Sealed container, container for cryopreservation, method for manufacturing a sealed container, method for manufacturing a frozen liquid, and method for manufacturing a liquid

Abstract

To provide an encapsulated container, a freezing preservation container, a production method of an encapsulated container, a production method of frozen liquid, and a production method of liquid capable of suppressing breakage of the encapsulated container during steps including a freezing step for freezing with liquid nitrogen or the like in an extremely low temperature environment, a transport step, and a thawing step.SOLUTION: An encapsulated container 10 comprises a container main body 11 having a bottom 13 and a trunk 12. The container main body is filled with liquid Lq so as to leave an air layer Ar in a part of the container main body, and the container main body is sealed. When a bottom part of the container main body is installed on a horizontal plane Hp in parallel, a flat ratio of a liquid level Ls viewed from a normal line direction of the liquid level of the liquid is 0.1 or smaller.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to sealed containers, cryopreservation containers, methods for making sealed containers, methods for making frozen liquids, and methods for making liquids. [Background technology]

[0002] Liquids containing biological samples such as biological cells are typically stored in cryopreservation containers made of resin. The liquid stored in the cryopreservation container is frozen and preserved using liquid nitrogen, a cryogenic freezer, or other means. When the frozen liquid is actually used, the cryopreservation container is thawed by immersing it in a warm bath at, for example, 37°C to 40°C. For example, Patent Document 1 discloses a cell cryopreservation container constructed by welding the peripheral edge of a first resin sheet-shaped member and the peripheral edge of a second resin sheet-shaped member together.

[0003] Resin cell cryopreservation containers are vitrified by freezing them in an ultra-low temperature environment using liquid nitrogen or the like. Therefore, there is a risk of the cell cryopreservation container being damaged when being frozen in an ultra-low temperature environment using liquid nitrogen or the like. Alternatively, there is a risk of the cell cryopreservation container being damaged if it is accidentally dropped or exposed to the external environment during transportation or when it is removed from liquid nitrogen or the like and thawed. In particular, the cell cryopreservation container is prone to damage if a large stress is applied around the liquid surface during freezing. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-42212 Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure provides a sealed container, a container for cryopreservation, a method for manufacturing a sealed container, a method for manufacturing a frozen liquid, and a method for manufacturing a liquid that can prevent damage to the sealed container during the process from freezing in an ultra-low temperature environment using liquid nitrogen or the like, through a transportation process, to a thawing process. [Means for solving the problem]

[0006] The sealed container of this embodiment is a sealed container comprising a container body having a bottom and a body, wherein the container body is filled with liquid so that an air layer remains in a portion of the container body, the container body is sealed, and the bottom of the container body is placed parallel to a horizontal plane, and when viewed from the normal direction of the liquid surface, the flatness of the liquid surface is 0.1 or less.

[0007] In the sealed container according to this embodiment, the container body may include a thermoplastic resin, and the barrel portion may have a storage portion filled with the liquid and a welded sealing portion.

[0008] In the sealed container according to this embodiment, the container body may contain polyethylene.

[0009] In the sealed container according to this embodiment, the body portion has a storage portion in which the liquid is filled and a welded sealing portion, and the thermal conductivity of the sealing portion may be lower than the thermal conductivity of the storage portion.

[0010] The cryopreservation container of this embodiment is a cryopreservation container comprising a container body having an opening, a bottom, and a body portion, the body portion having a portion to be sealed, and when the inner surfaces of the portion to be sealed are tightly attached to each other, the flattening ratio of the horizontal cross section of the body portion at a position 13 mm to 50 mm away from the portion to be sealed is 0.1 or less.

[0011] The cryopreservation container of this embodiment is a cryopreservation container comprising a container body having an opening, a bottom, and a body, the body having a portion to be sealed, the container body being filled with 1 mL to 100 mL of liquid to tightly seal the inner surfaces of the portion to be sealed, and the flattening ratio of the horizontal cross section of the body at the position of the liquid level when the bottom of the container body is placed parallel to a horizontal plane is 0.1 or less.

[0012] The method for manufacturing a sealed container according to this embodiment includes the steps of preparing a cryopreservation container according to this embodiment, filling the cryopreservation container with a liquid, and sealing the intended sealing portion of the cryopreservation container.

[0013] A method for manufacturing a sealed container according to this embodiment includes the steps of providing a sealed container according to this embodiment and freezing the sealed container.

[0014] A method for manufacturing a sealed container according to this embodiment includes the steps of preparing a sealed container according to this embodiment in a frozen state, and heating the sealed container to obtain a thawed liquid. [Effects of the Invention]

[0015] According to this embodiment, damage to the sealed container can be prevented during the process from freezing in an ultra-low temperature environment using liquid nitrogen or the like, through the transportation process, and up to the thawing process. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a perspective view of a sealed container according to one embodiment. [Figure 2] FIG. 2 is a front view of a sealed container according to one embodiment. [Figure 3] FIG. 3 is a plan view showing the liquid level in a sealed container according to one embodiment. [Figure 4] FIG. 4 is a cross-sectional view showing a method for measuring the length of the major axis and the minor axis of the liquid surface. [Figure 5]FIG. 5 is a perspective view showing a cryopreservation container according to one embodiment. [Figure 6] FIG. 6 is a front view showing a cryopreservation container according to one embodiment. [Figure 7] 7(a) and 7(b) are perspective views showing parts of cryopreservation containers according to modified examples. [Figure 8] 8(a)-(c) are schematic diagrams showing the conditions during freezing of the sealed container. [Figure 9] FIG. 9 is a schematic diagram showing a liquid filled in a sealed container. [Figure 10] FIG. 10 is a graph showing the relationship between the container temperature and the stress during freezing. [Figure 11] FIG. 11 is a graph showing the relationship between the flattening of the liquid surface and the maximum value of stress during freezing. [Figure 12] Figures 12(a) and (b) are graphs showing the measured freezing rate of water near the side wall of a sealed container at the liquid surface (position A) during freezing and at a position 3 mm below the liquid surface (position B). [Figure 13] FIG. 13 is a front view showing a sealed container according to a first modified example. [Figure 14] FIG. 14 is a schematic view showing the state during freezing of the sealed container according to the first variant. [Figure 15] FIG. 15 is a front view showing a sealed container according to a second modified example. [Figure 16] FIG. 16 is a front view showing a sealed container according to a third modified example. DETAILED DESCRIPTION OF THE INVENTION

