Cap, low-temperature storage container, method of preparing biological sample and method of managing biological sample

Abstract

To provide a storage container, a cap and methods of preparing a biological sample and of managing a biological sample allowing for preparing a biological sample even outside a clean bench.SOLUTION: A cap (10), to be fitted to an opening (21) of a container body (20) provided in a low-temperature storage container (1), comprises a lid portion (11) having a puncture area (15) where an injection needle can be punctured, to have low-temperature endurance.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present disclosure relates to a cap, a cryopreservation container, a biological sample preparation method, and a biological sample management method. [Background technology]

[0002] Biological samples used in regenerative medicine and the like are frozen and stored in order to maintain their physiological state. As described in Patent Document 1, a cryopreservation container for sealing and cryopreserving a biological sample, which includes a tube body and a cap, is known. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2020-150797 A Summary of the Invention [Problem to be solved by the invention]

[0004] Until now, when preparing biological samples in cryopreservation containers, the preparation was carried out in a clean bench because opening the cap could cause contamination.

[0005] An object of the present disclosure is to provide a storage container, a cap, etc. that allows preparation of a biological sample in a low-temperature storage container without opening the cap. [Means for solving the problem]

[0006] In order to solve the above problems, a cap according to one embodiment of the present disclosure is attached to an opening of a container body of a low-temperature storage container for storing biological samples, and includes a lid portion that is resistant to low temperatures and has a puncture area that can be punctured with an injection needle.

[0007] Moreover, a low-temperature storage container according to one aspect of the present disclosure includes the cap and a container body having an opening to which the cap is attached.

[0008] Furthermore, a biological sample preparation method according to one embodiment of the present disclosure includes an extraction step of extracting liquid stored inside a cryogenic storage container through a first injection needle by puncturing a cap attached to an opening of the cryogenic storage container storing a biological sample with the first injection needle, and an injection step of injecting new liquid into the cryogenic storage container through the second injection needle by puncturing the cap with the second injection needle.

[0009] Furthermore, a biological sample management method according to one embodiment of the present disclosure includes a step of storing a biological sample stored in a low-temperature storage container equipped with a first cap at a temperature lower than -40°C, and a step of replacing the first cap with a second cap that is resistant to low temperatures and has a puncture area that can be punctured with an injection needle in order to transport the biological sample. Effect of the Invention

[0010] According to one aspect of the present disclosure, biological samples can be prepared outside a clean bench. [Brief description of the drawings]

[0011] [Figure 1] 1 is a cross-sectional view showing an overview of a cryopreservation container and a biological sample preparation method according to a first embodiment of the present disclosure. [Diagram 2] 4 is a cross-sectional view showing a configuration of a second cap according to the first embodiment. FIG. [Diagram 3] 11 is a cross-sectional view showing a state in which the cap is attached to the container body and the extension portion is folded. FIG. [Figure 4] FIG. 4 is a plan view showing the upper surface of the second cap. [Diagram 5] FIG. 11 is a cross-sectional view showing the configuration of a second cap according to the second embodiment. [Figure 6] FIG. 11 is a plan view showing the configuration of a second cap according to the third embodiment. [Figure 7]FIG. 11 is a cross-sectional view showing the configuration of a second cap according to a fourth embodiment. [Figure 8] 13 is a flow chart showing a biological sample preparation method and a biological sample management method according to embodiment 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] [Embodiment 1] An embodiment of the present disclosure will be described in detail below. First, the technical concept underlying the present disclosure will be described. Until now, when cells to be used for cell transplantation were prepared, the work was performed in a sterile space such as a clean bench. Therefore, equipment for forming a sterile space such as a clean bench was required, and it was considered difficult to perform cell transplantation in a small hospital that does not have large-scale equipment.

[0013] The inventors of the present disclosure have overturned this stereotype and come up with a method for performing cell transplantation without using equipment for forming a sterile space. Figure 1 is a cross-sectional view showing an outline of a cryopreservation container and a biological sample preparation method according to a first embodiment of the present disclosure. Cells 42 to be used for cell transplantation are stored and frozen in a cryopreservation container 3 capable of long-term cryopreservation at extremely low temperatures such as in liquid nitrogen.

