JP7526460B2 - Sample cooling method, method for producing cooled material, and cooling tool - Google Patents

Description

The present invention relates to a method for cooling a sample, a method for producing a cooled object, and a cooling tool.

Conventionally, biological samples such as cells have been stored at low temperatures using liquid nitrogen or a freezer. In order to store biological samples at low temperatures, the biological samples may be cooled prior to storage, and a common cooling method has been to directly immerse a container such as a cryovial containing the biological sample in liquid nitrogen using tweezers or the like.

TOWA LAB Co., Ltd. "2-4620-01 Ice Rack(R) for 1.5mL and 0.5mL tubes IR-1" [online], [searched on July 8, 2019], Internet <URL: https://axel.as-1.co.jp/asone/d/2-4620-01/>

However, because typical cryovials have screw-type lids, when exposed to extremely low temperatures such as liquid nitrogen, the container contracts and negative pressure is created inside, allowing liquid nitrogen to enter the container through the small gap that forms between the screw and the container body. Furthermore, liquid nitrogen can be contaminated with bacteria, mycoplasma, or other types of cells, and this can adhere to biological samples, preventing them from being frozen and stored in a clean state.

In addition, various tools for handling tubes in general, called ice racks (e.g., Non-Patent Document 1), are commercially available, but these are designed for performing biological sample preparation work on ice and are not used to cool biological samples for the purpose of low-temperature preservation as described above. Even if they were used, the cooling stability and reliability would be insufficient when handling rare samples.

The present invention has been made to solve the above problems, and aims to provide a sample cooling method, a method for producing a cooled material, and a cooling tool that can cool biological samples while preventing contamination of the biological samples.

The inventors conducted intensive research to solve the above-mentioned problems, and discovered that when cooling biological samples, contamination of the biological samples can be prevented by cooling the biological samples using a pre-cooled cooling device rather than by directly immersing the container in liquid nitrogen, and thus completed the present invention.
That is, the present invention has the following aspects.

(1) A block having a recess for receiving a container for storing a biological sample,
The block is made of a metal or a material containing a metal.
A sample cooling method for cooling the biological sample, comprising the steps of:
a block cooling step of cooling the block to a temperature of −60° C. or lower; and a sample containing step of fitting a container for containing a biological sample into the recess of the block cooled to a temperature of −60° C. or lower, and placing the biological sample in the internal space of the recess.
(2) The sample cooling method according to (1), wherein the total volume of the internal space of the recess is 30 volume % or less relative to the total volume of the block, which is the sum of the parts occupied by the internal space of the recess and the material constituting the block.
(3) The sample cooling method according to (1) or (2), wherein a mass ratio of the block to a mass of an object to be cooled including the biological sample contained in the container (mass of the object to be cooled:mass of the block) is 1:10 to 1:100,000.
(4) The sample cooling method according to any one of (1) to (3), wherein the mass of the block is 300 g or more.
(5) The cooling tool further includes a lid for covering the recess, the lid being made of a metal or a material containing a metal,
A sample cooling method according to any one of (1) to (4), comprising the steps of:
a block cooling step of cooling the block to a temperature of −60° C. or lower;
A lid cooling step of cooling the lid to a temperature of −60° C. or lower;
a sample receiving step of fitting a container for receiving a biological sample into the recess of the block cooled to a temperature of -60°C or lower and placing the biological sample in an internal space of the recess; and a lid installation step of, after the sample receiving step, installing the lid, cooled to a temperature of -60°C or lower, on the block so as to cover the recess.
(6) The sample cooling method according to (5) above, wherein mating surfaces of the block and the lid of the cooling tool are in surface contact with each other.
(7) The sample cooling method according to (5) or (6), wherein when the lid is placed on the block so as to cover the recess, a lid recess is provided in the lid at a position facing the recess, into which a lid of the container for storing a biological sample is fitted.
(8) A plurality of the recesses and a plurality of the lid recesses are provided,
The sample cooling method according to (7), wherein the lid recesses are individually formed at positions facing the recesses of the block.
(9) The sample cooling method according to any one of (1) to (8), wherein the cooling rate of the object to be cooled, including the biological sample, after being accommodated in the internal space of the recess of the block, in a temperature range of 0°C to -60°C is 20°C/min or more.
(10) The sample cooling method according to any one of (1) to (9), wherein the material constituting the block has a thermal conductivity of 50 W/m·K or more.
(11) The sample cooling method according to any one of (1) to (10), further comprising vitrifying the biological sample.
(12) A method for producing a cooled biological sample, comprising cooling the biological sample by the sample cooling method according to any one of (1) to (11) above, and obtaining the cooled biological sample.
(13) A cooling tool used in the sample cooling method according to any one of (1) to (11),
a block having a recess for receiving a container for receiving a biological sample;
A cooling tool, wherein the block is made of a metal or a material containing a metal.
(14) The cooling tool according to (13), wherein a total volume of the internal space of the recess is 30 volume % or less with respect to a total volume of the block, which is the sum of the portion occupied by the internal space of the recess and the material constituting the block.
(15) The cooling tool according to (13) or (14) above, wherein the mass of the block is 300 g or more.
(16) The cooling tool according to any one of (13) to (15), further comprising a lid for covering the recess, the lid being made of a metal or a material containing a metal.
(17) The cooling tool according to (16), wherein the lid has a lid recess at a position facing the recess when the lid is placed on the block so as to cover the recess, into which a lid of the container for storing a biological sample is fitted.
(18) A plurality of the recesses and a plurality of the lid recesses are provided,
The cooling tool according to (17), wherein the lid recess is individually formed at a position facing each of the plurality of recesses of the block.
(19) The cooling tool according to any one of (16) to (18), wherein the mass of the lid body is 200 g or more.
(20) The cooling tool according to any one of (16) to (19), wherein mating surfaces of the block and the lid are in surface contact with each other.
(21) The cooling tool according to any one of (13) to (20), wherein the material constituting the block has a thermal conductivity of 50 W/m·K or more.

The present invention provides a sample cooling method, a method for producing a cooled material, and a cooling tool that can cool a biological sample while preventing contamination of the biological sample.

1A is a perspective view showing the configuration of a cooling tool according to one embodiment of the present invention, and FIG. 1B is a cross-sectional view showing the configuration of a cooling tool according to one embodiment of the present invention. 1A is a cross-sectional view showing the configuration of a cooling tool according to one embodiment of the present invention, and FIG. 1B is a top view showing the configuration of the cooling tool according to one embodiment of the present invention. 1A is a perspective view showing the configuration of a cooling tool according to one embodiment of the present invention, and FIG. 1B is a cross-sectional view showing the configuration of a cooling tool according to one embodiment of the present invention. 1A is a cross-sectional view showing the configuration of a lid provided in a cooling tool according to one embodiment of the present invention, and FIG. 1B is a top view showing the configuration of a lid provided in a cooling tool according to one embodiment of the present invention. 1 is a cross-sectional view showing a configuration of a cooling tool according to one embodiment of the present invention. 1A is a perspective view showing the configuration of a cooling tool according to one embodiment of the present invention, and FIG. 1B is a cross-sectional view showing the configuration of a cooling tool according to one embodiment of the present invention. 1A is a perspective view showing the configuration of a cooling tool according to one embodiment of the present invention, and FIG. 1B is a cross-sectional view showing the configuration of a cooling tool according to one embodiment of the present invention. 1A is a perspective view showing the configuration of a cooling tool according to one embodiment of the present invention, and FIG. 1B is a cross-sectional view showing the configuration of a cooling tool according to one embodiment of the present invention. 1A is a perspective view showing the configuration of a cooling tool according to one embodiment of the present invention, and FIG. 1B is a cross-sectional view showing the configuration of a cooling tool according to one embodiment of the present invention. 1A to 1C are diagrams illustrating an example of a procedure of a sample cooling method according to an embodiment of the present invention. 1A to 1C are diagrams illustrating an example of a procedure of a sample cooling method according to an embodiment of the present invention. 1A to 1C are diagrams illustrating an example of a procedure of a sample cooling method according to an embodiment of the present invention. 1A to 1C are diagrams illustrating an example of a procedure of a sample cooling method according to an embodiment of the present invention. FIG. 11 is a diagram showing an example of a sample accommodation step in the sample cooling method of the embodiment of the present invention. FIG. 2 is a diagram illustrating the shape of a block produced in this embodiment. 1A to 1C are diagrams illustrating the shape of a lid body produced in this embodiment.

Below, we will explain a sample cooling method, a method for producing a cooled object, and a cooling tool according to one embodiment of the present invention, with appropriate reference to the drawings.

<Cooling equipment>
The cooling device of the embodiment is a cooling device used in the sample cooling method of the embodiment, and includes a block having a recess for inserting a container for holding a biological sample, the block being made of metal or a material containing metal.
The sample cooling method of the embodiment will be described later.

First Embodiment
1 is a perspective view and a cross-sectional view showing the configuration of a cooling tool 1A of an embodiment. The cooling tool 1A includes a block 10. The block 10 is provided with a recess C into which a container for storing a biological sample is fitted. The recess C is formed in the center of the upper surface of the cylindrical block 10 and opens upward. The block 10 has a side wall portion 11 and a bottom portion 12 that surround the recess C.

In this example, the shape of the block 10 is shown as a cylinder, but the shape of the block is not particularly limited and may be a polygonal column, a sphere, or other shape. Considering the stability when the cooling tool is placed on a table or other stand and used, the block preferably has a flat bottom, and more preferably has a cylinder shape.

The block 10 is made of a metal or a material containing a metal. By making the block out of the material, the thermal conductivity of the block is increased, the cooling rate of the biological sample is improved, and the biological sample can be cooled in a good condition (for example, vitrified in terms of physical state). The faster the cooling rate of the biological sample, the better the preservation state of the biological sample tends to be.
The metal constituting the block is not particularly limited, and examples thereof include copper, aluminum, gold, silver, iron, stainless steel, and alloys containing these. The metal may be used alone or in combination of two or more types. From the viewpoints of high thermal conductivity and ease of processing and availability, copper or aluminum is preferred as the metal. From the viewpoint of increasing the thermal conductivity of the block and further improving the cooling rate of the biological sample, copper is more preferred as the metal.
The metal in the material containing a metal is not particularly limited, and examples thereof include those exemplified above as the metal. The content of the metal relative to the total mass of the material constituting the block may be 50% by mass or more, 80% by mass or more, or 90% by mass or more. As the material containing a metal, a material containing carbon fiber and a metal is preferable from the viewpoint of improving thermal conductivity.