[0017] Each embodiment will be described in detail below with reference to the drawings. The figures shown below are schematic illustrations. Therefore, the size and shape of each part are appropriately exaggerated for ease of understanding. Furthermore, appropriate modifications can be made within the scope of the technical concept. In the figures shown below, identical parts are denoted by the same reference numerals, and some detailed descriptions may be omitted. Furthermore, the numerical values, such as dimensions, and material names of each component described in this specification are examples of embodiments and are not limited thereto. They may be selected and used as appropriate. In this specification, terms specifying shapes or geometric conditions, such as parallel, orthogonal, and vertical, are used in their strict sense and also include substantially the same state. For convenience of explanation, the terms "upper" and "lower" may be used, but the up-down direction may be reversed.

[0018] In this specification, when a certain component such as a certain member or a certain region is described as being "on (or under)" another component such as another member or another region, it does not necessarily mean that the component is directly above (or directly below) the other component unless otherwise specified. When a certain component such as a certain member or a certain region is described as being "on (or under)" another component such as another member or another region, it also includes the case where another component is included between the other component and the component above (or below) the other component.

[0019] [Sealed container] First, the configuration of a sealed container according to this embodiment will be described with reference to Figures 1 to 3. Figure 1 is a perspective view showing a sealed container 10 according to this embodiment. Figure 2 is a front view showing the sealed container 10 according to this embodiment. Figure 3 is a plan view showing the liquid level Ls in the sealed container 10 according to this embodiment.

[0020] As shown in Figures 1 and 2, the sealed container 10 according to this embodiment is a vial-type container, and includes a container body 11 having a bottom 13 and a body 12. The container body 11 is filled with a liquid Lq such that an air layer Ar remains in a portion of the container body 11. The container body 11 is also sealed. When the bottom 13 of the container body 11 is placed on a horizontal plane Hp (i.e., a plane perpendicular to the direction in which gravity acts, a plane parallel to the XY plane in Figure 1), and viewed from the normal direction of the liquid surface Ls of the liquid Lq (i.e., the direction in which gravity acts, the Z direction in Figure 1), the oblateness of the liquid surface Ls is 0.1 or less.

[0021] A sealed portion 25 is formed at one end (first side end) of the body 12. A storage portion 26 is formed at the other end (second side end) of the body 12 closer to the sealed portion 25. The storage portion 26 stores liquid Lq. The storage portion 26 is cylindrical and is formed continuously between the sealed portion 25 and the bottom 13. A seal line 21 is formed at the lower end of the sealed portion 25 of the body 12. The container body 11 also has a central axis CL. The central axis CL is perpendicular to the horizontal plane Hp. The central axis CL may be a straight line connecting the centers of gravity of the container body 11 in each horizontal cross section.

[0022] Next, the detailed structure of the sealed container 10 will be further described.

[0023] As described above, the container body 11 contains the liquid Lq and has a cylindrical body 12 and a bottom 13. The body 12 has a sealing portion 25 and a storage portion 26. The storage portion 26 has a cylindrical or elliptical cylindrical shape. In this case, the horizontal cross-section (cross-section parallel to the XY plane) of the storage portion 26 gradually changes in shape along the vertical direction (Z direction). Specifically, the horizontal cross-section of the storage portion 26 is a perfect circle or an ellipse closer to a circle (with a smaller flattening ratio) toward the bottom 13. The horizontal cross-section of the storage portion 26 is a line segment or an ellipse closer to a line segment (with a larger flattening ratio) toward the sealing portion 25. The horizontal cross-section of the storage portion 26 may be uniform along the vertical direction (Z direction). In this embodiment, the container body 11 has a shape that is line-symmetrical in the left-right direction (X direction) when viewed from the front side (see FIG. 2).

[0024] The sealed portion 25 is a portion formed by sealing one end (the end on the positive side in the Z direction) of the container body 11 by heat sealing or the like. Specifically, the sealed portion 25 is formed by crushing one end of the container body 11A of the cryopreservation container 10A (described below) and sealing the opposing inner surfaces of the end. The sealed portion 25 is generally planar, with its main surfaces being generally rectangular and parallel to the ZX plane. The sealed portion 25 is a region located above the seal line 21 (opposite the bottom 13), and this entire region is sealed. Examples of methods for sealing the sealed portion 25 include ultrasonic sealing, heat sealing, and high-frequency sealing. Alternatively, the inner surface of the container body 11 may be sealed by applying an adhesive. In this embodiment, a sealing method without using an adhesive is preferred because it can prevent the adhesive from mixing with the liquid. Furthermore, a storage portion 26 is provided in the body 12, closer to the bottom 13 than the seal line 21.

[0025] The other end (second side end, negative end in the Z direction) of the container body 11 forms a closed bottom 13. During use, the liquid contained inside the container body 11 is taken out from this bottom 13. The bottom 13 may be made of a plate-like member that is approximately circular in plan view. The liquid can be easily aspirated from the sealed container 10 by piercing the bottom 13 with a syringe needle or the like and aspirating the liquid.

[0026] A flat stand portion 15 that supports the container body 11 is connected to the bottom portion 13 via a connecting portion 14. The connecting portion 14 is made of a thin, rod-like member that can be easily broken. A plurality of connecting portions 14 are provided at predetermined intervals along the circumferential direction of the bottom portion 13. In this embodiment, the connecting portions 14 are formed in two locations symmetrically about the central axis CL. The stand portion 15 is formed in a generally diamond shape in a plan view. The stand portion 15 is connected to the bottom portion 13 of the container body 11 via the connecting portion 14. The stand portion 15 is positioned parallel to the horizontal plane Hp.