[0014] When the cryopreserved cells are to be transported to an external facility (e.g., another hospital) for cell transplantation, an operator removes the cap 30 of the cryopreservation container 3 from the container body 20 and instead attaches the cap 10 to the container body 20. The entire container in this state is referred to as the cryopreservation container 1.

[0015] The cryopreservation container 1 is transported to an external facility at a low temperature. After that, the liquid containing the cells inside the cryopreservation container 1 is melted, and the cells are adjusted for cell transplantation. At this time, conventionally, the cell adjustment is performed in a clean bench with the cap removed from the container body. However, the cap 10 of the present disclosure has a puncture area 15 that can be punctured and penetrated with an injection needle. Therefore, by puncturing the puncture area 15 with an injection needle, it is possible to extract the liquid inside the container body 20 and to inject the liquid into the container body 20, and the cells can be adjusted even without equipment for forming a sterile space such as a clean bench.

[0016] In this specification, cells are given as an example of a biological sample to be stored in the low-temperature storage container 1, but the biological sample to be stored in the low-temperature storage container 1 is not limited to cells, and may be nucleic acids such as DNA, or organelles such as mitochondria, and is not particularly limited.

[0017] The cryopreservation containers 1 and 3 will be described in detail below.

[0018] (Cold storage container 3) The cryopreservation container 3 is a container capable of cryopreserving cells at extremely low temperatures for long periods of time. In general, cells are preferably stored at -40°C or lower, more preferably -70°C or lower, and even more preferably -196°C or lower. The lower the temperature, the longer the cells can be stored. The cryopreservation container 3 comprises a container body 20 and a cap 30 (first cap). The cryopreservation container 3 is sterilized in advance.

[0019] The container body 20 is a container capable of storing a biological sample. As shown in FIG. 1, the container body 20 has an opening 21 to which the cap 30 and the cap 10 can be attached. The container body 20 has a screw-fitting portion 22 that screws onto the cap 30 on the outer wall surface on the bottom side of the opening 21. In this embodiment, the container body 20 is a cylindrical container. The container body 20 stores a liquid 41 and cells 42. In this case, the liquid 41 is preferably a cell freezing liquid. For convenience, the liquid 41 and the cells 42 are described in a state of being separated into layers.

[0020] The container body 20 is preferably made of resin. For example, the material of the container body 20 may be a thermoplastic resin such as polyethylene resin, polypropylene resin, acrylonitrile-butadiene-styrene copolymer resin, polycarbonate resin, polyethylene terephthalate resin, or PFA (perfluoroalkoxy fluororesin). The resin container body 20 can realize a low-temperature storage container that is less likely to break compared to glass products. Since the toughness of glass products decreases at lower temperatures, if the container body 20 is made of glass, the risk of the container body 20 breaking during the process of transporting the low-temperature storage container 3 increases. On the other hand, since resin products have toughness even at low temperatures, the risk of breakage during transportation can be reduced by making the container body 20 of resin. The shape of the container body 20 is not particularly limited as long as the cap 30 can be attached and detached.

[0021] The cap 30 is a cap that seals the opening 21 of the container body 20. The cap 30 may have a structure that allows it to be fastened to the screw portion 22. The cap 30 may be made of resin, or may be made of the same material as the container body 20.

[0022] (Cold storage container 1) The cryopreservation container 1 is a container that can freeze and preserve cells 42 at a low temperature and allows the preparation of a biological sample without opening the cap. The cryopreservation container 1 includes a cap 10 and a container body 20. The cryopreservation container 1 is sterilized in advance.

[0023] (Cap 10) FIG. 2 is a cross-sectional view showing the configuration of cap 10 (second cap). As shown in FIG. 2, cap 10 is a cap that is attached to opening 21 and can be attached without capping. Cap 10 is capable of capping container body 20. Cap 10 comprises lid portion 11, insertion portion 12, protrusion portion 13, and extension portion 14. Cap 10 comprises insertion portion 12 and extension portion 14, and thus can be attached to container body 20 without capping using a capping machine. Cap 10 has elasticity and may be made of, for example, silicone rubber.