The thermal conductivity of the material constituting the block is preferably 50 W/m K or more, more preferably 80 W/m K or more, even more preferably 200 W/m K or more, and particularly preferably 300 W/m K or more. When the thermal conductivity of the material is equal to or more than the above lower limit, the cooling rate of the biological sample is improved, and the biological sample can be cooled in good condition.
There is no particular upper limit to the thermal conductivity of the material constituting the block, and it may be, for example, 5000 W/m·K or less, 1000 W/m·K or less, 800 W/m·K or less, or 500 W/m·K or less.
An example of the numerical range of the thermal conductivity may be, for example, 50 W/m·K or more and 5000 W/m·K or less, 80 W/m·K or more and 1000 W/m·K or less, 200 W/m·K or more and 800 W/m·K or less, or 300 W/m·K or more and 500 W/m·K or less.
The thermal conductivity of the material constituting the block may be a value measured in accordance with ASTM E1530.

The mass of the block may be, for example, 4 g or more, 300 g or more, 500 g or more, 700 g or more, 1000 g or more, or 1300 g or more. The greater the mass of the block, the greater the cooling and cold storage effects of the block, and the greater the cooling efficiency and cooling stability.
There is no particular upper limit to the mass of the block, but from the viewpoint of easy handling of the block, it may be 5000 g or less, 4000 g or less, 3500 g or less, 2500 g or less, or 2000 g or less.
An example of the numerical range of the mass of the above block may be 4 g or more and 5000 g or less, 300 g or more and 4000 g or less, 500 g or more and 3500 g or less, 700 g or more and 2500 g or less, 1000 g or more and 2000 g or less, or 1300 g or more and 2000 g or less.

The term "cooling stability" here means that when the block is not cooled, the temperature of the biological sample in the internal space of the recess is unlikely to change. Excellent cooling stability improves the quality of the cooled biological sample. In addition, the cooling tool can be easily handled in the cooling method.

It is preferable that the block has a metal or a material containing a metal exposed on at least a part, and preferably the entirety, of the surface of the inner wall surface 10c of the recess of the block, which allows for good heat absorption from the biological sample and efficient cooling of the biological sample.
It is also preferable that the block has a metal or a material containing a metal exposed on at least a part or the entire surface of the sidewall and/or bottom thereof, so that liquid nitrogen or cold air in a freezer can come into contact with the exposed parts of the block, thereby efficiently cooling the block itself.

In the cooling tool of the embodiment, it is preferable that the volume of the block made of metal or a material containing metal is larger than the total volume of the internal space of the recess. With such a configuration, the cooling and cold storage effects of the block that can cool the internal space of the recess are enhanced, and the cooling efficiency and cooling stability are improved.
The conventional tools shown in Non-Patent Document 1 and the like are made by combining plate-shaped members or molding plate-shaped materials, and are designed so that the volume occupied by the material is small. In such tools, the total volume of the material is small compared to the total volume of the internal space of the recess. This is thought to be because the conventional tools do not aim to improve the cooling efficiency or cooling stability of the biological sample or to improve the cooling speed, but rather emphasize the reduction of the required material to reduce costs and the reduction of weight.

In the embodiment of the block, the space between the inner wall surface of the recess of the block and the bottom surface, and the space between the inner wall surface of the recess of the block and the side wall surface are preferably occupied by a metal or a material containing a metal.

Taking the above into consideration, in the cooling device of the embodiment, the total volume of the internal space of the recess relative to the total volume of the block is preferably 30 volume % or less, preferably 1 volume % or more and 30 volume % or less, more preferably 5 volume % or more and 25 volume % or less, and even more preferably 10 volume % or more and 23 volume % or less.
Here, the total volume of the block refers to the sum of the internal spaces of the recesses and the portions occupied by the material constituting the block. When a block has multiple recesses, the total volume of the internal spaces of the recesses refers to the sum of the internal spaces of all the recesses in one block.
In addition, the internal space of the recess can be the area surrounded by the surface that defines the opening of the recess along the upper surface of the block (the surface on which the opening of the recess is formed) and the inner wall surface of the recess.

The shape of the recess can be appropriately determined taking into consideration the shape of the container so that the container can be fitted in. For example, the opening of the recess may be circular in plan view. In order to increase the cooling efficiency of the sample in the container, it is preferable that the shape of the recess is configured to minimize the gap that may occur between the fitted container and the inner wall surface 10c of the recess, and the shape of the recess and the outer shape of the portion of the container that fits into the recess and holds the biological sample can be approximately the same.

The inner diameter L1 of the recess can be determined appropriately taking into consideration the shape and size of the container to be fitted into the recess, and may be, for example, 5 to 50 mm, 7 to 40 mm, 8 to 17 mm, or 10 to 14 mm. Here, the inner diameter of the recess can be the maximum value of the inner diameters of the recess.

2 is a cross-sectional view and a top view showing the configuration of a cooling tool 1A' according to an embodiment. In the cooling tool 1A', the shape of the recess C is formed so that the inner diameter becomes narrower in the depth direction in accordance with the shape of a container for storing a biological sample. The maximum value of the inner diameter of a recess having such a shape is L1 in FIG. 2.
Considering that a container for accommodating a biological sample is inserted into the well, the maximum value of the inner diameter of the well usually coincides with the inner diameter of the opening of the well. In such a case, the inner diameter of the well may be interpreted as the diameter of the opening of the well.

If the planar shape of the recess is not a perfect circle, the inner diameter can be calculated by converting the shape into a perfect circle based on the area.

The depth of the recess can be determined appropriately taking into consideration the shape of the container in which the biological sample is to be placed, and may be, for example, 10 to 100 mm, 15 to 50 mm, or 30 to 40 mm.

In the cooling tool of the embodiment, the volume of the internal space per recess may be appropriately determined depending on the container for storing the biological sample, and may be, for example, 0.1 to ^{100 cm3} , 1 to 50 cm3, or 2 to ¹⁰ cm3.

From the viewpoint of improving the stability of cooling the biological sample, it is preferable that the side wall portion 11 of the block has a certain degree of thickness.
The minimum value L2 of the thickness of the side wall portion may be, for example, 1 to 150 mm, 3 to 100 mm, or 6 to 10 mm.
As an example, the ratio of the inner diameter L1 of the recess to the minimum thickness L2 of the side wall portion, (L1:L2), may be 1:30 to 30:1, 1:15 to 15:1, or 1:5 to 5:1.

Similarly, from the viewpoint of improving the stability of cooling to the biological sample, it is preferable that the bottom portion 12 of the block has a certain degree of thickness.
In the cooling tool 1A' shown in FIG. 2, the bottom portion 12 is within the area indicated by the dashed line.
The minimum thickness L3 of the bottom portion may be, for example, 1 to 30 mm, 2 to 15 mm, or 3 to 7 mm.
As an example, the ratio of the inner diameter L1 of the recess to the minimum thickness L3 of the bottom, (L1:L3), may be 1:30 to 30:1, 1:15 to 15:1, or 1:5 to 5:1.

As described below, if a block has multiple recesses, the thickness of the sidewall and other numerical values can be calculated by assuming that the block is divided equally based on the distance between adjacent recesses.

The cooling device of the embodiment has a good cooling effect and cooling stability for cooling biological samples, and a sample cooling method using the device can effectively prevent contamination of biological samples.

Second Embodiment
The cooling tool of the embodiment further includes a lid for covering the recess, and the lid is preferably made of a metal or a material containing a metal.
3 is a perspective view and a cross-sectional view showing the configuration of a cooling tool 1B of the embodiment. The cooling tool 1B is the cooling tool 1A further provided with a lid 20 that covers the recess C. The lid 20 is made of metal or a material containing metal.

The rest of the configuration is the same as the cooling tool 1A described above, and detailed explanations of parts that have the same configuration as the cooling tool 1A of the first embodiment described above will be omitted.

The lid 20 is removable and has a lid recess C' for fitting the lid of the container that contains the biological sample. The lid recess C' is located at a position facing the recess C when the lid 20 is placed above the block 10 so as to cover the recess C. The lid 20 has a lid side wall portion 21 and a top plate portion 22 that surround the lid recess C'.

The cooling tool 1B is provided with a lid 20, which allows the cooling speed of the biological sample to be improved by surrounding the biological sample with the lid, and also increases the cold storage effect in the lid portion, further improving the stability of cooling, compared to a cooling tool that does not have a lid 20.

The lid 20 is preferably configured so that when placed on the block 10, at least a portion of the lid side wall 21 and the block side wall 11 come into contact. With such a configuration, heat transfer occurs between the block 10 and the lid 20, and the contribution of the cooled lid 20 to cooling the biological sample increases. In addition, because the biological sample is covered by the lid and the block, the stability of cooling can be further improved.

In the cooling tool 1B, when the lid is placed on the block 10, the mating surface 10a (surface where the opening of the recess is formed) of the block 10 with the lid 20 comes into surface contact with the mating surface 20a (surface where the opening of the lid recess is formed) of the lid 20 with the block 10. This configuration further improves the efficiency of heat transfer via the mating surfaces 10a, 20a, and further increases the contribution of the cooled lid 20 to cooling the biological sample. This further improves the cooling rate of the biological sample, allowing the biological sample to be cooled in good condition. In addition, because the biological sample is sealed by the lid and the block, the stability of cooling can be further improved.

The surface contact area between mating surface 10a and mating surface 20a may be 50% to 100% of the area of the portion of the upper surface of block 10 where recess C is not formed, 80% to 100% of the area, or 90% to 100% of the area.

The material constituting the lid and the thermal conductivity of the material constituting the lid can be those exemplified in the above block.
The material constituting the block and the material constituting the lid may be the same or different from each other.