[0027] Such a sealed container 10 is produced using a cryopreservation container 10A, as described below. The cryopreservation container 10A is formed by integral molding with the end where the sealing portion 25 is provided left open. After the container body 11A of the cryopreservation container 10A is filled with liquid Lq, the open end is heat-sealed (i.e., the sealing portion 25 is formed). In this way, the sealed container 10 is formed. The sealed container 10 is positioned so that the sealing portion 25 faces vertically upward and the stand portion 15 faces vertically downward.

[0028] When using the sealed container 10, the connecting portion 14 is broken and the stand portion 15 is removed. Thereafter, a syringe needle is inserted into the bottom portion 13 to aspirate the liquid Lq, thereby allowing the liquid Lq to be extracted from the container body 11. Alternatively, the connecting portion 14 and the stand portion 15 may not be provided. In this case, for example, the container body 11 may be cut horizontally (in a direction parallel to the XY plane) to remove the bottom portion 13, and the liquid Lq may be extracted from the sealed container 10. The method for extracting the liquid Lq is not particularly limited, and examples include a method of aspirating and extracting the liquid using a syringe or the like. Alternatively, the bottom portion 13 side of the container body 11 may be opened, a lid (not shown) may be attached, and the liquid may be extracted from the sealed container 10 by removing the lid. The liquid may be extracted by aspirating and extracting the liquid Lq using a syringe or the like, as described above. Alternatively, the liquid Lq may be aspirated by piercing the lid with a syringe while the lid is still attached.

[0029] The sealing line 21 is formed between the storage portion 26 and the sealing portion 25. The sealing line 21 is a line that forms the boundary between the sealing portion 25 and the container body 11. In the sealed container 10 shown in Figures 1 and 2, the sealing line 21 is straight and parallel to the horizontal direction (X direction). However, this is not limited to this, and the sealing line 21 may be a curve with a predetermined curvature. Alternatively, the sealing line 21 may be a curve whose curvature changes in the length direction of the sealing line 21. Furthermore, the sealing line 21 may be bent along the way.

[0030] FIG. 3 shows the shape of the liquid surface Ls (hereinafter also referred to as the planar shape) when the bottom 13 of the container body 11 is placed on a horizontal plane Hp (a plane parallel to the XY plane; see FIGS. 1 and 2) and viewed from the normal direction (Z direction) of the liquid surface Ls of the liquid Lq. As shown in FIG. 3, in the planar shape of the liquid surface Ls, the liquid surface Ls has a major axis dl and a minor axis ds. Here, the major axis dl is defined as the longest distance between two parallel lines that contact the liquid surface Ls from both sides in a planar view. The minor axis ds is defined as the shortest distance between two parallel lines that contact the liquid surface Ls from both sides in a planar view. The major axis dl is equal to or longer than the minor axis ds. The major axis dl and the minor axis ds intersect at the central axis CL of the container body 11. The major axis dl and the minor axis ds may be perpendicular to each other. In this case, the major axis dl is parallel to the X axis, and the minor axis ds is parallel to the Y axis.

[0031] The oblateness of the liquid surface Ls is 0.1 or less, preferably 0.08 or less, and more preferably 0.06 or less. The oblateness can be calculated by the formula (de) / d, where d is the length of the major axis dl and e is the length of the minor axis ds. The oblateness is 0 if the planar shape of the liquid surface Ls is a perfect circle, and approaches 1 as the planar shape of the liquid surface Ls becomes closer to a line segment.

[0032] The lengths of the major axis dl and minor axis ds of the liquid level Ls can be determined as follows. For example, as shown in FIG. 4, the diameter da of the body portion 12 is measured at the position where the liquid level Ls is present. The diameter da of the body portion 12 may be measured, for example, with a vernier caliper. In this case, the diameter da of the body portion 12 is measured at eight locations (22.5 degrees apart) at equal angular intervals around the central axis CL. The thicknesses tb1 and tb2 of the body portion 12 are also measured at each location. The diameter da of the body portion 12 is subtracted by the thicknesses tb1 and tb2 of the body portion 12, and the maximum value of the subtraction values ​​(da - tb1 - tb2) is defined as the major axis dl and the minimum value as the minor axis ds. The thicknesses tb1 and tb2 of the body portion 12 may be measured, for example, by cutting the portion of the body portion 12 where the liquid level Ls is located horizontally and measuring the thicknesses tb1 and tb2 with a vernier caliper or the like.

[0033] By making the oblateness of the liquid surface Ls 0.1 or less, the freezing rate of the liquid Lq can be slowed down as described below, and the amount of frozen liquid Lq present near the liquid surface Ls decreases. As a result, pressure can be more easily released toward the air layer Ar, and the stress on the liquid Lq due to freezing decreases. This makes it possible to prevent damage to the container body 11 due to the stress caused by the freezing of the liquid Lq.

[0034] The oblateness of the liquid surface Ls may be greater than 0, preferably greater than 0.01, and more preferably greater than 0.02. Having the oblateness of the liquid surface Ls greater than 0 provides the following effects. For example, when the liquid Lq is a cellular medicine, the frozen liquid Lq is generally thawed by rapidly heating the sealed container 10 in a thermostatic bath at 37°C. This is to prevent damage to cells caused by ice crystals, and it is preferable to thaw as quickly as possible so as to pass through the temperature range where ice crystals form in the shortest time possible. Therefore, by shifting the planar shape of the frozen liquid surface Ls from a perfect circle, the thawing rate of the frozen liquid Lq can be increased. Note that the greater the oblateness of the liquid surface Ls, the faster the freezing rate of the liquid Lq and the thawing rate of the frozen liquid Lq.

[0035] The material constituting the container body 11 is preferably a material that can be thermally welded by heat sealing or the like. The material constituting the container body 11 is preferably a material that does not affect the liquid Lq when it comes into contact with the liquid Lq. The material constituting the container body 11 is appropriately selected depending on the type of liquid Lq contained in the sealed container 10. Furthermore, the material constituting the container body 11 is also appropriately selected depending on the intended use of the sealed container 10. Specifically, when the sealed container 10 is to be endowed with predetermined properties such as strength, flexibility, water vapor permeability, heat resistance, and light transmittance, it is preferable to appropriately select a material that has such properties. Suitable materials for such materials include thermoplastic resins, particularly polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), ethylene-vinyl acetate copolymer, fluororesin, and cyclic olefin copolymer (COC). In particular, polyethylene, a thermoplastic resin, is preferably used as the material constituting the container body 11. This is because polyethylene has a low melting point, allowing for thermal welding such as heat sealing at low temperatures. Also, polyethylene has a small Young's modulus, so the stress generated when the sealed container 10 is frozen is small.

