation jig was in the liquid nitrogen (the state in which the joint portion 10 was below the liquid surface of the liquid nitrogen), the fitting between the cap member 3 and the handle 5 was loosened, and the body 2 was separated from the cap member 3. Then, the deposition part 4 of the body 2 was immersed in the thawing solution kept warm at 37° C.
Each of the cryopreservation jigs of Examples 1 and 2 and Comparative Examples 1 to 3 was subjected to evaluation of the state of air bubble attachment to the deposition part at five seconds after the deposition part was immersed in the thawing solution in the thawing step. The evaluation was performed by counting the number of air bubbles (diameter: 30 μm or more) attached to the surface of the deposition part 4 in the region from 2 mm to 7 mm from the tip (area: width of 1.5 mm×length of 5 mm). In each freezing and thawing method, the evaluation was repeated three times. Table 1 shows the results.
The above results show that the use of the freezing and thawing method of the present invention can reduce or prevent air bubble attachment to the deposition part and the cell or tissue during thawing, thus facilitating recovery of the cell or tissue. In addition, the cell or tissue can be thawed in a very short time, which is expected to lead to high viability.
In the following, each fixture in the thawing step was examined in detail.
Aluminum was subjected to metal cutting to produce a fixture of Example 3 in the form including one slit 14 with the protruding irregular structures 15 (illustrated in
Aluminum was subjected to metal cutting to produce a fixture of Example 4 in the form including one slit 14 with the protruding irregular structures 15 in which the width T2 of the slit 14 gradually decreased in the insertion direction from the slit opening (illustrated in
Aluminum was subjected to metal cutting to produce a fixture of Example 5 in the form including the protruding irregular structure 15 only on one inner side surface of the slit (illustrated in
A fixture of Example 6 was produced as in Example 3, except for processing ABS resin by a sheet lamination 3D printer.
A sheet lamination 3D printer was used with ABS resin to produce a fixture of Example 7 in the form including the bent structure and the protruding irregular structure 15 extending on the inner side surfaces of the slit (illustrated in
Aluminum was subjected to metal cutting to produce a fixture of Example 8 in the form including one slit 14 and the liquid surface gauge portion 21 having a quadrangular prism shape attached to the upper surface of the base 25 (illustrated in
Aluminum was subjected to metal cutting to produce a fixture of Example 9 in the form including the slit 14 with the tapered structure (illustrated in
A fixture of Example 10 was produced as in Example 9, except for processing polyamide resin by a 3D printer.
A polyamide resin was processed by a 3D printer to produce a fixture of Example 11 including the insertion portion 26 for inserting a cryopreservation jig on the upper surface (top surface) of the base 25 and the slit opening 16 at a lateral portion of the insertion portion 26 in the form illustrated in the schematic top view of
For each of the fixtures of Examples 3 to 11, a cryopreservation jig as illustrated in
AA: The fixing was particularly securely completed, and was particularly highly evaluated.
A: The fixing was securely completed with stability.
B: The fixing was stably completed.
C: The fixing was completed but was slightly unstable.
Aluminum was subjected to metal cutting to produce a fixture of Example 12 in which the slit 14 had the tapered structure illustrated in
Aluminum was subjected to metal cutting to produce a fixture of Example 12 in which the slit 14 had the tapered structure illustrated in
Aluminum was subjected to metal cutting to produce a fixture of Example 13 in which the slit 14 had the tapered structure and the bent structure illustrated in
For each of the fixtures of Examples 12 to 14, a cryopreservation jig as illustrated in
An equilibrated mouse 8-cell stage embryo was dropped with a preservation solution (0.1 μL) to the above-described cryopreservation jig under a transmission microscope. The preservation solution had a composition containing DMSO (15 vol %), ethylene glycol (15 vol %), and sucrose (17 mass %) in Medium 199 available from Sigma-Aldrich. Then, the strip including the deposition part was inserted into the cap member under a transmission microscope, and was subsequently immersed in liquid nitrogen for freezing. After the freezing, the cap member-side of the cryopreservation jig was fixed to the slit 14 in the liquid nitrogen, and the fixing and holding state in the form illustrated in
AA: The fixing was particularly securely completed, and was particularly highly evaluated.
A: The fixing was securely completed with stability.
B: The fixing was stably completed.
C: The fixing was completed but was slightly unstable.
Each of the fixtures of Examples 3 to 14 was used to fix and hold the cryopreservation jig in the form illustrated in
Subsequently, the working efficiency in separating the cap member from the body of the cryopreservation jig in the form illustrated in
These results show that use of the fixture of the present invention makes it possible to stably fix and hold the cryopreservation jig prior to transfer of the cell or tissue to the thawing solution. When the cryopreservation jig is fixed and held using the fixture of the present invention prior to transfer of the cell or tissue to the thawing solution, an operator can easily confirm that the cell or tissue deposited on the strip of the cryopreservation jig is below the liquid surface of the liquid nitrogen, and also can perform rapid thawing.
The present invention can be applied to cryopreservation and thawing of cells or tissues, such as cells or tissues for embryo transfer and artificial insemination of domestic animals (e.g., cattle) and other animals, and for human artificial insemination; iPS cells; ES cells; commonly used culture cells; cells or tissues, including embryos and eggs, harvested from living bodies for the purpose of examination or implantation; and cells or tissues cultured in vitro.
1 cryopreservation jig
2 body
3 cap member
4 deposition part
5 handle
6 marking
7 fitting structural member
8 fitting contact portion
9 fixing portion
10 joint portion
11 cell
12 preservation solution
13 fixture
14 slit
15 irregular structure
16 slit opening
17 hollow portion
18 liquid nitrogen
19 liquid surface
20 freezing container
21 liquid surface gauge portion
22 upper limit marking
23 lower limit marking
24 thawing solution
25 base
26 insertion portion
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-109751 | Jun 2019 | JP | national |
| 2019-191857 | Oct 2019 | JP | national |
| 2020-011785 | Jan 2020 | JP | national |
| 2020-084840 | May 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/021895 | 6/3/2020 | WO |
