Cell culture kit, method for producing cell culture body, and cell culture body accommodated in container

Abstract

A cell culture kit (100) comprises a culture container (110), a lid (130), and a ring-shaped member (170). A culture space (111) for culturing cells is formed in the culture container (110), and the culture container (110) includes an opening (114a) that communicates with the culture space (111). The lid (130) is attachable to and detachable from the culture container (110) so as to close the opening (114a). The ring-shaped member (170) has a ring shape with an open top and bottom, and is removably accommodated in the culture space (111). The height of the ring-shaped member (170) is equal to or less than the height of the culture space (111). The ring-shaped member (170) includes a held part (172) that forms a protrusion that is to be held for taking out the ring-shaped member (170).

Description

Cell culture kit, method for producing cell cultures, and cell cultures contained in a container

[0001] This disclosure relates to a cell culture kit, a method for producing cell cultures, and cell cultures contained in a container.

[0002] Non-patent document 1 discloses a technique for culturing cells on a silk film, which is a protein-based biomaterial. Non-patent document 1 describes that in the pretreatment step of the silk film before culturing cells on the silk film, 70% ethanol (EtOH bath) is prepared in a Petri dish, and when sterilizing the silk film, a stainless steel ring is placed on the silk film placed in the well.

[0003] Brian D. Lawrence, et al., "Silk Film Culture System for in vitro Analysis and Biomaterial Design," [online], [Accessed September 13, 2024], Internet <URL: https: / / www.jove.com / t / 3646 / silk-film-culture-system-for-in-vitro-analysis-and-biomaterial-design>

[0004] When a culture sheet (e.g., silk film) is fixed to the bottom of a culture vessel (e.g., a well) by the weight of a ring-shaped member (e.g., a stainless steel ring), it is usually necessary to remove the ring-shaped member from the culture vessel before removing the culture sheet from the culture vessel (e.g., a well). However, Non-Patent Document 1 does not contain any description regarding removing the stainless steel ring from the well after culturing cells on the silk film.

[0005] One of the objectives of this invention is to provide a cell culture kit, etc., that allows for easy removal of a ring-shaped member for fixing a culture sheet from the culture vessel.

[0006] According to one aspect of this disclosure, the following cell culture kit, method for producing a cell culture, and cell culture contained in a container are provided.

[0007] 1. A cell culture kit comprising: a culture vessel having a culture space formed inside for culturing cells and an opening communicating with the culture space; a lid that can be attached to the culture vessel so as to close the opening; a culture sheet for culturing the cells; and a ring-shaped member that is open at the top and bottom and is removably housed in the culture space, wherein the ring-shaped member has a height less than or equal to the height of the culture space and includes a holding portion that forms a projection or recess for holding the ring-shaped member for removal. 2. The cell culture kit according to 1. wherein the ring-shaped member is housed in the culture vessel with a gap between it and the inner surface of the culture vessel. 3. The cell culture kit according to 1. or 2. wherein the holding portion is the projection and includes opposing surfaces for gripping and holding with an instrument. 4. The cell culture kit according to 3. wherein the opposing surfaces are composed of opposing surfaces along the circumferential direction of the ring-shaped member. 5. The specific gravity of the ring-shaped member is greater than 1.0. A cell culture kit as described in any one of the following. 6. The material of the ring-shaped member includes at least one of the following: PEEK (polyether ether ketone), PET (polyethylene terephthalate), PBT (polybutylene terephthalate), PS (polystyrene), PC (polycarbonate), mPPE (modified polyphenylene ether), PPS (polyphenylene sulfide), PSU (polysulfone), PAR (polyarylate), LCP (liquid crystal polymer), ABS (acrylonitrile butadiene styrene), PES (polyethersulfone), POM (polyacetal), PI (polyimide), PA (polyamide, including nylon 6 and nylon 66), PGA (polyglycolic acid), PLA (polylactic acid), AS resin (acrylonitrile styrene copolymer resin), MS resin (methyl methacrylate styrene copolymer resin), PMMA (methacrylic resin), acrylic resin, polyurethane resin, fluororesin (PTFE (polytetrafluoroethylene), PVDF (polyvinylidene fluoride), etc.), polyphenyl ether resin, polyetherimide resin, vinyl chloride resin, glass, ceramics, and metal. A cell culture kit as described in any one of items 1 through 5.7. The cell culture kit according to any one of 1 to 6, wherein the ring-shaped member, when placed on the culture sheet placed inside the culture container, sandwiches at least a portion of the culture sheet between itself and the bottom of the culture container. 8. The cell culture kit according to any one of 1 to 7, wherein the culture space can be sealed when the lid is attached to the culture container. 9. The cell culture kit according to 2, wherein the holding portion is the projection and includes opposing surfaces for gripping and holding with an instrument, the ring-shaped member, when placed on the culture sheet placed inside the culture container, sandwiches at least a portion of the culture sheet between itself and the bottom of the culture container, and the culture space can be sealed when the lid is attached to the culture container. 10. A method for producing a cell culture comprising: placing a culture sheet for culturing cells inside a culture vessel having a culture space formed inside for culturing cells and an opening communicating with the culture space; placing a ring-shaped member, which is open at the top and bottom and is removablely housed in the culture space, on top of the culture sheet; introducing a culture medium into the culture space; introducing cells into the culture space; placing a removable lid on top of the culture vessel so as to close the opening; and culturing the introduced cells to obtain a cell culture on the culture sheet, wherein the ring-shaped member has a height less than or equal to the height of the culture space and includes a holding portion that forms a projection or recess for holding the ring-shaped member for removal. 11. The method for producing a cell culture according to 10, further comprising freezing the cell culture.12. A cell culture body contained in a container, comprising: a culture vessel having a culture space formed inside for culturing cells and including an opening communicating with the culture space; a lid that can be attached to the culture vessel so as to close the opening; a culture sheet disposed inside the culture vessel to which cell culture bodies are attached; and a ring-shaped member that is open at the top and bottom and is removably housed in the culture space, wherein the ring-shaped member has a height less than or equal to the height of the culture space and includes a holding portion that forms a projection or recess for holding the ring-shaped member for removal, and at least a portion of the culture sheet to which the cell culture bodies are not attached is sandwiched between the bottom of the culture vessel. 13. A cell culture body contained in the container according to 12., wherein the cell culture bodies are frozen by being immersed in a cryopreservation solution.