[0024] The cap 10 is resistant to low temperatures. The cryopreservation container 1 is frozen and preserved until the cells 42 are adjusted. Since the cap 10 is resistant to low temperatures, deterioration of the cap 10 in a low-temperature environment is reduced, and the reliability of the storage container can be improved.

[0025] Furthermore, the cap 10 may have low temperature resistance of -40°C or less. Low temperature resistance here means that the material has a dynamic elastic modulus of a predetermined value or less even at -40°C or less. Specifically, the material preferably has a dynamic elastic modulus of 20 MPa or less at -100°C to 100°C. The dynamic elastic modulus here is measured according to the measurement method of JIS K 6250. Examples of silicone rubber having such low temperature resistance include KE-183-U and KE-186-U manufactured by Shin-Etsu Chemical Co., Ltd., and the silicone rubber can be used as the material of the cap 10.

[0026] In general, it is considered preferable to store biological samples at a temperature of −40° C. or lower to prevent deterioration. The cap 10 reduces deterioration even at −40° C. or lower, which is suitable for storing biological samples, and can seal the container body 20 at room temperature and in low-temperature environments.

[0027] The cap 10 may preferably have low temperature resistance of -70°C or less. A temperature of -70°C or less is a suitable temperature for storing cells, and when storing cells in the cryopreservation container 1, it is preferable that the temperature when replacing the cap 30 with the cap 10 is also -70°C or less. In addition, when the cryopreservation container 1 is transported while being cooled using dry ice, the temperature inside the refrigerator storing the cryopreservation container 1 is also approximately -70°C. By making the cap 10 resistant to low temperatures of -70°C or less, it becomes possible to transport the cryopreservation container 1 storing cells and to perform preparations for transportation at a temperature suitable for storing the cells.

[0028] The lid part 11 is a part that seals the opening 21. As shown in Fig. 2, the lid part 11 has a puncture area 15. The lid part 11 has a shape and size that match the opening 21. When the container body 20 is cylindrical, the lid part 11 may be circular. When viewed in a plan view, the lid part 11 has an area that is the same as or larger than the opening area of ​​the opening 21.

[0029] (Insertion part 12) The insertion part 12 is a part that is connected to the lid part 11 and is inserted into the opening 21 when the cap 10 is attached to the opening 21. The insertion part 12 is connected to the container body 20 side of the lid part 11. The insertion part 12 is substantially cylindrical or a part of it, and has a shape that can be fitted into the opening 21 of the container body 20. In this embodiment, the insertion part 12 is substantially cylindrical.

[0030] 2, the outer wall surface of the insertion part 12 is in contact with the inner wall surface of the container body 20. By bringing the inner wall surface of the container body 20 and the outer wall surface of the insertion part 12 into contact with each other, liquid leakage can be prevented and the cap 10 can be made difficult to come off.

[0031] The insertion part 12 may have any taper angle θ with respect to the inner wall of the container body 20 when the cap 10 is attached to the opening 21, and the taper angle θ may be greater than 0° and within 3.5°. The insertion part 12 may have the taper angle θ only at its tip and in the vicinity of the tip. In the example shown in FIG. 2, an inclined surface having a taper angle θ is formed on about 1 / 3 of the tip side of the insertion part 12. By the insertion part 12 having the taper angle θ, the insertion part 12 made of a soft material can be easily inserted into the container body 20. By setting the taper angle θ to be greater than 0° and within 3.5°, the insertion part 12 can be easily inserted into the container body 20, and the possibility of the liquid for preparing the cells 42 entering the gap between the container body 20 and the insertion part 12 can be reduced. If the liquid enters the gap, it will have an adverse effect on the success or yield of the cell preparation, so it is preferable to set the taper angle θ to be within 3.5°. In addition, by providing a taper angle θ only at the tip of the insertion portion 12 and in the vicinity of the tip, it is possible to achieve both the function of the insertion portion 12 of "preventing liquid leakage and making it difficult for the cap 10 to be removed" and the function of the taper angle θ of making it easy to insert the insertion portion 12 into the container body 20.

[0032] The insertion part 12 may be made of an elastic material. When the insertion part 12 has elasticity, the function of the insertion part 12, "preventing liquid leakage and making it difficult for the cap to come off," can be further improved by bringing the insertion part 12 into elastic contact with the inner wall surface of the opening 21.