The mass of the lid body may be, for example, 3 g or more, 100 g or more, 200 g or more, or 700 g or more. The greater the mass of the lid body, the greater the cooling and cold storage effects of the lid body, and the greater the cooling efficiency and cooling stability.
There is no particular upper limit to the mass of the lid body, but from the viewpoint of easy handling of the lid body, it may be 4000 g or less, 3000 g or less, 2000 g or less, or 1500 g or less.
An example of the numerical range of the mass of the lid body may be 3 g or more and 4000 g or less, 100 g or more and 3000 g or less, 200 g or more and 2000 g or less, or 700 g or more and 1500 g or less.

It is preferable that the lid has a metal or a material containing a metal exposed on at least a part, preferably the entirety, of the inner wall surface 20c of the lid recess. By exposing the material on the inner wall surface of the lid recess of the lid, heat absorption from the biological sample is improved, and the biological sample can be cooled efficiently.
In addition, it is preferable that the lid has a metal or a material containing a metal exposed on at least a part, preferably the entirety, of the surface of the side wall and/or top plate of the lid, so that liquid nitrogen or cold air from a freezer can come into contact with the exposed parts of the lid, thereby efficiently cooling the lid itself.

As with the above block description, it is preferable that the volume of the lid, which is made of metal or a material containing metal, is larger than the total volume of the internal space of the lid recess. This configuration enhances the cooling and cold storage effects of the lid, improving cooling efficiency and cooling stability.

In consideration of the above, in the lid of the embodiment, it is preferable that the space between the inner wall surface of the lid recess and the top plate of the lid, and the space between the inner wall surface of the lid recess and the lid side wall of the lid are occupied by metal or a material containing metal.

Taking the above into consideration, the total volume of the internal space of the lid recess relative to the total volume of the lid is preferably 30 volume % or less, preferably 1 volume % or more and 30 volume % or less, more preferably 5 volume % or more and 25 volume % or less, and even more preferably 10 volume % or more and 20 volume % or less.
Here, the total volume of the lid refers to the sum of the internal space of the lid recess and the material that constitutes the lid. When the lid has multiple lid recesses, the total volume of the internal space of the lid recesses refers to the sum of the internal spaces of all the lid recesses in one lid.
In addition, the internal space of the lid body recess can be a portion surrounded by a surface that defines the opening of the lid body recess along the upper surface of the lid body (the surface on which the opening of the lid body recess is formed) and the inner wall surface of the lid body recess.

The shape of the lid recess can be appropriately determined taking into consideration the shape of the container lid so that the container lid can be fitted into it. For example, the opening of the lid recess may be circular in plan view. In order to increase the cooling efficiency of the sample in the container, it is preferable that the shape of the lid recess is configured to minimize the gap that may occur between the fitted container lid and the inner wall surface 20c of the lid recess, and the shape of the lid recess and the outer shape of the container lid that fits into the lid recess can be approximately the same.

Generally, the outer diameter of the container lid is larger than the outer diameter of the container body, so the inner diameter of the lid recess of the lid may be larger than the inner diameter of the recess of the block.

The inner diameter L4 of the lid recess can be determined appropriately taking into consideration the shape and size of the container lid to be fitted into the lid recess, and may be, for example, 5 to 60 mm, 10 to 50 mm, or 12 to 25 mm. Here, the inner diameter of the lid recess can be the maximum value of the inner diameters of the recesses.

4 is a cross-sectional view and a top view showing the configuration of the lid of the cooling tool 1B of the embodiment. The maximum value of the inner diameter of the lid recess having such a shape is L4 in FIG.
Considering that a lid of a container for storing a biological sample is fitted into the lid recess, the maximum value of the inner diameter of the lid recess usually coincides with the inner diameter of the opening of the lid recess. In such a case, the inner diameter of the lid recess may be interpreted as the diameter of the opening of the lid recess.

If the planar shape of the lid recess is not a perfect circle, the inner diameters can be calculated by converting the shape into a perfect circle based on the area.

The depth of the lid recess can be determined appropriately taking into consideration the shape of the lid of the container in which the biological sample is stored, and may be, for example, 5 to 50 mm, 7 to 30 mm, or 10 to 20 mm.

In the cooling tool of the embodiment, the volume of the internal space per one lid recess may be determined appropriately depending on the container for storing the biological sample, and may be, for example, 0.1 to ³⁰ cm3, 0.5 to ²⁰ cm3, or 1 to ¹⁰ cm3.

From the viewpoint of improving the stability of cooling the biological sample, it is preferable that the lid side wall 21 of the lid has a certain degree of thickness.
The minimum thickness L5 of the lid side wall portion may be, for example, 1 to 150 mm, 3 to 50 mm, or 5 to 20 mm.
As an example, the ratio of the inner diameter L4 of the lid recess to the minimum value L5 of the thickness of the side wall portion (L4:L5) may be 1:30 to 30:1, 1:15 to 15:1, or 1:5 to 5:1.

Similarly, from the viewpoint of improving the stability of cooling the biological sample, it is preferable that the top plate portion 22 of the lid has a certain degree of thickness.
In the cover 20 shown in FIG. 4, the top plate portion 22 is within the area indicated by the dashed line.
The minimum value L6 of the thickness of the top plate portion may be, for example, 1 to 30 mm, 2 to 20 mm, or 3 to 15 mm.
As an example, the ratio of the inner diameter L4 of the lid recess to the minimum value L6 of the thickness of the top plate portion (L4:L6) may be 1:30 to 30:1, 1:15 to 15:1, or 1:5 to 5:1.

As described below, if the lid has multiple lid recesses, the thickness and other values of the lid side wall can be calculated assuming that the lid is divided into equal parts based on the distance between adjacent lid recesses.

According to the cooling device of the embodiment, the lid improves the cooling effect and cooling stability of the biological sample, and the sample cooling method using the lid can effectively prevent contamination of the biological sample.

Third Embodiment
5 is a cross-sectional view showing another example of a cooling tool according to an embodiment. The cooling tool 1C includes a block 100 having a rectangular prism shape, and the block 100 is provided with a plurality of recesses C for receiving containers for storing biological samples.

The rest of the configuration is the same as the cooling tool 1A described above, and detailed explanations of parts that have the same configuration as the cooling tool 1A of the first embodiment described above will be omitted.

The cooling device 1C has multiple recesses C, allowing a large number of biological samples to be cooled efficiently.

The number of recesses C per block is not particularly limited, but may be, for example, 2 to 100, 5 to 50, or 7 to 20.

Fourth Embodiment
6A and 6B are a perspective view and a cross-sectional view showing another example of the cooling tool of the embodiment. The cooling tool 1D is the cooling tool 1C further including a lid 200 for covering the recess C.
As shown in FIG. 6, the cover recess C' of the cooling tool 1D is formed across a position facing a plurality of recesses C.

The rest of the configuration is the same as the cooling tool 1C described above, and detailed explanations of parts that have the same configuration as the cooling tool 1C of the first embodiment described above will be omitted.

Next, a modification of this embodiment will be described.
7, the lid 210 has a plurality of lid recesses C', which are individually formed at positions facing the plurality of recesses C of the block 100. With this configuration, each of the lid side walls 21 arranged around each lid recess C' exerts a cooling effect on the biological sample, and the cooling efficiency for the biological sample can be improved as compared to the lid 200 of the cooling tool 1D.

The number of cover recesses C' per cover is not particularly limited, but may be, for example, 2 to 100, 5 to 50, or 7 to 20.

Fifth Embodiment
8 is a perspective view and a cross-sectional view showing another example of the cooling tool of the embodiment. The cooling tool 1F includes a block 10 having a rectangular prism shape, and a first positioning portion 41 is formed on a mating surface 10a of the block 10 with the lid body 20, and a second positioning portion 42 is formed on a mating surface 20a of the lid body 20 with the block 10. The first positioning portion 41 has a convex shape, and the second positioning portion 42 has a concave shape. When the lid body 20 is attached, the first positioning portion 41 and the second positioning portion 42 mesh with each other, preventing the block 10 and the lid body 20 from being misaligned.

Next, a modification of this embodiment will be described.
The combination of the recesses and protrusions of the positioning parts arranged on the lower and upper sides may be reversed. The positions of the positioning parts are not particularly limited.
For example, as in a cooling tool 1G shown in Fig. 9, the first positioning portion 43 may have a concave shape, and the second positioning portion 44 may have a convex shape. Furthermore, the positioning portions 43, 44 are formed along the peripheral portions of the mating surfaces 10a, 20a, respectively.

The cooling tool of the embodiment described above can be suitably used in the sample cooling method of the embodiment described below. The cooling tool of the embodiment is an excellent device that can cool biological samples with high quality and prevent contamination of the biological samples, and can easily achieve this.

<Sample cooling method>
<First embodiment of sample cooling method>
The sample cooling method of the embodiment is a method for cooling the biological sample using the cooling tool of the embodiment, and includes the following "block cooling step" and "sample containing step".
a block having a recess for receiving a container for receiving a biological sample;
The block is made of a metal or a material containing a metal.
A sample cooling method for cooling the biological sample, comprising the steps of:
a block cooling step of cooling the block to a temperature of −60° C. or lower; and a sample containing step of fitting a container for containing a biological sample into a recess of the block cooled to a temperature of −60° C. or lower, and placing the biological sample in the internal space of the recess.

Examples of cooling tools of the embodiment include those exemplified in the cooling tools of the above embodiment, and detailed explanations will be omitted here.

Figure 10 is a schematic diagram showing the steps of the sample cooling method of this embodiment. The sample cooling method of this embodiment will be explained below with reference to the figure.

(Block cooling process)
The block cooling step of the embodiment is a step of cooling the block of the cooling tool of the embodiment to a temperature of −60° C. or lower. The method of cooling the block is not particularly limited.

In this embodiment, as shown in FIG. 10(a), the bottom 12 of the block 10 of the cooling tool 1A is brought into contact with liquid nitrogen 50. In the block cooling process, it is preferable to cool the block so that the liquid nitrogen does not come into direct contact with the inner wall surface 10c of the recess. By doing so, even if the liquid nitrogen is contaminated by bacteria, mycoplasma, or other types of cells, there is no opportunity for contaminants that may be contained in the liquid nitrogen to come into contact with the container holding the biological sample, and contamination of the biological sample can be effectively prevented.