[0008] According to this disclosure, it becomes possible to easily remove the ring-shaped member from the culture vessel.

[0009] This is a perspective view showing an example of the configuration of a cell culture kit according to this disclosure. This is a side cross-sectional view showing an example of a culture vessel according to this disclosure. This is a diagram showing an example of the flow of a method for manufacturing a cell culture according to this disclosure. This is a side cross-sectional view showing an example of a culture vessel after step 3 according to this disclosure has been completed. This is a side cross-sectional view showing an example of a cell culture kit after culturing cells according to this disclosure. This is a diagram showing an example of a method for using a cell culture according to this disclosure. This is a perspective view showing another example of a ring-shaped member.

[0010] The embodiments relating to this disclosure will be described below with reference to the drawings. In all drawings, similar components are denoted by the same reference numerals, and their descriptions are omitted where appropriate. The drawings are schematic diagrams and do not correspond to the actual dimensional ratios. Terms indicating directions such as up, down, left, and right are used for illustrative purposes only and are not intended to limit this disclosure.

[0011] [Embodiment 1] The cell culture kit 100 is a kit for producing a cell culture containing cells.

[0012] (Cell cultures) Cell cultures are, for example, three-dimensional cell cultures such as cell sheets, spheroids, and organoids, but are not limited to three-dimensional cell cultures.

[0013] A cell sheet is a structure in which cells are physically and functionally connected to each other via adhesion molecules, the extracellular matrix, and other means, resulting in a sheet-like structure.

[0014] A cell sheet may be a monolayer structure consisting of one cell layer, or a layered structure consisting of two or more cell layers. While there are no particular limitations on the layered structure, examples include multilayer structures with two, three, four, or five layers. Multilayer cell sheets may be obtained through culture, or by stacking monolayer cell sheets.

[0015] The thickness of the cell sheet is not particularly limited, but for example, it is between 0.001 mm and 2.0 mm. The lower limit is more preferably 0.01 mm, even more preferably 0.015 mm, and particularly preferably 0.02 mm. On the other hand, the upper limit is more preferably 1.5 mm, even more preferably 1.2 mm, and particularly preferably 1.0 mm. The thickness of the cell sheet may also be between 0.02 mm and 0.03 mm. By setting the thickness of the cell sheet within the above range, a high cell activity rate within the cell sheet and excellent shape retention ability advantageous for cell transplantation can be achieved.

[0016] The area of ​​the cell sheet is not particularly limited, but for example, 0.1 cm² ２ More than 1000cm ２ The following applies. The lower limit is more preferably 0.2 cm. ２ More preferably 0.25 cm ２ Particularly preferably 0.3 cm ２ On the other hand, the upper limit is more preferably 900 cm. ２ More preferably 800 cm ２ , particularly preferably 500 cm ２ That is the case.