[0033] (Puncture area 15) The puncture region 15 is a region in the lid 11 where an injection needle can be punctured and penetrated. The puncture region 15 is preferably a region where an injection needle can be punctured and penetrated multiple times. The puncture region 15 is defined inside the connection portion of the insertion part 12 to the lid 11 when viewed from above. The puncture region 15 in the lid 11 allows the cells 42 to be prepared without opening the cap 10 of the cryopreservation container 1, reducing the possibility of contamination. Therefore, the cells 42 can be prepared outside a clean bench.

[0034] The hardness of the puncture region 15 may be 30 or more and 60 or less at 25°C, as measured with a durometer type A. The cells 42 are prepared at room temperature. If the hardness of the puncture region 15 is high, the injection needle cannot penetrate the puncture region 15, and if the hardness is low, the strength of the lid portion 11 decreases. Therefore, a puncture region 15 having a hardness of 30 to 60 at 25°C can realize a lid portion 11 that can be easily punctured while maintaining the function of the lid portion 11.

[0035] The thickness of the puncture region 15 may be set appropriately depending on the hardness. If the thickness of the puncture region 15 is too thick, it becomes difficult to penetrate the needle, and if the thickness is too thin, the strength of the cover part 11 decreases. If the hardness is as described above, the thickness of the puncture region 15 may be about 2 mm.

[0036] It is preferable that the puncturing region 15 can be punctured seven or more times to adjust the cells 42. In general, when one vial is punctured multiple times, it is preferable that each time the vial is punctured at a different location in order to prevent the occurrence of coring. Therefore, in order to prevent the same location from being punctured, the puncturing region 15 may have a puncturing mark 17 indicating the location to be punctured.

[0037] FIG. 4 is a plan view showing the upper surface of the cap 10. The puncture mark 17 may be displayed in a linear manner to divide the puncture region 15 into eight equal parts as shown in FIG. 4. The puncture mark 17 may be displayed three-dimensionally by projections and recesses. In order to improve visibility, the puncture mark 17 is preferably displayed on the surface of the cover part 11 opposite to the surface that contacts the opening 21. By dividing the puncture site with the puncture mark 17, in other words, by providing puncture marks that divide the puncture region 15 into a plurality of sections, the risk of puncturing the same site can be reduced and the occurrence of coring can be prevented. The number of sections into which the puncture region 15 is divided and the shape of each section can be arbitrary.

[0038] The puncture mark 17 may include a number. For example, the numbers "1" to "8" may be displayed on the eight equally divided puncture areas 15 shown in FIG. 4. The puncture site marked with "1" may be designated as the first puncture site, the puncture site marked with "2" may be designated as the second puncture site, and this may be repeated until the puncture site marked with "8" is designated as the eighth puncture site. Since the operator can puncture while associating the number of punctures with the puncture area 15, the risk of puncturing the same site can be further reduced.

[0039] (Protrusion 13) The protrusion 13 is a portion for allowing the operator to recognize the range of the puncture region 15. The protrusion 13 is located on at least a part of the outer edge of the puncture region 15 on the surface of the cover 11 opposite to the side on which the insertion portion 12 is located. As shown in FIG. 4, the protrusion 13 is located on at least a part of the outer edge of the puncture region 15 in a top view. In FIG. 4, an annular region where the insertion portion 12 is connected to the cover 11 is shown by a dashed line. Since the puncture region 15 is an area within the range surrounded by the insertion portion 12, the protrusion 13 is located at a position overlapping with the inner edge of the annular region or located inside that position.

[0040] When puncturing the puncture region 15, if the puncture position is the position where the insertion part 12 is located, there is a risk of non-penetration or foreign matter being generated as the needle passes through the insertion part 12. By providing the protrusion 13 on at least a part of the outer edge of the puncture region 15, the range of the puncture region 15 can be recognized by the operator, and the possibility of puncturing the portion where the insertion part 12 is located can be reduced when the injection needle is punctured into the cover part 11.

[0041] The protrusion 13 may be annular in shape corresponding to the outer edge of the puncture region 15, or may be a collection of a plurality of protruding shapes provided partially along the outer edge.