[0036] The properties of the container body 11, such as thickness, strength, light transmittance, heat resistance, and gas barrier properties, can be adjusted as appropriate depending on the use of the sealed container 10 and the type of liquid Lq contained in the container.

[0037] The container body 11 may have a single-layer structure or a multi-layer structure. When the container body 11 has a multi-layer structure, only the inner surface of the container body 11 may be made of the resin material described above, and the other layers may have an aluminum layer containing aluminum. Furthermore, the outer surface of the container body 11 may be surface-coated with a material such as silica, if necessary. In this case, barrier properties can be imparted to the sealed container 10.

[0038] The liquid Lq contained in the sealed container 10 may be a medicinal solution such as a pharmaceutical product. Specific examples of such medicinal solutions include antirheumatic drugs, insulin preparations, sugar solutions such as glucose, electrolyte correction solutions such as sodium chloride and potassium lactate, protein preparations, antibody drugs, contrast agents, protease inhibitors, fat emulsions, antibiotics, anticancer drugs, heparin calcium anesthetics, and peritoneal dialysis solutions. Specific examples of medicinal solutions include so-called premixed preparations prepared by dissolving preparations such as analgesics, antipyretics, antiemetics, antitussives, antihistamines, antiallergic drugs, bronchodilators, steroids, antiarrhythmic drugs, and antiepileptic drugs in sterile water such as RO water or distilled water or physiological saline. Furthermore, the medicinal liquid may be a biological medicine such as a vaccine for influenza, tetanus, pneumococcus, polio, Japanese encephalitis, rubella, measles, yellow fever, Hib, hepatitis, chickenpox, rabies, rotavirus, mumps, cervical cancer, MQ, DT, DPT, etc. Furthermore, the medicinal liquid may be a biological cell such as a bone marrow or lymphocyte. The pharmaceutical solution may also be, for example, a cell preparation, specifically, hepatoma cells, hepatocytes (liver parenchymal cells), Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells such as epidermal keratinocytes, epithelial cells such as tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells, mammary gland cells, pericytes, muscle cells such as smooth muscle cells and cardiac muscle cells, kidney cells, pancreatic islet cells of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, bone cells, or stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. Examples of stem cells include bone marrow undifferentiated mesenchymal stem cells, hematopoietic stem cells, vascular stem cells, neural stem cells, small intestinal stem cells, adipose stem cells, skin stem cells, periodontal tissue stem cells, ciliary body stem cells, corneal limbal stem cells, and visceral stem cells.

[0039] Alternatively, the liquid Lq contained in the sealed container 10 may be a food-related liquid, specifically, a liquid such as a beverage or seasoning.

[0040] The sealed container 10 according to this embodiment can be used, for example, as a medical container or a food container for storing the liquid Lq as described above.

[0041] [Container for cryopreservation] Next, a cryopreservation container 10A for producing the above-mentioned sealed container 10 will be described with reference to Figures 5 and 6. Figures 5 and 6 are diagrams showing the cryopreservation container 10A before the container body 11A is sealed.

[0042] 5 and 6, a cryopreservation container 10A according to this embodiment includes a container body 11A. The container body 11A has an opening 20 formed at one end (the end on the positive side in the Z direction). Furthermore, at least the inner surface of the opening 20 of the container body 11A is weldable.

[0043] The container body 11A is capable of containing the liquid Lq and has a body 12A and a bottom 13A located at the other end (the end on the negative side in the Z direction) of the container body 11A. The body 12A is substantially cylindrical, and its horizontal cross section (a plane parallel to the XY plane) is substantially circular. The horizontal cross section of the body 12A may be substantially uniform along the vertical direction (Z direction).

[0044] The opening 20 communicates with the outside. The opening 20 has a circular shape when viewed from above (the positive side in the Z direction). The periphery of the opening 20 is sealed by welding to form a sealed portion 25 (see Figures 1 and 2). The body portion 12A also has a portion to be sealed 24. The portion to be sealed 24 is formed along the circumferential direction near the opening 20 before the opening 20 is welded (see Figures 5 and 6). The portion to be sealed 24 forms the sealed portion 25 after the inner surfaces of the portion to be sealed are welded together with each other in close contact.

[0045] As shown in Figures 5 and 6, the portion to be sealed 24 is located at one end of the container body 11A on the opening 20 side (the end on the positive side in the Z direction). The portion to be sealed 24 is sealed by welding using heat sealing or the like. The portion to be sealed 24 may be formed over the entire circumferential direction of one end of the container body 11A. The portion to be sealed 24 may be formed on a part of the circumferential direction of one end of the container body 11A. The portion to be sealed 24 may have a generally cylindrical shape as a whole.

[0046] The height H1 (distance in the Z direction) of the intended sealing portion 24 may be 1 mm or more and 50 mm or less, and preferably 1.6 mm or more and 20 mm or less. By making the height H1 of the intended sealing portion 24 1 mm or more, the periphery of the opening 20 of the container body 11A can be effectively welded. Furthermore, by making the height H1 of the intended sealing portion 24 50 mm or less, the size of the container can be reduced, improving handleability. Furthermore, when the height H1 of the intended sealing portion 24 is 1.6 mm or more and 20 mm or less, the opening 20 can be sealed using a normal heat sealing machine (such as an impulse sealing machine).

[0047] A seal mark 27 is provided at the lower end of the portion to be sealed 24. The seal mark 27 may be provided in a line shape over the entire circumferential direction. Alternatively, the seal mark 27 may be provided on a portion of the circumferential direction. The seal mark 27 may be a convex or concave portion formed on the surface of the container body 11A. Alternatively, the seal mark 27 may be a colored line formed on the surface of the container body 11A. This seal mark 27 is used as a mark for the position where the portion to be sealed 24 is welded. In other words, the opening 20 side of the seal mark 27 is welded. Furthermore, a storage section 26A that stores the liquid Lq is provided on the body portion 12A closer to the bottom 13A than the seal mark 27.