The temperature of the cooled block is −60° C. or lower, and may be −70° C. or lower, −80° C. or lower, −100° C. or lower, −120° C. or lower, or −170° C. or lower. By cooling the block to a temperature equal to or lower than the upper limit, the biological sample placed in the internal space of the recess in the subsequent sample holding step can be cooled to the same temperature.
The lower limit of the temperature of the cooled block is preferably −273° C. or higher, more preferably −200° C. or higher, and even more preferably −196° C. or higher, taking into consideration the versatility of materials suitable for low temperature resistance for the cooling means and the container in which the biological sample is stored.
An example of the numerical range of the temperature of the cooled block may be -273°C or more and -60°C or less, -273°C or more and -70°C or less, -200°C or more and -80°C or less, -200°C or more and -100°C or less, -200°C or more and -120°C or less, or -196°C or more and -170°C or less.

The cooling means for cooling the block is not particularly limited, and for example, liquid nitrogen (boiling point: -196°C), dry ice (boiling point: -78.5°C), a freezer (for example, -85°C or less, -90°C or less, -150°C or less, etc.) can be used.
In the block cooling step, it is preferable to cool the block so that the cooling means does not come into contact with the inside of the recess.
From the viewpoint of increasing the cooling rate of the biological sample and improving the preservation state of the biological sample, it is preferable to cool the block at a low temperature, and among the above cooling means, it is preferable to use liquid nitrogen. On the other hand, when importance is placed on performing the cooling operation more easily, it is preferable to use dry ice or a freezer among the above cooling means.

When the above-mentioned cooling means is used, the block cooling step of the embodiment can be exemplified by the following steps.
A block cooling step of contacting the block of the cooling tool of the embodiment with liquid nitrogen or its cold air and cooling it to a temperature of -60°C or lower.
A block cooling step of contacting the block of the cooling tool of the embodiment with dry ice or its cold air to cool it to a temperature of -60°C or lower.
A block cooling step of placing the block of the cooling tool of the embodiment in a freezer and cooling it to a temperature of −60° C. or lower.

The duration of the block cooling step may be appropriately determined so that the temperature of the block is equal to or lower than the above-specified temperature. The cooling operation may be continued even after the temperature of the block is equal to or lower than the above-specified temperature.

(Sample storage process)
Next, after the block cooling step, the sample accommodation step is carried out.
The sample accommodation step of the embodiment is a step of fitting a container for accommodating a biological sample into the recess of the block cooled to a temperature of −60° C. or lower in the cooling step, and placing the biological sample in the internal space of the recess. When the biological sample is placed in the internal space of the recess, heat is rapidly removed from the biological sample, which is at a higher temperature than the cooled block, and the biological sample is rapidly cooled.

As shown in FIG. 10(b), a container 62 containing a biological sample 60 may be inserted into the recess C (Pattern A), or as shown in FIG. 12(b)-(c), a container 62 not containing a biological sample 60 may be inserted into the recess C, and then the biological sample 60 may be inserted into the container 62 (Pattern B).

In the case of the above-mentioned pattern A, the sample accommodation step of the embodiment can be exemplified by the following steps.
A sample containing step of fitting a container containing a biological sample into a recess of the block cooled to a temperature of −60° C. or lower, and placing the biological sample in the internal space of the recess.

In the case of the above-mentioned pattern B, the sample accommodation step of the embodiment can be exemplified by the following steps.
A sample containing step of fitting a container into a recess of the block cooled to a temperature of −60° C. or lower, containing a biological sample 60 in the container, and placing the biological sample in the internal space of the recess.

In this specification, the term "biological sample" generally refers to an organism, something derived from an organism, or something collected from an organism. Biological samples include, for example, cells, cell masses, spheroids, tissues, organs, organoids, fertilized eggs, early embryos, microorganisms, etc. The species of organisms in these biological samples are not particularly limited, and examples include bacteria, fungi, animals, plants, algae, etc.

The type of cell is not particularly limited, but preferred examples include germ cells, stem cells, etc. Examples of germ cells include sperm, sperm cells, spermatocytes, egg cells, oocytes, etc. Examples of stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), tissue stem cells, etc.
Moreover, a suitable example of a biological sample for the sample cooling method of the embodiment is a pathological specimen collected from a living body.

As the "container" for storing the biological sample, any suitable one commonly used in this field may be used, with cryopreservation tubes (also called cryovials) being preferred. The container is preferably made of a low-temperature resistant material, such as polypropylene or polyethylene.

From the viewpoint of more effective cooling of the biological sample, it is preferable that the volume of the gap (see, for example, gap G in FIG. 14 ) formed between the container containing the biological sample and the inner wall surface of the recess when the container is fitted into the recess of the block is small. For example, the volume of the gap may be ⁵⁰⁰ mm3 or less, 400 ^mm3 or less, or ³⁰⁰ mm3 or less.

Considering that heat exchange occurs between the biological sample and the cooling tool during the sample storage process, in order to improve the stability of cooling, it is preferable that the mass of the block be larger than the mass of the object to be cooled, including the biological sample contained in the container.
The object to be cooled is contained within the container, and includes, in addition to the biological sample, for example, a vitrification liquid (described below) and cooling aids such as a cryoplate.
Taking the above into consideration, the mass ratio of the block to the mass of the object to be cooled, including the biological sample contained in the container (mass of the object to be cooled: mass of the block) is preferably 1:10 to 1:100,000, more preferably 1:50 to 1:50,000, and even more preferably 1:100 to 1:10,000.

As an example of the method for producing a cooled substance of the embodiment, a method for producing a cooled substance of a biological sample is exemplified, which includes cooling the biological sample by the sample cooling method of the embodiment to obtain the cooled substance of the biological sample.
As one embodiment of the present invention, there can be provided a method for producing a cooled biological sample, the method including cooling the biological sample by a sample cooling method of the embodiment to obtain a cooled biological sample.
As an example of a method for producing a cooled material of the embodiment, a method for producing a cooled material of a biological sample is exemplified, which includes, for example, using a cooling device of the embodiment, a block cooling step and a sample containing step, in which the biological sample is cooled by the sample containing step, and a cooled material of the biological sample is obtained.
The steps and cooling tools in this production method are the same as those exemplified in the sample cooling method, and detailed explanations will be omitted.

The sample cooling method of the embodiment can be suitably applied as a method for cooling a biological sample and cryopreserving the cooled biological sample.
The method for producing a cooled substance according to the embodiment can be suitably applied to a method for storing the obtained cooled substance at low temperatures.

In the sample holding step, the temperature of the biological sample before being placed in the well may be higher than that of the block in the sample holding step.
When the biological sample is cooled, the biological sample may be in an uncooled state before being placed in the well in the sample holding step. The temperature of the biological sample placed in the well in the sample holding step may be higher than 0° C., and may be, for example, 1° C. to 50° C., or 5° C. to 40° C. In normal indoor operations that do not involve cooling or heating, the temperature of the biological sample is usually the same as room temperature (15 to 30° C.).

In order to cool or freeze a biological sample (hereinafter, cells are taken as an example of a biological sample) in a high-quality state, it is important to suppress the growth of ice crystals within the biological sample (e.g., inside a cell) during the cooling process. If ice crystals grow inside a cell, this causes physical damage to the intracellular structure.
Known methods for cooling biological samples include the slow freezing method and the vitrification method. In the slow freezing method, a sample is cooled at a temperature drop rate of about 1° C. per minute (for example, 1 to 2° C. per minute), which causes ice crystals to form outside the cells and freezes and dehydrates the cells, thereby preventing the growth of ice crystals inside the cells. On the other hand, in the vitrification method, a biological sample treated with a vitrification liquid or the like is rapidly cooled using a cryogen such as liquid nitrogen, so that ice crystals are not formed and the sample is stored at a low temperature in a vitrified state. Compared to the slow freezing method, the vitrification method requires a shorter time to pass through the temperature range where ice grows, and is less likely to cause physical damage due to the formation of ice crystals.

The vitrification method and slow freezing method described above both have the advantage that the vitrified biological specimen can be made viable again by raising the temperature. The sample cooling method of the embodiment can be suitably applied to either method. The sample cooling method of the embodiment is suitable as a method for vitrifying a biological specimen by cooling the biological specimen.

When the sample cooling method of the embodiment is applied to a vitrification method for vitrifying a biological sample, for example, a composition obtained by immersing the biological sample in advance in a vitrification liquid as a cooling object used in a sample containing step can be placed in the internal space of the recess of the block cooled to a temperature of −60° C. or lower in the cooling step.
The components contained in the vitrification liquid may be any known component, such as alcohols such as ethylene glycol, propylene glycol, butylene glycol, and glycerin; sugars such as glucose, fructose, trehalose, and sucrose; dimethyl sulfoxide, polylysine, glycine betaine, proline, and fructan. Examples of the sugars include monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and polysaccharides. Examples of the vitrification liquid include, but are not limited to, those containing at least one compound selected from the group consisting of ethylene glycol, propylene glycol, butylene glycol, glycerin, dimethyl sulfoxide, polylysine, glycine betaine, proline, fructan, glucose, fructose, trehalose, and sucrose.

When the sample cooling method of the embodiment is applied to the slow freezing method, the slowly frozen biological sample can be further cooled to a lower temperature. For example, a biological sample that has been slowly frozen in advance can be placed in the internal space of the recess of the block that has been cooled to a temperature of -60°C or lower in the cooling step as the biological sample used in the sample storage step. For example, a biological sample that has been slowly frozen in a slow freezing device can be transferred to a cooling tool (placed in the internal space of the recess of the block that has been cooled to a temperature of -60°C or lower in the cooling step). By combining the slow freezing method with the sample cooling method of the embodiment, the slowly frozen biological sample can be stored at a high quality at a low temperature while preventing contamination of the biological sample.

In addition, even without using vitrification methods, etc., the very act of freezing a biological sample by rapid cooling makes it difficult for ice crystals to form, and the biological sample can be kept in a high-quality state. Therefore, the sample cooling method of the embodiment is an excellent method from the viewpoint of being able to rapidly cool a biological sample.

For example, in cases where the purpose is to analyze biomolecules contained in a biological sample, and the survival of the biological sample after heating is not an issue, it is not necessary to use the slow freezing method or vitrification method. As an example, a biological sample collected from an organism can be used as is as the biological sample used in the sample storage process, and the biological sample can be frozen to a high quality while preventing contamination.