[0042] (extending part) 3 is a cross-sectional view showing a state in which the cap 10 is attached to the container body 20 and the extension portion 14 is folded. The extension portion 14 is a portion for reducing the possibility that the cap 10 will come off the container body 20.

[0043] The extension portion 14 is connected to the outer edge of the lid portion 11, and when the cap 10 is attached to the opening 21, it elastically folds back toward the container body 20 side, thereby adhering closely to the outer wall surface that forms the opening 21.

[0044] As shown in Fig. 2, immediately after the cap 10 is attached to the container body 20, the extension portion 14 extends from the surface of the lid portion 11 opposite the side where the insertion portion 12 is located. Fig. 3 shows the state in which the extension portion 14 is folded back toward the container body 20 from this state. This state is the state in which the attachment of the cap 10 is complete. By folding back the extension portion 14 in this manner, the possibility of the cap 10 coming off the container body 20 can be reduced.

[0045] The extension portion 14 is an elastic member having a shape that can be fitted into the outer wall surface of the opening 21, and may be tubular. In this embodiment, the extension portion 14 is cylindrical.

[0046] [Embodiment 2] Other embodiments of the present disclosure will be described below. For convenience of explanation, the same reference numerals will be given to members having the same functions as those described in the above embodiment, and the description thereof will not be repeated.

[0047] 5 is a cross-sectional view showing the configuration of the cap 10A (second cap). The cryopreservation container 1A includes the cap 10A and a container body 20. The cap 10A is a cap that is attached to an opening 21 of the container body 20. The material of the cap 10A may be the same as that of the cap 10. The cap 10A has the same low temperature resistance as the cap 10.

[0048] The lid portion 11A of the cap 10A has a puncture region 15A that can be punctured and penetrated by an injection needle, similar to the puncture region 15. The insertion portion 12A is connected to the lid portion 11A and has the same function as the insertion portion 12. However, the surface of the lid portion 11A on which the puncture region 15A is formed (referred to as the top surface 16) is located closer to the bottom of the container body 20 than the tip of the opening 21. Therefore, the part of the lid portion 11A that covers the tip of the opening 21 and the part that connects the top surface 16 (referred to as the connecting portion 18) is inserted inside the opening 21, and the outer wall surface of the connecting portion 18 is in contact with the inner wall surface of the container body 20. Therefore, when the cap 10A is attached to the opening 21, the puncture region 15A is located closer to the bottom of the container body 20 (not shown) than the edge of the opening 21. The puncture region 15A is defined inside the insertion section 12A in top view.

[0049] When adjusting the cells 42 contained in the cryopreservation container 1A, it is preferable that the injection needle reach the bottom of the container body 20. For example, when the length of the injection needle is 38 mm and the length from the opening 21 to the bottom is 39 mm, the needle tip does not reach the bottom, and the liquid 41 stored at the bottom cannot be extracted.

[0050] According to the above configuration, for example, when the puncture region 15A is positioned so as to sink 5 mm toward the bottom side from the edge of the opening 21, the needle tip can reach the bottom of the container body 20. In this way, even if the injection needle is short compared to the length of the container body 20, it is possible to easily extract the liquid 41 and the cells 42 that have settled to the bottom. In order to increase safety and reliability, it is preferable that the position up to 32 mm from the needle tip is in the space between the top surface 16 and the bottom of the container body 20.

[0051] [Embodiment 3] FIG. 6 is a plan view showing the configuration of the cap 10B (second cap). The cryopreservation container 1B includes the cap 10B and a container body 20 (see FIG. 1). The cap 10B is a cap that is attached to an opening 21 of the container body. The lid portion 11B of the cap 10B includes a puncture region 15B that can be punctured and penetrated multiple times with an injection needle, similar to the puncture region 15. In FIG. 6, the annular region where the insertion portion 12 is connected to the lid portion 11 is shown by a dashed line. The material of the lid portion 11B may be the same as that of the cap 10. The cap 10B has the same low-temperature resistance as the cap 10.

[0052] The puncture region 15B has a plurality of puncture marks 17B indicating the puncture site. As shown in FIG. 6, the puncture marks 17B may be circular, and the puncture marks 17B may be spaced apart from one another. The puncture marks 17B may be displayed three-dimensionally by projections and recesses. The puncture marks 17B are displayed on the surface of the cover portion 11B opposite to the surface that contacts the opening 21. By having the puncture marks 17B, the puncture site becomes more clear, reducing the risk of puncturing the same site and preventing the occurrence of coring.