[0048] In FIG. 6, the position of the liquid level Ls (also referred to as the expected liquid level position) when the container body 11A is filled with the liquid Lq (see FIGS. 1 and 2), the inner surfaces of the intended sealing portions 24 are tightly attached, and the bottom portion 13A of the container body 11A is placed parallel to the horizontal plane Hp is indicated by the symbol Pl. When the inner surfaces of the intended sealing portions 24 are tightly attached, the oblateness of the horizontal cross section of the body portion 12A at the expected liquid level position Pl is 0.1 or less, preferably 0.08 or less, and more preferably 0.06 or less. By making the oblateness of the horizontal cross section of the body portion 12A at the expected liquid level position Pl 0.1 or less, the freezing rate of the liquid Lq in the sealed container 10 can be slowed. As a result, damage to the container body 11 due to stress caused by the freezing of the liquid Lq can be suppressed.

[0049] The expected liquid level position Pl may be the position of the liquid level Ls of the liquid Lq when 1 mL or more of the liquid Lq is filled into the container body 11A and the inner surfaces of the intended sealing portion 24 are tightly attached to each other. By filling the container body 11A with 1 mL or more of the liquid Lq, the size of the container can be reduced and handleability can be improved. The expected liquid level position Pl may be the position of the liquid level Ls of the liquid Lq when 100 mL or less, more preferably 62.23 mL or less, even more preferably 50 mL or less, even more preferably 10 mL or less, even more preferably 5 mL or less, even more preferably 2 mL or less, and even more preferably 1.2 mL or less of the liquid Lq is filled and the inner surfaces of the intended sealing portion 24 are tightly attached to each other. Furthermore, by filling the container body 11A with 100 mL or less of the liquid Lq, the size of the container can be reduced and handleability can be improved.

[0050] Furthermore, the expected liquid level position Pl may be at a position 13 mm or more and 50 mm or less away from the intended sealing portion 24. Note that the distance H2 from the intended sealing portion 24 refers to the distance measured parallel to the central axis CL from the point of the seal mark 27 closest to the bottom 13A. By positioning the expected liquid level position Pl 15 mm or more away from the intended sealing portion 24, it is possible to keep the temperature rise of the liquid Lq to within 1°C when the intended sealing portion 24 is sealed at 250°C, for example. By positioning the expected liquid level position Pl 50 mm or less away from the intended sealing portion 24, it is possible to reduce the size of the container and improve handleability.

[0051] As shown in FIG. 7(a), the intended sealing portion 24 may be thinner than the storage portion 26A. In FIG. 7(a), the intended sealing portion 24 has a substantially uniform thickness T2 around the entire circumference of the opening 20 of the container body 11A. Specifically, for example, the thickness T2 of the intended sealing portion 24 in the body portion 12A is 250 μm or more and 1 mm or less. Furthermore, the thickness T4 of the portion of the body portion 12A other than the intended sealing portion 24 is 1 mm or more and 3 mm or less. Alternatively, as shown in FIG. 7(b), the thickness T3 of the intended sealing portion 24 may gradually decrease from the storage portion 26A toward the opening 20. In this case, the thin intended sealing portion 24 is heat-sealed, and the sealed container 10 can maintain its hermeticity when stored at an extremely low temperature (approximately −150°C), such as in the vapor phase of liquid nitrogen.

[0052] [Operation of this embodiment] Next, the operation of this embodiment having such a configuration will be described.

[0053] First, as shown in Figures 5 and 6, a cryopreservation container 10A is produced with the opening 20 open. The cryopreservation container 10A may be produced by, for example, injection molding.

[0054] Next, a predetermined amount of liquid Lq is filled into the container body 11A of the cryopreservation container 10A through the opening 20, and the intended sealing portion 24 is sealed by welding. At this time, the liquid Lq is filled so that some air remains in the container body 11A. The seal mark 27 is used as a mark for the position where the intended sealing portion 24 is to be welded. At this time, the intended sealing portion 24 located above the seal mark 27 (opposite the bottom portion 13) on the container body 11A is welded. This forms the sealed portion 25, and a sealed container 10 is obtained (see Figures 1 and 2). When this sealed container 10 is placed parallel to the horizontal plane Hp and viewed from the normal direction of the liquid level Ls of the liquid Lq, the oblateness of the liquid level Ls is 0.1 or less.

[0055] Examples of devices for welding the intended sealing portion 24 in this way include a one-side heating heat sealing machine, a two-side heating heat sealing machine, an ultrasonic sealing machine, a high-frequency sealing machine, a sealing machine using hot air, etc. Alternatively, a non-heating adhesion method (such as photocuring) may be used.

[0056] Next, the sealed container 10 containing the liquid Lq is placed on a rack or the like (not shown). Thereafter, the sealed container 10 is placed in a storage container filled with liquid nitrogen. In the storage container, the sealed container 10 is frozen with liquid nitrogen. The sealed container 10 is stored or transported in an environment of at least 0°C or below, preferably -80°C or below, more preferably -150°C or below (temperature in an ultra-low temperature freezer or in the vapor phase of liquid nitrogen), and particularly preferably -196°C (liquid nitrogen temperature). When the sealed container 10 is frozen with liquid nitrogen, the sealed container 10 is frozen in the vapor phase present above the liquid nitrogen surface.

[0057] In this embodiment, a method for producing frozen liquid Lq is also provided, which includes the steps of preparing the sealed container 10 and freezing the sealed container 10.

[0058] During this time, as shown in FIG. 8(a), the liquid Lq in the sealed container 10 freezes gradually from the bottom 13 side (lower side) closest to the liquid nitrogen. As the freezing progresses, the unfrozen liquid Lq is surrounded by frozen liquid (hereinafter also referred to as frozen liquid Lf) (FIG. 8(b)). After that, all of the liquid Lq surrounded by the frozen liquid Lf also freezes. At this time, stress is applied to the frozen liquid Lf present near the liquid surface Ls (FIG. 8(c)).