As an indicator that the biological sample is rapidly cooled, the cooling rate of the cooling object including the biological sample in the temperature range of 0°C to -60°C after it is accommodated in the internal space of the recess of the block may be 20°C/min or more, 30°C/min or more, 40°C/min or more, 50°C/min or more, 80°C/min or more, 90°C/min or more, 100°C/min or more, 130°C/min or more, 140°C/min or more, 150°C/min or more, 160°C/min or more, 170°C/min or more, 200°C/min or more, 250°C/min or more, 280°C/min or more, or 300°C/min or more.
The upper limit of the cooling rate of the biological sample in the temperature range of 0°C to -60°C is not particularly limited, but as an example, it may be 500°C/min or less, 480°C/min or less, 460°C/min or less, 440°C/min or less, 420°C/min or less, 400°C/min or less, 380°C/min or less, 360°C/min or less, 350°C/min or less, 340°C/min or less, 330°C/min or less, or 320°C/min or less.
The upper and lower limits can be freely combined, and the numerical range of the cooling rate of the biological sample in the temperature range of 0°C to -60°C may be, for example, 20°C/min or more and 500°C/min or less, 30°C/min or more and 500°C/min or less, 40°C/min or more and 500°C/min or less, 50°C/min or more and 500°C/min or less, 80°C/min or more and 500°C/min or less, 90°C/min or more and 480°C/min or less, or 100°C/min or more and 460°C/min or less. The cooling rate may be 130°C/min or more and 440°C/min or less, 140°C/min or more and 420°C/min or less, 150°C/min or more and 400°C/min or less, 160°C/min or more and 380°C/min or less, 170°C/min or more and 360°C/min or less, 200°C/min or more and 350°C/min or less, 250°C/min or more and 340°C/min or less, 280°C/min or more and 330°C/min or less, or 300°C/min or more and 320°C/min or less. If the cooling rate is within the above range, the quality of the cooled biological sample is improved, which is preferable.
In addition, since the above cooling rate values are values during cooling, they may be treated as negative (-) values (for example, 20° C./min or more is -20° C./min or more).
The above cooling rate value can be calculated as the average value of the cooling rates in the temperature range of 0°C to -60°C.

In order to increase the cooling rate of the biological sample, the biological sample in the sample receiving step may be directly or indirectly attached or placed on a cooling aid such as a cryoplate.
An example of a form in which a biological sample is indirectly attached to or placed on a cooling aid is a form in which a biological sample is attached to or placed on a cooling aid via any processing liquid such as a vitrification liquid, culture medium, or buffer solution.
The cooling aid may be of any size so long as it can be placed in the container, but considering the use of the lid, it is preferable that the cooling aid is of a size that can be completely accommodated in the internal space of the recess. An example of the cooling aid is made of a metal or a material containing a metal. An example of the cryoplate is a plate made of a metal or a material containing a metal. Examples of the metal or material containing a metal include those exemplified as the cooling tool above. The cryoplate may have a recess on its surface for placing a biological sample thereon.

The block cooled in the block cooling step can easily be kept at a low temperature until the subsequent sample storage step is performed, so cooling the block is not essential in the sample storage step.
Not performing the cooling process of the block in the sample accommodation step has the advantage that the cooling method of the embodiment can be performed easily. In addition, not using a cooling means makes it easier to keep the cooling tool clean. For example, a pre-cooled cooling tool can be brought into an operating room without a cooling means and used to cool a specimen sample in the operating room.

On the other hand, it is preferable to cool the block during the sample storage step, since this makes it easier to maintain the temperature of the block at a lower temperature. For example, as shown in FIG. 11, the block 10 may be left in contact with liquid nitrogen 50 or its cold air even during the sample storage step. When a hotter biological sample is stored in the block, the block absorbs the heat of the biological sample, causing the temperature of the block to rise, but by cooling the block with a cooling means during the sample storage step, the cooling effect on the biological sample can be improved.

The temperature of the biological sample in the internal space of the recess in the sample holding step is equal to or higher than the temperature of the cooled block. The temperature of the biological sample may be, for example, −20° C. or lower, −50° C. or lower, −60° C. or lower, −70° C. or lower, −80° C. or lower, −100° C. or lower, −120° C. or lower, or −170° C. or lower. The lower limit of the temperature of the biological sample may be −273° C. or higher, −200° C. or higher, or −196° C. or higher, depending on the cooling means, etc.
An example of the numerical range of the temperature of the biological sample may be, for example, -273°C or more and -20°C or less, -273°C or more and -50°C or less, -200°C or more and -60°C or less, -200°C or more and -70°C or less, -196°C or more and -80°C or less, -196°C or more and -100°C or less, -196°C or more and -120°C or less, or -196°C or more and -170°C or less.
The time for performing the sample containing step may be determined as appropriate, for example, until the temperature of the biological sample reaches the above-mentioned temperature.
An example of a sample holding process according to the embodiment is a sample holding process in which a container for holding a biological sample is inserted into the recess of the block cooled to a temperature of -60°C or lower, the biological sample is placed in the internal space of the recess, and the biological sample is cooled to a temperature of -273°C or higher and -20°C or lower.

The temperature of the biological sample shown here can be exemplified as the temperature of the cooled biological sample in the method for producing a cooled substance of the embodiment.
The temperature of the biological sample shown here can be exemplified as the temperature of the cooled biological sample or the cooled biological sample that is stored at a low temperature in the sample storage step described below.

Note that the block cooled to a temperature of -60°C or lower during the sample storage process does not include the temperature of the block after the biological sample is placed inside.

(Sample preservation process)
The sample cooling method of the embodiment may further include a sample preservation step of preserving the biological sample at a temperature of −60° C. or lower after the sample accommodation step.

As a method for preserving the biological sample, a method commonly used in this field can be appropriately used. After the sample receiving step, the container containing the biological sample may be removed from the cooling device and the container containing the biological sample may be preserved. For example, the container may be preserved in liquid nitrogen in a tank filled with liquid nitrogen (liquid phase preservation), 2) in a tank filled with liquid nitrogen at the bottom so as not to come into direct contact with liquid nitrogen (gas phase preservation), or 3) in a freezer. The above gas phase preservation is preferred from the viewpoint of improving the preservation state of the biological sample.

Alternatively, after the sample receiving step, the container containing the biological sample can be stored without being removed from the cooling tool, with the container containing the biological sample remaining as a cooling tool having a block fitted into the recess. For example, 1) the cooling tool can be stored in liquid nitrogen in a tank filled with liquid nitrogen (liquid-phase storage), 2) the cooling tool can be stored in a tank whose lower part is filled with liquid nitrogen so as not to come into direct contact with liquid nitrogen (gas-phase storage), or 3) the cooling tool can be stored in a freezer, and the above-mentioned gas-phase storage is preferred from the viewpoint of improving the preservation state of the biological sample.

(Cooling equipment sterilization process)
The sample cooling method of the embodiment may further include a cooling tool sterilization step of sterilizing the cooling tool before the sample containing step.
The cooling device can be sterilized by any method commonly used in the art. For example, the cooling device can be sterilized by heating the device or by steam heating under high pressure. Examples of the high pressure steam sterilization method include a high pressure steam sterilization method in which a cooling tool is sterilized by irradiating the tool with radiation, and a radiation sterilization method in which a cooling tool is sterilized by irradiating the tool with radiation. The high pressure steam sterilization method includes an autoclave sterilization method using a so-called autoclave device (for example, 121°C, 2 atm). preferable.
For example, the cooling tool can be stored in a packaging material such as an autoclave bag and sterilized by autoclave or the like, and the packaging material can be opened just before the sample storage step, and the sample storage step can be performed. At this time, the block cooling step may be carried out while the cooling tool is still contained in the packaging material.
The sample cooling method of the embodiment preferably further includes a cooling tool sterilization step, which further reduces the chance of contamination of the biological sample.

The above steps can be performed, for example, in the following order: cooling tool sterilization step, block cooling step, sample accommodation step, and sample storage step.
It should be noted that the above-mentioned cooling tool sterilization step and sample preservation step are not essential steps in the sample cooling method of the embodiment.

The cooling rate can be easily increased by directly contacting a container holding a biological sample with a cooling means such as liquid nitrogen, but as mentioned above, there is a risk that contaminants may contaminate the biological sample via the cooling means such as liquid nitrogen.
On the other hand, according to the sample cooling method of the present embodiment, the biological sample is cooled using the cooling tool of the embodiment, so that contamination of the biological sample can be effectively prevented.
According to the sample cooling method of this embodiment, by cooling the block in advance in the block cooling process, the cooling rate of the sample in the subsequent sample storage process is improved, making it possible to rapidly cool biological samples and achieve high-quality low-temperature preservation.
The block of the cooling device of the embodiment is made of metal or a material containing metal, so it has high thermal conductivity and can cool biological samples at an excellent cooling rate without direct contact with the cooling means, making it possible to rapidly cool the biological sample and achieve high-quality low-temperature preservation.
In other words, the sample cooling method of the embodiment is an extremely excellent method that can preserve biological samples at low temperatures with high quality and prevent contamination of the biological samples, and can achieve this in a simple manner.

<Second embodiment of sample cooling method>
The sample cooling method of the embodiment is a sample cooling method for cooling a biological sample using a cooling tool of the embodiment equipped with a lid, and includes the following "block cooling step,""lid cooling step,""sample containing step," and "lid installation step."
The following are examples of sample cooling methods according to the embodiment.
The biological sample storage device includes a block having a recess for receiving a container for storing a biological sample therein, and a cover for covering the recess, the block being made of a metal or a material containing a metal,
The lid is made of a metal or a material containing a metal.
A sample cooling method for cooling the biological sample, comprising the steps of:
a block cooling step of cooling the block to a temperature of −60° C. or lower;
A lid cooling step of cooling the lid to a temperature of −60° C. or lower;
a sample receiving step of fitting a container for receiving a biological sample into the recess of the block cooled to a temperature of -60°C or lower and placing the biological sample in an internal space of the recess; and a lid installation step of, after the sample receiving step, installing the lid, cooled to a temperature of -60°C or lower, on the block so as to cover the recess.