[0053] The puncture marks 17B may be any shape that indicates the range of each puncture site, and may be a shape having three or more sides such as a triangle or a rectangle, or an ellipse, without being limited to a circle. The number and arrangement of the puncture marks 17B in the puncture region 15B are also not particularly limited. The number of puncture marks 17B formed may be determined according to the intended use of the cryopreservation container 1B.

[0054] [Embodiment 4] FIG. 7 is a cross-sectional view showing the configuration of cap 10C (second cap). Low-temperature storage container 1C includes cap 10C and container body 20. Cap 10C is a cap that is attached to opening 21 of container body 20. Cap 10C includes lid portion 11C and cap screwing portion 70. Lid portion 11C of cap 10C includes puncture area 15C that can be punctured and penetrated by an injection needle, similar to puncture area 15. Lid portion 11C may be made of the same material as lid portion 11. Cap 10C has the same low-temperature resistance as cap 10.

[0055] The cap screwing portion 70 is located on the inner wall surface facing the outer wall surface of the container body 20 when the cap 10C is attached to the container body 20, and screws into the screwing portion 22 of the container body 20. The cap 10C has a lid upper portion 71 that covers the outer edge of the lid portion 11C when the cap 10C is viewed in plan. The cap screwing portion 70 and the lid upper portion 71 may be made of a hard resin, and may be made of the same material as the container body 20.

[0056] Lid upper part 71 is located on the surface of lid part 11C opposite to the surface in contact with opening 21, and covers the outer edge of lid part 11C. Lid upper part 71 can elastically bring lid part 11C into close contact with the edge of opening 21 in order to seal opening 21. Lid upper part 71 may be annular in plan view.

[0057] The inner edge of the annular portion of lid upper part 71 in plan view is located at least inside the inner wall of container body 20 in top view. The area of ​​lid part 11C that is not covered by lid upper part 71 becomes puncture area 15C. Lid upper part 71 and lid part 11C may be bonded together.

[0058] According to the above-mentioned configuration, the cap screwing portion 70 screws into the screwing portion 22, so that the cap 10C can be prevented from coming off. Furthermore, when the cap is closed, the lid portion 11C is elastically and closely attached to the opening 21 by the lid portion upper portion 71, so that leakage can be prevented.

[0059] [Embodiment 5] [Biological sample management method and biological sample preparation method] An embodiment of the biological sample management method and biological sample preparation method will be described with reference to Figures 1 and 8. Figure 8 is a flow chart showing the method of managing and preparing the cell 42.

[0060] The cells 42 contained in the cryopreservation container 3 equipped with the cap 30 are stored at a temperature lower than -40°C, preferably lower than -70°C (S1). At this time, the cryopreservation container 3 contains a cell freezing liquid (liquid 41) together with the cells 42. When the cells 42 are stored for a long period of time, they may be stored at -80°C to -196°C, and the storage temperature may be set appropriately depending on the storage period. A previous step may be a step in which the cells 42 are frozen by a slow temperature change (-1°C / min).

[0061] In order to transport the cells 42, the low-temperature storage container 3 is replaced with the cap 10 from the cap 30 (S2). At this time, the container body 20 is not capped as shown in Fig. 1, so the replacement work is performed in a clean bench. To prevent the cells 42 from dissolving during the replacement work, the work may be performed while maintaining the temperature using a constant temperature container such as a low-temperature bath.

[0062] The cryopreservation container 1 closed with the cap 10 is placed in an insulated container such as a cooler box and transported to a destination such as a hospital (S3). Dry ice may be placed in the insulated container as a cooling agent.

[0063] According to the above-mentioned management methods (S1 to S3), cells 42 are cryopreserved for long-term storage using a cryopreservation container 3 equipped with a cap 30, and by replacing the cap with 10 when the cells 42 are transported, the cells 42 can be provided in a manner that allows the cells 42 to be prepared even outside a clean bench.