[0059] The stress generated when the sealed container 10 is frozen is not uniform across the area. In the case of a vial-shaped sealed container 10, the stress is greatest in the area that freezes slowest because the force has nowhere to escape. In other words, the sealed container 10 starts freezing from the bottom 13, and the area near the liquid surface Ls freezes last. This is because, in a liquid nitrogen storage container, the temperature on the bottom 13 side is low and heat transfer from the air layer Ar to the liquid Lq is small.

[0060] Generally, the sealed container 10 is vitrified by freezing in an ultra-low temperature environment using liquid nitrogen or the like, and is easily broken by impact. For example, the sealed container 10 may be accidentally dropped or collide with a hard object while being stored or transported in a storage container, or while being removed from the storage container and thawed. In this case, the vitrified sealed container 10 may be broken.

[0061] In contrast, according to the present embodiment, by setting the oblateness of the liquid surface Ls during freezing to 0.1 or less, the freezing rate of the liquid Lq present near the liquid surface Ls can be slowed. This reduces the amount of frozen liquid Lf present near the liquid surface Ls. In this case, the pressure caused by the freezing of the liquid Lq surrounded by the frozen liquid Lf is more likely to escape upward from the liquid surface Ls, thereby minimizing the stress caused by freezing. As a result, damage to the container body 11 due to the stress caused by the freezing of the liquid Lq can be suppressed. As described above, according to the present embodiment, damage to the container body 11 when freezing the liquid Lq in an ultra-low temperature environment using liquid nitrogen or the like can be suppressed. Furthermore, damage to the container body 11 can be suppressed when the sealed container 10 is accidentally dropped or comes into contact with the external environment and subjected to an impact during transportation of the sealed container 10 or when the sealed container 10 is removed from liquid nitrogen or the like and thawed.

[0062] On the other hand, if the flattening ratio of the liquid surface Ls during freezing exceeds 0.1, the freezing rate of the liquid Lq will increase. In this case, the amount of frozen liquid Lf present near the liquid surface Ls will increase. As a result, stress caused by the freezing of the liquid Lq surrounded by the frozen liquid Lf will be applied to the body portion 12 near the liquid surface Ls (the area circled in Figure 8(c)), which is the part that freezes last. In this case, there is a risk that the container body 11 will be more susceptible to damage.

[0063] When the liquid Lq is to be used, the sealed container 10 is removed from the storage container and thawed. At this time, the sealed container 10 is pulled out of the storage container. Then, the sealed container 10 is immersed in a thermostatic bath at, for example, 37°C to 40°C and rapidly thawed.

[0064] When the oblateness of the liquid surface Ls is greater than 0, the thawing speed of the frozen liquid Lf is increased by deviating the planar shape of the frozen liquid surface Ls from a perfect circle compared to when the planar shape of the liquid surface Ls is a perfect circle. This allows the frozen liquid Lf to pass through the temperature range where ice crystals form during thawing in the shortest time, particularly when the liquid Lq is a cellular medicine. As a result, damage to cells caused by ice crystals can be reduced.

[0065] In this embodiment, a method for producing liquid Lq is also provided, which includes the steps of preparing a sealed container 10 in a frozen state and heating the sealed container 10 to obtain thawed liquid Lq.

[0066] Next, we will explain why the freezing speed of the liquid Lq differs depending on the oblateness of the liquid surface Ls.

[0067] First, as shown in Figure 9, assume that the liquid Lq filled in the sealed container 10 is cylindrical with a circumferential length X. In this case, the circumferential length X is approximated as 2πr using an imaginary radius r. The imaginary radius is the radius when the flattening ratio a = 0 (= perfect circle). If the height of this liquid Lq is 1, then the semimajor axis (dl / 2) of the liquid surface Ls is ((1-a) / (2-a)) × 2r. The semiminor axis (ds / 2) of the liquid surface Ls is (2 / (2-a)) × r.

[0068] In this case, the surface area A of the liquid Lq is

number

[0069] Also, the volume V of the liquid Lq is

number

[0070] From the above, the surface area / volume (A / V) of the liquid Lq is

number

[0071] On the other hand, if we consider that the temperature T of a cylindrical sealed container (surface area: A, volume: V, specific heat: c, density: ρ) freezing in the open air (temperature: T∞, heat transfer coefficient: h) follows the lumped heat capacity model (see http: / / www.kz.tsukuba.ac.jp / ~abe / ohp-heat / chapter4.pdf, etc.), the following formula holds (when the initial conditions are t=0 and T=T0):

number

[0072] Substituting the above A / V value into this, we get

number

[0073] Here, in the range of 0≦a≦1,

number

[0074] As described above, according to this embodiment, the bottom 13 of the container body 11 is placed parallel to the horizontal plane Hp, and when viewed from the normal direction of the liquid surface Ls of the liquid Lq, the oblateness of the liquid surface Ls is 0.1 or less. This makes it possible to slow down the freezing rate of the liquid Lq in the sealed container 10. Therefore, when the liquid Lq freezes, the amount of frozen liquid Lq present near the liquid surface Ls can be reduced. This makes it easier for pressure to escape above the liquid surface Ls, thereby reducing the stress caused by freezing. As a result, it is possible to prevent the container body 11 from being damaged by the stress caused by the freezing of the liquid Lq.

[0075] [Example] Next, a specific example of this embodiment will be described.

[0076] Example 1 A cryopreservation container having the shape shown in Figures 5 and 6 was fabricated. This cryopreservation container was made of polyethylene. Next, the cryopreservation container was filled with liquid (water) in an amount equal to or greater than half the volume of the cryopreservation container. At this time, the liquid level was 14 mm from the seal mark toward the bottom. The flatness of the liquid level was 0.03. Next, the intended sealing portion was welded using a one-side heating impulse sealer. The sealed container thus obtained was placed in the vapor phase of a dry shipper containing liquefied nitrogen. The sealed container was then cooled in the liquid nitrogen vapor phase until the temperature of the sealed container reached -150°C, freezing the liquid.