The sample cooling method of the second embodiment is the sample cooling method of the first embodiment described above, further including a lid cooling step and a lid installation step, and detailed description of parts having the same configuration as the sample cooling method of the first embodiment described above will be omitted.

Examples of cooling tools with a lid include those exemplified in the cooling tools of the above embodiment, and detailed explanations will be omitted here.

Figure 13 is a schematic diagram showing the steps of the sample cooling method of this embodiment. The sample cooling method of this embodiment will be explained below with reference to the figure.

(Block cooling process)
The block cooling step of the embodiment may be the same as that described in the block cooling step of the first embodiment.

(Lid cooling process)
The lid cooling step of the embodiment is a step of cooling the lid of the cooling tool of the embodiment to a temperature of −60° C. or lower.
Although the cooling method of the block is not particularly limited, in this embodiment, as shown in Fig. 13(a), the lid 20 of the block 10 of the cooling tool 1B is brought into contact with liquid nitrogen 50. In the lid cooling step, it is preferable to cool the lid so that the liquid nitrogen does not come into contact with the inner wall surface 20c of the lid recess. By doing so, even if the liquid nitrogen is contaminated by bacteria, mycoplasma, other types of cells, etc., the contaminants that may be contained in the liquid nitrogen will not have an opportunity to come into contact with the lid of the container containing the biological sample, and contamination of the biological sample can be effectively prevented.

The temperature of the cooled lid may be the temperature exemplified as the temperature of the cooled block in the block cooling process.

The cooling means for cooling the lid can be the same as those exemplified as the cooling means for the blocks in the block cooling process.

In the case where the cooling means described in the block cooling step is used in the lid cooling step in the embodiment, the following steps can be exemplified.
A step of contacting the lid of the cooling tool of the embodiment with liquid nitrogen or its cold air and cooling it to a temperature of -60°C or lower.
A step of contacting the lid of the cooling tool of the embodiment with dry ice or its cold air and cooling it to a temperature of -60°C or lower.
A step of placing the lid of the cooling tool of the embodiment in a freezer and cooling it to a temperature of -60°C or lower.

The duration of the lid cooling step may be appropriately determined so that the temperature of the lid is equal to or lower than the above-specified temperature. The cooling operation may be continued even after the temperature of the lid is equal to or lower than the above-specified temperature.

(Sample storage process)
Next, after the block cooling step, the sample accommodation step is carried out (FIG. 13(b)).
The sample accommodation step of the embodiment may be the same as that described in the first embodiment.

(Lid installation process)
Next, after the block cooling step and the sample accommodation step, the lid installation step is carried out (FIG. 13(c)).
The lid installation step in the embodiment is a step of installing the lid, which has been cooled to a temperature of −60° C. or lower, on the block so as to cover the recess.

As shown in FIG. 13(c), when the lid 20 is placed on the block 10, the container 62 containing the biological sample 60 and the container lid 63 are surrounded by the cooled block 10 and lid 20, allowing the biological sample 60 to be cooled very efficiently.

Here, it is preferable that the mating surface 10a of the block 10 and the mating surface 20a of the lid 20 are in surface contact. Since the biological sample 60 is housed on the side of the block 10, the efficiency of heat transfer via the mating surfaces 10a, 20a is improved, and the contribution of the cooled lid 20 to cooling the biological sample is further increased. This further improves the cooling speed of the biological sample, allowing the biological sample to be stored at a low temperature in good condition. In addition, since the biological sample is sealed by the lid and the block, the stability of cooling can be further improved.

Note that the lid cooled to a temperature of -60°C or lower during the lid installation process does not include the temperature of the lid after it is installed on the block.

Considering that heat exchange occurs between the biological sample and the cooling device equipped with the lid during the lid installation process, in order to improve the stability of cooling, it is preferable that the total mass of the lid and block be larger than the mass of the object to be cooled, including the biological sample contained in the container.
Taking the above into consideration, the total mass ratio of the block and lid to the mass of the object to be cooled, including the biological sample contained in the container (mass of the object to be cooled:total mass of the block and lid) is preferably 1:10 to 1:200,000, preferably 1:50 to 1:70,000, and more preferably 1:100 to 1:20,000.

As an example of the method for producing a cooled substance of the embodiment, a method for producing a cooled substance of a biological sample is exemplified, which includes cooling the biological sample by the sample cooling method of the embodiment to obtain the cooled substance of the biological sample.
As an example of a method for producing a cooled material of the embodiment, a method for producing a cooled biological sample is exemplified, which uses a cooling tool of the embodiment, includes a block cooling step, a lid cooling step, a sample containing step, and a lid installation step, and includes cooling the biological sample by the sample containing step and the lid installation step, thereby obtaining a cooled biological sample.
The steps and cooling tools in this manufacturing method are the same as those exemplified in the sample cooling method, and detailed explanations will be omitted.

The sample cooling method of the present embodiment is suitably applicable to a method for cooling a biological sample and cryopreserving the cooled biological sample.
The method for producing a cooled substance according to the embodiment can be suitably applied to a method for storing the obtained cooled substance at low temperatures.

(Sample preservation process)
The sample cooling method of the embodiment may further include a sample preservation step of preserving the biological sample at a temperature of −60° C. or lower after the sample accommodation step.
The sample preservation step of the embodiment may be the same as that described in the sample accommodation step of the first embodiment. When the container containing the biological sample is not removed from the cooling tool and the cooling tool is stored with the block having the container containing the biological sample fitted into the recess, the block may be stored with a lid placed on it. By placing the lid, the cooling stability of the sample can be improved.

(Cooling equipment sterilization process)
The sample cooling method of the embodiment may further include a cooling tool sterilization step of sterilizing the cooling tool including the block and the lid before the sample containing step.
The cooling tool sterilization step of this embodiment may include operations similar to those described in the cooling tool sterilization step of the first embodiment above.
For example, when the cooling device is packed in a packaging material such as an autoclave bag, the block and the lid may be packed in the same packaging material, or each may be packed in a separate packaging material and sterilized. .

The above steps can be performed in the following order: cooling tool sterilization step, block cooling step, lid cooling step, sample accommodation step, lid installation step, and sample preservation step. The block cooling step and the lid cooling step may be performed simultaneously, or the block cooling step may be performed after the lid cooling step.
Alternatively, the steps may be performed in the following order: cooling tool sterilization step, block cooling step, sample accommodation step, lid cooling step, lid installation step, and sample storage step.
It should be noted that the above-mentioned cooling tool sterilization step and sample preservation step are not essential steps in the sample cooling method of the embodiment.

According to the sample cooling method of this embodiment, the sample is cooled from both the lid and the block, thereby improving the cooling efficiency of the biological sample in the subsequent lid installation process, and achieving high-quality cooling and low-temperature storage of the biological sample.
The lid is made of metal or a material containing metal, and therefore has high thermal conductivity, enabling high-quality cooling and low-temperature storage by rapid cooling of biological samples.
In other words, the sample cooling method of the embodiment is an extremely excellent method that can cool and cryopreserve a biological sample with high quality, prevent contamination of the biological sample, and achieve this in a simple manner.

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

1. Preparation of Blocks The blocks and lids prepared in this example were of the following two types (aluminum and copper).

FIG. 15 and Table 1 show the shapes of the blocks produced in this example and the associated numerical values.
FIG. 16 and Table 2 show the shape of the lid body produced in this example and the associated numerical values.
The aluminum block and aluminum lid made of aluminum, and the copper block and copper lid made of copper have the same shape.

1. Measurement of the cooling rate of a sample placed in a recess in a block (reagents, equipment, and devices)
The reagents, instruments and equipment used in this measurement are as follows.
Vitrification solution (PVS2) (composition: MS liquid medium containing 0.4 M sucrose, 300 g/L glycerin, 150 g/L ethylene glycol, and 150 g/L dimethyl sulfoxide)
- Cryovial (polypropylene, WHEATON, model number W985861)
- Cryoplate [aluminum, manufactured by the Genetic Resource Center of the National Agriculture and Food Research Organization, No. 3 Type, 7 mm x 37 mm x 0.5 mm, with 10 or 12 bale-shaped depressions (long diameter 2.5 mm, short diameter 1.5 mm, depth 0.75 mm) on the surface]
Sensor [sheathed thermocouple (T thermocouple Class 1, Okazaki Manufacturing Co., Ltd.), measurement data acquisition (imported into data logger (TR-V500, Keyence Corporation) every 1/100 seconds)]

[Experimental Example 1]
In the cryovial, only 50 μL of vitrification liquid (PVS2) was placed as a measurement object instead of an actual sample. A thin hole was made in the lid of the cryovial, and a sensor was inserted therein to measure the temperature change of the vitrification liquid in the cryovial.
The series of experiments were carried out in a laboratory at room temperature of 25° C. Therefore, the temperatures of the vitrification liquid and the cryovials before being placed in the block were room temperature (about 25° C.).
A polystyrene foam container was filled with liquid nitrogen (-196°C) to a depth of 1-2 cm, and the aluminum block was placed therein and brought into contact with the liquid nitrogen to cool the aluminum block to -196°C (corresponding to the block cooling step). At this time, care was taken to prevent the liquid nitrogen from entering the recessed portion of the aluminum block.
Next, while the aluminum block was still in contact with liquid nitrogen, the cryovial containing the above-mentioned measurement sample was placed in the recess of the block (corresponding to the sample containing step), the measurement sample was cooled, and its temperature change was immediately measured.