[0064] The cells 42 are thawed at the destination, such as a hospital (S4). Unlike when frozen, the cells 42 are preferably warmed by rapid temperature change. The cells 42 may be thawed in a hot bath, or may be thawed in a few minutes using a known thawing device.

[0065] The operator inserts the injection needle into the cap 10 of the cryopreservation container 1 and injects physiological saline (S5). Thus, the injection process is a process in which the injection needle is inserted into the cap 10 to inject new liquid into the cryopreservation container 1 via the injection needle (second injection needle). This process produces a mixture of the cell freezing liquid and physiological saline, reducing the concentration of the cell freezing liquid. The amount of liquid injected at this time depends on the capacity of the cryopreservation container 1 and the amount of freezing liquid, but may be approximately 0.5 ml.

[0066] Thereafter, the operator puts the cryopreservation container 1 into a centrifuge to separate the mixed liquid from the cells 42, and then pierces the cap 10 with a needle to extract the mixed liquid (S6). After centrifugation, the cells 42 settle at the bottom of the container body 20, so that the mixed liquid and the cells 42 are separated into layers, and the operator extracts the upper layer of the mixed liquid. In this manner, the extraction step is a step of extracting the liquid stored inside the cryopreservation container 1 through the injection needle (first injection needle) by piercing the cap 10 with the injection needle. The adjustment method shown in this embodiment is merely an example, and the order of the injection step and the extraction step is not particularly limited, and the extraction step may be performed before the injection step. The injection step may be performed continuously, or the extraction step may be performed continuously. In addition, the first injection needle and the second injection needle may be the same injection needle or different injection needles.

[0067] According to the above steps (S5 and S6), the cells 42 can be adjusted without opening the cap 10 of the cryopreservation container 1, so that the cells 42 can be adjusted outside a clean bench.

[0068] The operator then inserts a syringe needle into the cap 10 of the cryopreservation container 1 and injects saline (S7). This step produces a diluted mixture of the mixture and saline, further reducing the concentration of the cell freezing solution. The amount of injection at this time may be more than S5, and may be approximately 1.5 ml, depending on the capacity of the cryopreservation container 1 and the amount of freezing solution.

[0069] After the cryopreservation container 1 is centrifuged to separate the diluted mixture from the cells 42, the operator inserts a needle into the cap 10 to extract the diluted mixture (S8). Before moving to the next step, steps S7 and S8 may be repeated multiple times as necessary. The injection and extraction steps may use the same needle.

[0070] Thereafter, the operator inserts an injection needle into the cap 10 of the cryopreservation container 1 and injects GCSF (Granulocyte Colony Stimulating Factor) (S9). The injection of GCSF is preferably performed using a microsyringe. This step is a step of injecting GCSF (medicine) at least once among the multiple injection steps after performing the extraction step and the injection step multiple times. In this way, the cells 42 can be adjusted without opening the cap 10 of the cryopreservation container 1.

[0071] Thereafter, the operator inserts an injection needle into the cap 10 of the cryopreservation container 1 and injects collagen (S10).

[0072] Thereafter, the operator inserts an injection needle into the cap 10 of the cryopreservation container 1 and extracts the cells 42 together with the collagen (S11). The diameter (gauge) of the needle used at this time may be larger than the needles used in the previous extraction and injection steps so as not to damage the cells 42. The extracted cells 42 are used as appropriate.

[0073] Note that the management and adjustments in S1 to S11 are all based on the assumption that one cryopreservation container is used, but they are also applicable to the case where multiple cryopreservation containers are used. For example, in S6, multiple cryopreservation containers may be centrifuged simultaneously, and then the mixed liquid may be extracted from the multiple cryopreservation containers in sequence.

[0074] 〔summary〕 The cap according to aspect 1 of the present disclosure is attached to the opening of a container body of a cryopreservation container for storing biological samples, and comprises a lid portion that is resistant to low temperatures and has a puncture area that can be punctured with an injection needle.

[0075] The cap according to embodiment 2 of the present disclosure has low temperature resistance of -40°C or lower in embodiment 1.

[0076] The cap according to aspect 3 of the present disclosure is in accordance with aspects 1 and 2, wherein the hardness of the puncture region is 30° or more and 60° or less at 25°C.