[0077] Example 2 A sealed container was prepared in the same manner as in Example 1, except that the flatness of the liquid surface was 0.06, and the liquid was frozen in a liquid nitrogen vapor phase.

[0078] (Comparative Example 1) A sealed container was prepared in the same manner as in Example 1, except that the flatness of the liquid surface was 0.11, and the liquid was frozen in a liquid nitrogen vapor phase.

[0079] [Calculation of stress during freezing] For each of these three types of sealed containers (Example 1, Example 2, Comparative Example 1), strain was measured at the location including the liquid surface during freezing, and the stress during freezing was calculated. Stress is the force acting per unit area, and according to material mechanics, it can be calculated by multiplying strain by Young's modulus. However, since Young's modulus changes significantly due to temperature changes during freezing, it is difficult to directly apply the above relationship. Therefore, to calculate the stress during freezing, the stress increase calculated by multiplying the strain due to freezing at a certain moment by the Young's modulus at that temperature was added up until freezing was complete, and the result was calculated as the stress due to freezing (see the formula below).

[0080]

number

[0081] Strain was measured using a strain gauge (Kyowa Electric Industry Co., Ltd., KFGS) attached to the sealed container with adhesive (Kyowa Electric Industry Co., Ltd., CC33-A) and a strain measuring instrument (Kyowa Electric Industry Co., Ltd., EDX-10). The strain excluding the thermal shrinkage of the sealed container was taken as the strain during freezing. Specifically, the strain when the sealed container without water was frozen alone was measured in advance, and this was subtracted from the result when the container was filled with water to obtain the strain during freezing.

[0082] The temperature was measured by attaching a thermocouple (molded surface sensor, manufactured by Toa Denki Co., Ltd.) to the sealed container near the strain gauge and using a temperature logger (GL220, manufactured by A&D Co., Ltd.).

[0083] Young's modulus was calculated based on JIS K 7161-1:2014, "Plastics - Determination of Tensile Properties." Specifically, test specimens measuring 5 mm wide and 70 mm high were cut from sealed containers. Tensile tests were then performed using a Tensilon universal testing machine (A&D, RTG-1210) with a chuck distance of 25 mm and a tensile speed of 20 mm / min. Young's modulus was calculated by dividing the stress by the strain. Stress was calculated by dividing the load by the area on which the load was applied, and strain was calculated by dividing the elongation by the chuck distance. Young's modulus values ​​were calculated at 25°C and in liquid nitrogen (approximately -196°C), and the Young's modulus at the time of freezing, E(T(t)), was calculated using the approximate formula E = E - B exp (-Tg / T) (E: Young's modulus at 0 K, B: constant, Tg: glass transition temperature, T: temperature). The Young's modulus at each temperature may be experimentally determined.

[0084] The results are shown in Figure 10. As is clear from Figure 10, the sealed container of Comparative Example 1 experienced a large stress during freezing, while the sealed containers of Examples 1 and 2 experienced a small stress during freezing.

[0085] Figure 11 is a graph showing the relationship between the flattening ratio of the liquid surface and the maximum value of the stress during freezing. The maximum value of the stress during freezing was determined based on the results in Figure 10. As is clear from Figure 11, the stress during freezing rises sharply with a flattening ratio of around 0.1 as the threshold value.

[0086] [Relationship between flattening ratio and freezing speed] To investigate the relationship between oblateness and freezing rate, the freezing rate of water near the sidewall of the sealed container was measured at the liquid surface (Position A) during freezing and at a position 3 mm below the liquid surface (Position B). The results are shown in Figures 12(a) and 12(b). Figure 12(a) is a graph for the sealed container of Example 1, and Figure 12(b) is a graph for the sealed container of Comparative Example 1.

[0087] 12(a), in the sealed container of Example 1, the freezing rate of water near the side wall was almost the same at the liquid surface (position A) and at a position 3 mm below the liquid surface (position B). When the time it takes for water to reach 0°C at position A is defined as TA and the time it takes for water to reach 0°C at position B is defined as TB, TB / TA = 107%.

[0088] 12(b), in the sealed container of Comparative Example 1, the freezing rate of water near the side wall was faster at the liquid surface (position A) than at a position 3 mm below the liquid surface (position B). When the time it takes for water to reach 0°C at position A is TA and the time it takes for water to reach 0°C at position B is TB, TB / TA = 116%.

[0089] This confirmed that the freezing rate of the liquid surface increased as the flattening ratio increased, which is thought to have caused an increase in stress during freezing.

[0090] [Variations] Next, various modifications of this embodiment will be described with reference to Figures 13 to 16. Figures 13 to 16 are diagrams showing modifications of this embodiment. In Figures 13 to 16, the same parts as those in the embodiment shown in Figures 13 to 16 are given the same reference numerals, and detailed descriptions thereof will be omitted.

[0091] (First Modification) Fig. 13 shows a sealed container 10 according to a first modified example. In Fig. 13, the sealed container 10 includes a container body 11 having a bottom 13 and a body 12. The container body 11 is filled with liquid Lq so that an air layer Ar remains in a portion of the container body 11, and the container body 11 is sealed at a sealing portion 25. When the bottom 13 of the container body 11 is placed parallel to a horizontal plane Hp and viewed from the normal direction of the liquid surface Ls of the liquid Lq, the oblateness of the liquid surface Ls is 0.1 or less.

[0092] As shown in FIG. 13, in the horizontal cross section, the perimeter of the liquid Lq gradually increases from the bottom 13 to the liquid level Ls. In this case, it is possible to further reduce the stress caused by freezing of the liquid Lq. This is because, when the perimeter of the liquid Lq increases from the bottom 13 to the liquid level Ls, the shape of the liquid Lq surrounded by the frozen liquid Lf becomes a shape in which the area on the liquid level Ls side increases (see FIG. 14). In this case, the component of stress that escapes to the air layer Ar increases (see the arrow in FIG. 14), making it difficult for the stress caused by freezing to be applied to the container body 11. It is preferable that the value of "perimeter of the liquid Lq at the liquid level Ls" / "perimeter of the liquid Lq at the bottom 13" be 1.06 or more.