[0017] (Cells) Cells are clinically useful cells for treating or preventing symptoms associated with defects and dysfunction of cells, tissues, or organs, or cultureable cells used in non-clinical trials, etc., and are not particularly limited as long as they are cells isolated from a living organism.

[0018] Examples of cells include living tissue cells, mesenchymal stem cells capable of differentiating into cells belonging to mesenchymal tissues, pluripotent stem cells capable of differentiating into various living tissues, and differentiated stem cells and progenitor cells. The cells may be adherent cells or suspension cells.

[0019] Specific examples of living tissue cells include, for instance, fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiomyocytes, myoblasts, osteocytes, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, kidney cells, gingival cells, periosteal cells, skin cells, and endothelial cells.

[0020] Specific examples of mesenchymal stem cells include, for instance, adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, oral tissue-derived mesenchymal stem cells, and skin tissue-derived mesenchymal stem cells.

[0021] Specific examples of pluripotent stem cells include induced pluripotent stem cells, embryonic stem cells, nuclear transfer embryonic stem cells, embryonic tumor cells, and embryonic germ cells. These cells may be cultured individually or in combination of two or more types. These cells may be appropriately selected from known types depending on the intended use of the cells.

[0022] The origin of the cells is not particularly limited, but examples include mammals, birds, amphibians, fish, insects, plants, and microorganisms. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and chickens.

[0023] Figure 1 is a perspective view showing an example configuration of a cell culture kit 100 according to this disclosure. The cell culture kit 100 comprises, for example, a culture container 110, a lid 130, a culture sheet 150, and a ring-shaped member 170.

[0024] (Regarding the culture vessel 110) Figure 2 is a lateral cross-sectional view showing an example of the culture vessel 110.

[0025] The culture vessel 110 has a culture space 111 formed inside for culturing cells in a culture medium, and includes an opening 114a that communicates with the culture space 111. The culture vessel 110 according to this embodiment is a bottomed cylindrical shape and includes a bottom portion 112, a side wall portion 113, an opening 114 that forms the opening 114a, and a leg portion 115.

[0026] Furthermore, the shape of the culture vessel 110 is not limited to a cylindrical shape in which the bottom 112 and side walls 113 are circular when viewed from above (for example, in the direction normal to the bottom 112). For example, the culture vessel 110 may be a bottomed cylindrical shape in which the bottom 112 and side walls 113 are of a predetermined shape such as an ellipse, rectangle, or polygon when viewed from above, or it may be any other shape.

[0027] The bottom portion 112 is flat and includes an upper surface 112a and a lower surface 112b.

[0028] The upper surface 112a is the inner surface of the bottom portion 112 and forms the culture space 111.

[0029] The lower surface 112b is the outer surface of the bottom 112. For example, when the culture container 110 is placed on a mounting surface such as a table or a constant temperature incubator, the lower surface 112b faces the mounting surface.

[0030] The side wall portion 113 extends upward from the bottom portion 112. More specifically, the side wall portion 113 extends upward from the outer edge of the bottom portion 112. The inner surface of the side wall portion 113 (in this embodiment, the inner circumferential surface) together with the upper surface 112a forms the culture space 111. The side wall portion 113 has a first locking portion 113a at the upper part of its outer surface (in this embodiment, the outer circumferential surface). The first locking portion 113a in this embodiment is a helical projection.

[0031] The opening 114 is, for example, at the upper end of the side wall portion 113 and forms an opening 114a that communicates with the culture space 111. That is, the culture vessel 110 includes the opening 114a that communicates with the culture space 111. Further, the opening 114a according to the present embodiment is provided above the culture vessel 110.

[0032] The leg portion 115 is a portion that extends downward from the lower surface 112b and has a flat lower surface. The outer shape of the leg portion 115 according to the present embodiment is substantially circular when viewed from above. By providing the leg portion 115, the lower surface 112b faces the placement portion with an interval corresponding to the length of the leg portion.

[0033] Note that the shape and configuration of the leg portion 115 are not limited to this. The leg portion 115 may be, for example, a polygonal shape such as a hexagon. The leg portion may not be provided, and in this case, the lower surface 112b may, for example, contact the placement portion.

[0034] The culture vessel 110 may generally be, for example, those referred to as a dish, a flask, a plate, a chamber slide, a petri dish, a tube, a tray, a culture bag, a roller bottle, etc. The dish may be, for example, those referred to as a Petri dish, a tissue culture dish, a multi-dish, etc., but is not limited thereto. The flask may be, for example, those referred to as a tissue culture flask, etc., but is not limited thereto. The plate may be, for example, those referred to as a microplate, a microwell plate, a multi-plate, a multi-well plate, etc., but is not limited thereto. Note that the general names of the culture vessel 110 are not limited to those exemplified here.