[0077] The cap of aspect 4 of the present disclosure is any one of aspects 1 to 3 and comprises an insertion portion connected to the lid portion and inserted into the opening when the cap is attached to the opening, and a protrusion portion located on at least a part of the outer edge of the puncture area on the side of the lid portion opposite the side on which the insertion portion is located.

[0078] The cap of aspect 5 of the present disclosure is any one of aspects 1 to 4, and is provided with an insertion portion connected to the lid portion and inserted into the opening when the cap is attached to the opening, and when the cap is attached to the opening, the angle of the insertion portion with respect to the inner wall of the low-temperature storage container is greater than 0° and not greater than 3.5°.

[0079] A cryogenic storage container according to a sixth aspect of the present disclosure is a cryogenic storage container having a cap according to any one of the first to fifth aspects and a container body having an opening to which the cap is attached.

[0080] The biological sample preparation method according to aspect 7 of the present disclosure includes an extraction step of extracting liquid stored inside a cryogenic storage container storing a biological sample via a first injection needle by puncturing a cap attached to an opening of the cryogenic storage container with the first injection needle, and an injection step of injecting new liquid into the cryogenic storage container via the second injection needle by puncturing the cap with the second injection needle.

[0081] A biological sample preparation method according to aspect 8 of the present disclosure is the same as aspect 7, in which the biological sample is a cell, the extraction step and the injection step are each performed multiple times, and a drug is injected at least once of the multiple injection steps.

[0082] A biological sample management method according to aspect 9 of the present disclosure includes a step of preserving a biological sample stored in a cryogenic storage container equipped with a first cap at a temperature lower than -40°C, and a step of replacing the first cap with a second cap that is resistant to low temperatures and has a puncture area that can be punctured with an injection needle in order to transport the biological sample.

[0083] According to the cap, low-temperature storage container, biological sample preparation method, or biological sample management method of the present disclosure, the biological sample in the low-temperature storage container can be prepared without opening the cap. Therefore, the present disclosure can be utilized in regenerative medicine (dental pulp regeneration, etc.) and contribute to the SDGs. [Explanation of symbols]

[0084] 1, 1A, 1B, 1C, 3 Cold storage container 30 Cap (1st Cap) 10, 10A, 10B, 10C Cap (Second Cap) 11, 11A, 11B, 11C Lid 12, 12A Insertion Part 13 Protrusion 14 Extension 15, 15A, 15B, 15C puncture area 20 Container body 21 Opening 22 Threaded joint 41 Liquid 42 Cells (biological samples)

Claims

1. The device is attached to the opening of a container body of a cryogenic storage container for storing biological samples, It has low temperature resistance, A cap having a lid portion having a puncture area that can be punctured with an injection needle.

2. The cap according to claim 1, which has low temperature resistance of -40°C or less.

3. The cap according to claim 1 , wherein the hardness of the puncture region is 30° or more and 60° or less at 25° C.

4. an insertion portion that is connected to the lid portion and is inserted into the opening when the cap is attached to the opening; The cap according to claim 1 , further comprising a protrusion located on at least a part of an outer edge of the puncture area on a surface of the lid portion opposite to the side on which the insertion portion is located.

5. an insertion portion connected to the lid portion and inserted into the opening when the cap is attached to the opening; The cap according to claim 1 , wherein when the cap is attached to the opening, the insertion portion forms an angle with the inner wall of the cryogenic storage container that is greater than 0° and is within 3.5°.

6. A cap according to any one of claims 1 to 5, and a container body having an opening to which the cap is attached.

7. a step of inserting a first injection needle into a cap attached to an opening of a cryopreservation container storing a biological sample, thereby extracting a liquid stored inside the cryopreservation container through the first injection needle; and an injection step of injecting new liquid into the low-temperature storage container through the second injection needle by puncturing the cap with the second injection needle.

8. The biological sample is a cell, The extraction step and the injection step are each performed a plurality of times; The biological sample preparation method according to claim 7 , wherein an agent is injected in at least one of the multiple injection steps.

9. storing a biological sample contained in a cryopreservation container having a first cap at a temperature lower than −40° C.; and replacing the first cap with a second cap that is resistant to low temperatures and has a puncture area that can be punctured with an injection needle in order to transport the biological sample.

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