[0093] (Second Modification) Fig. 15 shows a sealed container 10 according to a second modified example. In Fig. 15, the sealed container 10 includes a container body 11 having a bottom 13 and a body 12. The container body 11 is filled with liquid Lq so that an air layer Ar remains in a portion of the container body 11, and the container body 11 is sealed at a sealing portion 25. When the bottom 13 of the container body 11 is placed parallel to a horizontal plane Hp and viewed from the normal direction of the liquid surface Ls of the liquid Lq, the oblateness of the liquid surface Ls is 0.1 or less.

[0094] 15, the seal line 21 and the upper edge 25a of the sealed container 10 are each inclined with respect to a plane parallel to the horizontal plane Hp. In this case, since the seal line 21 extends at an angle with respect to a plane parallel to the horizontal plane Hp, a large area can be secured for the sealed portion 25, and the welding strength of the sealed portion 25 can be increased.

[0095] (Third Modification) Fig. 16 shows a sealed container 10 according to a third modified example. In Fig. 16, the sealed container 10 includes a container body 11 having a bottom 13 and a body 12. The container body 11 is filled with liquid Lq so that an air layer Ar remains in a portion of the container body 11, and the container body 11 is sealed at a sealing portion 25. When the bottom 13 of the container body 11 is placed parallel to a horizontal plane Hp and viewed from the normal direction of the liquid surface Ls of the liquid Lq, the oblateness of the liquid surface Ls is 0.1 or less.

[0096] 16, the thermal conductivity of the sealing portion 25 of the sealed container 10 is lower than that of the accommodating portion 26. For example, the thermal conductivity of the sealing portion 25 may be lower than that of the accommodating portion 26 by making the materials of the sealing portion 25 and the accommodating portion 26 different from each other. Alternatively, the thermal conductivity of the sealing portion 25 may be lower than that of the accommodating portion 26 by making the thickness of the sealing portion 25 and the thickness of the accommodating portion 26 different from each other.

[0097] Here, thermal conductivity is defined as the heat transfer coefficient / thickness. Thermal conductivity is measured along the longitudinal axis of the sealed container 10 (the direction of arrow Ax in Figure 16). Thermal conductivity can be measured using methods such as the heat flow meter method (JIS A1412-2), the laser flash method (JIS R 1611), or the cyclic heating method (ISO 22007-3). For known materials, literature values ​​may be used as the thermal conductivity. For example, the thermal conductivity of low-density polyethylene (PE) is approximately 0.33 W / m K, the thermal conductivity of cyclic olefin copolymer (COC) is approximately 0.16 W / m K, and the thermal conductivity of polypropylene (PP) is approximately 0.13 W / m K. The thickness of the sealed portion 25 refers to the thickness of one of the opposing surfaces of the container body 11 when they are sealed together. The thickness of the storage portion 26 refers to the thickness of the wall of the container body 11 that constitutes the storage portion 26.

[0098] In this way, by making the thermal conductivity of the sealing portion 25 lower than that of the storage portion 26, safety is enhanced when the sealed container 10 is thawed. That is, when thawing a frozen sealed container 10, the liquid level Ls is placed in a thermostatic bath, so the sealed portion 25 side containing the air layer Ar is often grasped. On the other hand, if the frozen product is accidentally touched directly with the hand, the moisture in the hand will instantly freeze and adhere to the skin, which is dangerous. By making the thermal conductivity of the sealing portion 25 lower than that of the storage portion 26, the moisture in the hand is less likely to freeze, enhancing safety.

[0099] The components disclosed in the above embodiments and modifications may be combined as needed, or some components may be omitted from all the components disclosed in the above embodiments and modifications. [Explanation of symbols]

[0100] 10 Sealed containers 10A cryopreservation container 11, 11A Container body 12, 12A Torso 13, 13A bottom 14 Connection part 15 Stand section 20 Opening 21 Seal line 24 Sealing section 25 Sealed part 26 Storage section 27 Sticker Mark

Claims

1. A sealed container comprising: The container comprises a polyethylene container body having a bottom and a body, The container body is filled with a liquid so that an air layer remains in a part of the container body, and the container body is sealed. The sealed container has a liquid surface flatness of 0.01 to 0.1 when the bottom of the container body is placed parallel to a horizontal plane and viewed from the normal direction of the liquid surface, and is frozen under liquid nitrogen and thawed by immersion in a warm bath at 37 to 40°C.

2. A sealed container as described in claim 1, wherein the body has a storage section in which the liquid is filled and a welded sealing section.

3. the body portion has a container portion filled with the liquid and a welded sealing portion; 3. The sealed container according to claim 1, wherein the heat transferability of the sealing portion is less than the heat transferability of the containing portion.

4. A cryopreservation container comprising: The container comprises a polyethylene container body having an opening, a bottom, and a body; The body portion has a sealing portion, The container for cryopreservation has a horizontal cross-sectional flattening ratio of 0.01 to 0.1 at a position 13 mm to 50 mm away from the intended sealing portion when the inner surfaces of the intended sealing portion are tightly attached to each other, and is frozen under liquid nitrogen and thawed by immersion in a warm bath at 37 to 40°C.

5. A cryopreservation container comprising: The container comprises a polyethylene container body having an opening, a bottom, and a body; The body portion has a sealing portion, The container for cryopreservation is such that the container body is filled with 1 mL or more and 100 mL or less of liquid, the inner surfaces of the intended sealing portions are tightly attached to each other, and the flattening ratio of the horizontal cross section of the body portion at the position of the liquid surface when the bottom of the container body is placed parallel to a horizontal plane is 0.01 or more and 0.1 or less, the container is frozen under liquid nitrogen, and is thawed by immersing in a warm bath at 37 to 40°C.

6. A step of preparing a cryopreservation container according to claim 4 or 5; Filling the cryopreservation container with a liquid; and sealing the intended sealing portion of the cryopreservation container.

7. Providing a sealed container according to any one of claims 1 to 3; and freezing the sealed container under liquid nitrogen.

8. Providing a sealed container according to any one of claims 1 to 3 in a frozen state; and warming the sealed container in a hot bath at 37-40°C to obtain a thawed liquid.

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