[Experimental Example 2]
A cryovial was prepared with 50 μL of vitrification liquid (PVS2) placed in it as a measurement object in place of an actual sample. A thin hole was drilled in the lid of the cryovial, and a sensor was inserted into the hole to measure the temperature change of the vitrification liquid in the cryovial.
The series of experiments were carried out in a laboratory at room temperature of 25° C. Therefore, the temperatures of the vitrification liquid and the cryovials before being placed in the block were room temperature (about 25° C.).
Liquid nitrogen (-196°C) was poured into a polystyrene foam container to a depth of 1-2 cm, and the aluminum block and aluminum lid were placed therein and brought into contact with the liquid nitrogen to cool the aluminum block and aluminum lid to -196°C (corresponding to the block cooling step and lid cooling step). At this time, care was taken to prevent liquid nitrogen from entering the recessed portion of the aluminum block and the lid recessed portion of the aluminum lid.
Next, while the aluminum block was still in contact with liquid nitrogen, the cryovial containing the above-mentioned measurement sample was placed in the recess of the block (corresponding to the sample containing step), and the above-mentioned lid body, which had been cooled to -196°C, was immediately placed on the block (corresponding to the lid body placing step), the measurement sample was cooled, and its temperature change was immediately measured.
Here, a thin hole was previously drilled in part of the top plate of the lid, a sensor was inserted through the hole, and the mating surfaces of the block and the lid were in surface contact with each other.

[Experimental Example 3]
In the above-mentioned Experimental Example 1, the above-mentioned aluminum block was cooled to -80°C using a freezer (-80°C) instead of liquid nitrogen, and then the above-mentioned test object was placed in the recess of the above-mentioned block in the freezer. Except for this, the test object was cooled in the same manner as in the above-mentioned Experimental Example 1, and its temperature change was immediately measured.

[Experimental Example 4]
In the above-mentioned Experimental Example 2, the above-mentioned aluminum block and aluminum lid were cooled to -80°C using a freezer (-80°C) instead of liquid nitrogen, and then the above-mentioned test object was placed in the recess of the above-mentioned block in the freezer, and the aluminum lid was immediately placed on the block in the freezer. Except for this, the test object was cooled and its temperature change was immediately measured in the same manner as in the above-mentioned Experimental Example 2.

[Experimental Example 5]
The test object was cooled and its temperature change was immediately measured in the same manner as in the above-mentioned Experimental Example 1, except that a copper block was used instead of the aluminum block.

[Experimental Example 6]
The test object was cooled and its temperature change was immediately measured in the same manner as in Experimental Example 2, except that a copper block and a copper lid were used instead of the aluminum block and the aluminum lid in Experimental Example 2.

[Experimental Example 7]
The test object was cooled and its temperature change was immediately measured in the same manner as in the above-mentioned Experimental Example 3, except that a copper block was used instead of the aluminum block.

[Experimental Example 8]
The test object was cooled and its temperature change was immediately measured in the same manner as in Experimental Example 4, except that a copper block and a copper lid were used instead of the aluminum block and the aluminum lid in Experimental Example 4.

[Experimental Examples 9 to 16]
In each of the above experimental examples 1 to 8, the object to be measured was cooled and its temperature change was measured immediately in the same manner as in each of the above experimental examples 1 to 8, except that a cryoplate was used instead of a vitrification liquid as the object to be measured.

[Experimental Example 17]
In the above-mentioned Experimental Example 1, the test sample was cooled and its temperature change was measured immediately in the same manner as in the above-mentioned Experimental Example 1, except that no block (nor lid) was used and the cryovial containing the test sample was directly immersed in liquid nitrogen.

[Experimental Example 18]
In the above-mentioned Experimental Example 3, the measurement object was cooled and the temperature change was immediately measured in the same manner as in the above-mentioned Experimental Example 3, except that a block (and a lid) were not used and the cryovial containing the measurement object was placed directly in a freezer (-80°C).

[Experimental Example 19]
In the above-mentioned Experimental Example 9, the test sample was cooled and its temperature change was measured immediately in the same manner as in the above-mentioned Experimental Example 9, except that no block (nor lid) was used and the cryovial containing the test sample was directly immersed in liquid nitrogen.

[Experimental Example 20]
In the above-mentioned Experimental Example 11, the test sample was cooled and the temperature change was immediately measured in the same manner as in the above-mentioned Experimental Example 11, except that a block (and a lid) were not used and the cryovial containing the test sample was placed directly in a freezer (-80°C).

[Experimental Example 21]
In the above-mentioned Experimental Example 3, except that a commercially available rack for ceramic tubes (manufactured by Sumitomo Bakelite Co., Ltd., withstands up to −80° C., made of ABS resin) was used instead of the aluminum block, the test object was cooled and the temperature change was immediately measured in the same manner as in the above-mentioned Experimental Example 3.
The above-mentioned serum tube rack comes with a thin, transparent lid (0.33 mm thick) made of PVC (polyvinyl chloride), but the results were the same whether or not this lid was used.

[Experimental Example 22]
In the above-mentioned Experimental Example 11, except that a commercially available rack for ceramic tubes (manufactured by Sumitomo Bakelite Co., Ltd., withstands up to −80° C., made of ABS resin) was used instead of the aluminum block, the test object was cooled and the temperature change was immediately measured in the same manner as in the above-mentioned Experimental Example 11.
The above-mentioned serum tube rack comes with a thin, transparent lid (0.33 mm thick) made of PVC (polyvinyl chloride), but the results were the same whether or not this lid was used.

[Experimental Example 23]
The test sample was cooled and its temperature change was immediately measured in the same manner as in Experimental Example 3, except that a commercially available paper sample rack was used instead of the aluminum block.

[Experimental Example 24]
The test sample was cooled and its temperature change was immediately measured in the same manner as in Experimental Example 11 above, except that a commercially available paper sample rack was used instead of the aluminum block.

Table 3 shows the time (seconds) required for cooling and the cooling rate (°C/min) in the temperature range of 0°C to -60°C obtained from the measurement of the temperature change of the above measured objects.

In Experiments 17 and 19, where cooling was performed using liquid nitrogen (-196°C) without using any cooling equipment, the cooling rate itself was high, but it was difficult to prevent contamination of the biological samples.

In Experimental Examples 1 to 16, which used a cooling tool (block) made of metal material, the cooling speed was improved and contamination of biological samples could be prevented compared to Experimental Examples 21 to 24, which used a sample rack made of resin or paper that did not contain metal materials.

In addition, in the experimental examples in which a lid was used in addition to the block (Experimental Examples 2, 4, 6, 8, etc.), a significant improvement in cooling rate was observed compared to experimental examples in which a lid was not used (Experimental Examples 1, 3, 5, 7, etc.).

Compared to Experimental Examples 18 and 20, in which cooling was performed in a freezer without using a cooling tool, improved cooling rates were observed in Experimental Examples 3-4, 7-8, 11-12, and 15-16, in which cooling tools made of metal materials were used.

Regarding the type of metal material, the cooling speed was faster in the experimental examples using copper cooling tools (Experimental Examples 5 to 8, etc.) than in the experimental examples using aluminum cooling tools (Experimental Examples 1 to 4, etc.). In particular, Experimental Example 6, which used liquid nitrogen (-196°C) as the cooling means and a copper cooling tool block and lid, showed a particularly excellent cooling speed.

These results show that the use of the cooling device according to the embodiment can prevent contamination of biological samples and can effectively cool objects by achieving excellent cooling speeds.

2. Verification of shoot regeneration rate from cooled garlic shoot tips [Experimental Example 25]
(1) Test materials
Garlic (A. sativum) 'white' bulbs harvested by an agricultural producer in Aomori Prefecture in June and stored at room temperature for about two months after summer dormancy were used as test materials. After washing the bulbs with tap water, they were immersed in 70% (v/v) ethanol for 5 minutes and then in antiformin with 2% (v/v) available chlorine for 10 minutes for surface sterilization. After washing twice with sterile water, the shoot tips (about 2 mm) were excised from the bulbs and placed on agar medium (pH 5.8) containing 0.3 M sucrose. The medium had a composition of 1/2MS (Murashige and Skoog, 1962), and the agar concentration was set to 8.0 g/L. After placement, the shoot tips were pre-cultured for two days at 25°C under LED lighting (55 μmol m ^-2 s ^-1 ) with a day length of 16 hours.

(2-1) Treatment of garlic shoot tips by the V cryo-plate method This treatment was carried out according to the V cryo-plate method (previously reported in “Development of a Cryopreservation Procedure Using Aluminum Cryo-plates” Yamamoto, Shin-ichi; Rafique, Tariq; Priyantha, Wickramage Saman; Fukui, Kuniaki; Matsumoto, Toshikazu; Niino, Takao, Cryoletters, Volume 32, Number 3, June 2011, pp. 256-265).
2 μL of MS liquid medium containing 0.4 M sucrose and 3% (w/v) sodium alginate solution was dropped into each of the 10 wells on the cryoplate. Next, one shoot tip pre-cultured for 2 days was placed in each well. To fix the shoot tip to the well, 0.5 mL of MS liquid medium containing 0.4 M sucrose and 100 mM calcium chloride was poured onto the cryoplate, and a solidification reaction was carried out at 25° C. for 15 minutes to form an alginate gel, and a cryoplate with the shoot tip was obtained.
Next, the cryoplate with the shoot apices was transferred to a 55 mm diameter sterile plastic petri dish containing about 15 mL of MS liquid medium (LS liquid) containing 1.0 M sucrose and 2.0 M glycerin solution, and left to stand for 30 minutes (LS treatment) at 25° C. Furthermore, the cryoplate with the shoot apices was transferred to a 55 mm diameter petri dish containing about 15 mL of vitrification liquid PVS2 (Sakai et al., 1990) containing MS liquid medium, and subjected to osmotic dehydration treatment at 25° C. for 30 minutes.

(3) Cryopreservation of garlic shoot tips A cooled material of garlic shoot tips was produced using the same block and lid as in the above Experimental Example. Specifically, a cooled material of garlic shoot tips was produced using the aluminum block and aluminum lid of the Example in the same manner as in Experimental Example 10 above, except that a cryoplate with shoot tips treated by the V cryo-plate method was used instead of the cryoplate in Experimental Example 10.

(4) Warming of garlic shoot apices and shoot regeneration For plant regeneration, 1.8 mL of MS liquid medium containing 1.0 M sucrose solution was placed in a 2 mL cryovial, and the cryoplate with the cooled garlic shoot apices obtained above was immersed in it and heated to room temperature of 25° C. It was then left for 15 minutes to wash the shoot apices and adjust the osmotic pressure. The shoot apices were removed from the cryoplate and placed on 1/2 MS agar medium for re-culture.
Re-cultivation was performed under LED lighting (55 μmol m ^-2 s ^-1 ) at 25°C with a day length of 16 hours. The agar concentration was 8.0 g/L, and 0.2 mg/L of benzyladenine was added as a plant growth regulator. The above treatment solution and medium were adjusted to pH 5.8 and sterilized in an autoclave at 121°C for 15 minutes.
The reculture results were evaluated by counting the number of shoots that had normally grown shoot apices and developed leaves 30 days after placement on the bed, and this was taken as the regeneration rate (regeneration rate (%) = number of shoot apices with confirmed grown shoots/total number of shoot apices × 100). Each experiment was repeated three times with 10 shoot apices, and the average value was shown as the regeneration rate for each experiment.

[Experimental Example 26]
(2-2) Treatment of garlic stem buds by the D cryo-plate method This treatment was carried out according to the D cryo-plate method (previously reported: “Dehydration Improves Cryopreservation of Mat Rush (Juncus decipiens Nakai) Basal Stem Buds on Cryo-plates” Niino, Takao; Yamamoto, Shin-Ichi; Fukui, Kuniaki; Martinez, Carlos Roman Castillo; Arizaga, Miriam Valle; Matsumoto, Toshikazu; Engelmann, Florent, Cryoletters, Volume 34, Number 6, November 2013, pp. 549-560).
After the LS treatment in the (2-1)V cryo-plate method of the above-mentioned Experimental Example 25, the cryo-plate was air-dried for 2 hours at a wind speed of 0.6 m/sec on a petri dish in a clean bench (manufactured by Panasonic Healthcare, MCV-131BNF-PJ) instead of immersion in the vitrification liquid. The production of the cooled material, heating, and chute regeneration were carried out in the same manner as in the above-mentioned Experimental Example 25, except that.

[Experimental Example 27]
The production of the cooled material, heating and chute regeneration were carried out in the same manner as in Experimental Example 26, except that the above block and lid were cooled to -80°C using a freezer (-80°C) instead of liquid nitrogen.

Table 5 shows the results of the shot recall rate.

The results shown in Table 5 indicate that the plant samples of Experimental Examples 25 to 27 could be regenerated at a very high shoot regeneration rate, which is comparable to the results of the conventional method of directly immersing a cryovial in liquid nitrogen.
From the above, it has been demonstrated that by using the cooling device according to the embodiment, biological samples can be cooled and cryopreserved under very good conditions that prevent contamination of the biological samples and show an extremely high recovery rate.

3. Verification of oomycete recovery rate from cooled rapeseed seeds containing oomycetes [Experimental Example 28]
(1) Test material Sterilized rapeseed seeds were placed on PDA medium, inoculated with oomycetes, and cultured for one month at 25° C. The rapeseed seeds containing oomycetes that had penetrated and proliferated inside the seeds were used as test materials.

(2) Treatment of oomycetes by vitrification This treatment was carried out according to the vitrification method previously reported (A comparison of Vitrification and Droplet vitrification procedures for the cryopreservation of in vitro grown Black chokeberry shoot tips, Acta Horticulturae 908, 325-330, October 2011).
The rapeseed seeds on which the oomycetes had grown were placed in a 55 mm diameter sterile plastic petri dish containing about 15 mL of MS liquid medium (LS liquid) containing 1.0 M sucrose and 2.0 M glycerin solution, and left to stand for 30 minutes (LS treatment) at 25° C. Further, the rapeseed seeds were transferred to a 55 mm diameter petri dish containing about 15 mL of vitrification liquid PVS2 containing MS liquid medium, and subjected to osmotic dehydration treatment for 30 minutes at 25° C. One rapeseed seed was placed in each of 10 wells on a cryoplate.

(3) Cryopreservation of oomycetes A cooled product of oomycetes was produced in the same manner as in Experimental Example 10 above, except that a cryoplate with rapeseed seeds treated by the above-mentioned vitrification method was used instead of the cryoplate in Experimental Example 10 above.

(4) Heating and regeneration of oomycetes For regeneration of oomycetes, 1.8 mL of MS liquid medium containing 1.0 M sucrose solution was placed in a 2 mL cryovial, and the cryoplate with the cooled oomycetes obtained above was immersed in it and heated to room temperature of 25°C. The mixture was then left for 15 minutes to wash the rapeseed seeds containing the oomycetes and adjust the osmotic pressure. The rapeseed seeds were then placed in MS liquid medium containing 0.5 M sucrose solution and left for 15 minutes. The rapeseed seeds were removed from the cryoplate and placed on PDA medium for re-culture.
The re-cultivation was carried out under culture conditions of 25° C. The above-mentioned treatment liquid and medium were sterilized in an autoclave at 121° C. for 15 minutes.
The reculture results were evaluated by counting the number of rapeseed seeds in which oomycetes had normally grown after 3 days of placement on the bed, and this was taken as the regeneration rate (regeneration rate (%) = number of species with confirmed grown mycelia/total number of species x 100). Each experiment was repeated three times using 10 rapeseed seeds, and the average value was shown as the regeneration rate for each experiment.

Table 4 shows the time required for cooling and the cooling rate in the temperature range of 0°C to -60°C obtained from the measurement results of the temperature change of the above measured objects.

The results shown in Table 4 show that the cooling rate of the center of the cryoplate alone (Experimental Example 10) and the rapeseed seed sample placed on the cryoplate (Experimental Example 24) are almost the same.

Table 5 shows the results of oomycete regeneration rate.

The results shown in Table 5 indicate that the oomycete sample of Experimental Example 28 could be regenerated with a very high regeneration rate. Oomycetes are difficult to preserve, and have been difficult to preserve in the past. However, this experiment showed that they can be preserved in a very good condition with a high regeneration rate.
From the above, it has been shown that by using the cooling device according to the embodiment, biological samples can be cooled and cryopreserved under very good conditions that prevent contamination of the biological samples and achieve extremely high recovery rates.

The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited to each embodiment, but is limited only by the scope of the claims.

1A, 1B, 1B', 1C, 1D, 1E, 1F, 1G...cooling tool, 10, 100...block, 10a...mating surface, 10c...inner wall surface of recess, 11...side wall, 12...bottom, 20, 200, 210...lid, 20a...mating surface, 20c...inner wall surface of lid recess, 21...lid side wall, 22...top plate, 41, 43...first positioning portion, 42, 44...second positioning portion, 50...liquid nitrogen, 60...biological sample, 62...container, 63...container lid, C...recess, C'...lid recess, G...gap

Claims (21)

a block having a recess for receiving a vessel for storing a biological sample , and a lid for covering the recess ;
The block and the lid are made of a metal or a material containing a metal.
A sample cooling method for cooling the biological sample, comprising the steps of:
a block cooling step of cooling the block to a temperature of −60° C. or lower; and a sample containing step of fitting a container for containing a biological sample into the recess of the block cooled to a temperature of −60° C. or lower, and placing the biological sample in the internal space of the recess. The method for cooling a sample according to claim 1, wherein the total volume of the internal space of the recess is 30% or less by volume relative to the total volume of the block, which is the sum of the portion occupied by the internal space of the recess and the material constituting the block. The sample cooling method according to claim 1 or 2, wherein the mass ratio of the block to the mass of the object to be cooled, including the biological sample contained in the container (mass of the object to be cooled: mass of the block) is 1:10 to 1:100,000. The sample cooling method according to any one of claims 1 to 3, wherein the mass of the block is 300 g or more. Using the cooling tool,
A method for cooling a sample according to any one of claims 1 to 4, comprising the steps of:
a block cooling step of cooling the block to a temperature of −60° C. or lower;
A lid cooling step of cooling the lid to a temperature of −60° C. or lower;
a sample receiving step of fitting a container for receiving a biological sample into the recess of the block cooled to a temperature of -60°C or lower and placing the biological sample in an internal space of the recess; and a lid installation step of, after the sample receiving step, installing the lid, cooled to a temperature of -60°C or lower, on the block so as to cover the recess. The sample cooling method according to claim 5, wherein the mating surfaces of the block and the lid of the cooling tool are in surface contact with each other. The sample cooling method according to claim 5 or 6, wherein the lid has a lid recess in a portion facing the recess when the lid is placed on the block so as to cover the recess, into which a lid of the container that contains a biological sample is fitted. A plurality of the recesses and a plurality of the lid recesses are provided,
The sample cooling method according to claim 7 , wherein the cover recesses are individually formed at positions facing the plurality of recesses of the block. The sample cooling method according to any one of claims 1 to 8, wherein the cooling rate of the object to be cooled, including the biological sample, in the temperature range of 0°C to -60°C after being placed in the internal space of the recess of the block is 20°C/min or more. The sample cooling method according to any one of claims 1 to 9, wherein the thermal conductivity of the material constituting the block is 50 W/m·K or more. The sample cooling method according to any one of claims 1 to 10, in which the biological sample is vitrified. A method for producing a cooled biological sample, comprising cooling the biological sample by the sample cooling method according to any one of claims 1 to 11, and obtaining the cooled biological sample. A cooling tool for use in the sample cooling method according to any one of claims 1 to 11,
a block having a recess for receiving a container for receiving a biological sample;
A cooling tool, wherein the block is made of a metal or a material containing a metal. The cooling tool according to claim 13, wherein the total volume of the internal space of the recess is 30% or less by volume relative to the total volume of the block, which is the sum of the portion occupied by the internal space of the recess and the material constituting the block. The cooling device according to claim 13 or 14, wherein the mass of the block is 300 g or more. The cooling tool according to any one of claims 13 to 15, further comprising a lid that covers the recess, the lid being made of a metal or a material that contains a metal. The cooling tool according to claim 16, wherein the lid has a lid recess in a portion facing the recess when the lid is placed on the block so as to cover the recess, into which a lid of the container for storing a biological sample is fitted. A plurality of the recesses and a plurality of the lid recesses are provided,
The cooling tool according to claim 17 , wherein the lid recess is formed individually for each of the positions facing the recesses of the block. The cooling device according to any one of claims 16 to 18, wherein the mass of the lid is 200 g or more. The cooling tool according to any one of claims 16 to 19, wherein the mating surfaces of the block and the lid of the cooling tool are in surface contact with each other. The cooling tool according to any one of claims 13 to 20, wherein the thermal conductivity of the material constituting the block is 50 W/m·K or more.

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