[0035] The material of the culture vessel 110 may be, for example, resin, glass, metal, etc. The resin may be, for example, polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyoxymethylene (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), cyclic olefin polymer, AS resin (acrylonitrile-styrene copolymer resin), MS resin (methyl methacrylate-styrene copolymer resin), methacrylic resin (PMMA), acrylic resin, polyurethane resin, polytetrafluoroethylene (PTFE), etc., and may contain one or more of them. The glass may be soda glass, borosilicate glass, quartz glass, etc. The metal may be stainless steel, aluminum, etc. The culture vessel 110 may be manufactured so that the culture solution does not leak, and may be integrally formed, for example. Note that the material and manufacturing method of the culture vessel 110 are not limited to those exemplified here.

[0036] The area of the bottom 112 of the culture vessel 110 is not particularly limited, but culturing can be performed without problems as long as it is the area of a commercially available culture vessel. For example, it is 0.1 cm ２ or more and 1000 cm ２ or less. The lower limit value is more preferably 0.2 cm ２ , still more preferably 0.25 cm ２ , and particularly preferably 0.3 cm ２ . On the other hand, the upper limit value is more preferably 900 cm ２ , still more preferably 800 cm ２ , and particularly preferably 500 cm２ That is the case.

[0037] (Regarding the lid 130) The lid 130 is a removable component attached to the culture container 110 so as to close the opening 114a. The lid 130 covers the opening 114a by being attached, for example, above the culture container 110.

[0038] The lid 130 according to this embodiment includes a lid portion 131 and a hanging portion 132.

[0039] The lid portion 131 is flat. More specifically, the lid portion 131 is a circular flat plate.

[0040] The hanging portion 132 extends downward from the lid portion 131. More specifically, the hanging portion 132 extends downward from the lid portion 131 when the lid 130 is attached to the culture vessel 110. More specifically, the hanging portion 132 extends downward from the outer edge of the lid portion 131.

[0041] The inner surface of the hanging portion 132 is sized to fit loosely onto the outer surface of the side wall portion 113. In this embodiment, the inner diameter of the hanging portion 132 is larger than the outer diameter of the side wall portion 113. That is, the hanging portion 132 can be fitted onto the side wall portion 113.

[0042] Furthermore, the hanging portion 132 includes a second locking portion 132a on its inner surface (in this embodiment, the inner circumferential surface). The second locking portion 132a engages with the first locking portion 113a to detachably lock the culture container 110 and the lid 130.

[0043] The second locking portion 132a in this embodiment is a helical projection. As a result, the first locking portion 113a and the second locking portion 132a can fit together in a screw-like structure. Therefore, the culture container 110 and the lid 130 can be removably locked together by the first locking portion 113a and the second locking portion 132a. That is, the culture container 110 and the lid 130 each include a first locking portion 113a and a second locking portion 132a for locking together.

[0044] It is desirable that the culture container 110 and the lid 130 be in close contact with each other when the first locking portion 113a and the second locking portion 132a are locked together. In this embodiment, since the upper end of the side wall portion 113 and the lower surface of the lid portion 131 are both flat, the culture container 110 and the lid 130 can be in close contact with each other when they are tightened and locked together by the screw structure. This makes it difficult for the culture medium etc. contained in the culture space 111 to leak out to the outside.

[0045] The material of the lid 130 may be, for example, resin, glass, metal, etc. Examples of resins include polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), and tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer. The material may include one or more of the following: body (PFA), acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyacetal (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), cyclic olefin polymer, AS resin (acrylonitrile-styrene copolymer), MS resin (methyl methacrylate-styrene copolymer), methacrylic resin (PMMA), acrylic resin, polyurethane resin, polytetrafluoroethylene (PTFE), etc. The glass may be soda glass, borosilicate glass, quartz glass, etc. The metal may be stainless steel, aluminum, etc. The lid 130 should be manufactured so that the culture medium does not easily leak out when it is locked to the culture vessel 110, and may be integrally formed, for example. The material and manufacturing method of the lid 130 are not limited to those exemplified herein.

[0046] Note that the shape of the lid 130 is not limited to those described herein. For example, the lid portion 131 may be a flat plate of an appropriate shape such as a rectangle, or it may be a plate of an appropriate shape.

[0047] Furthermore, the first locking portion 113a and the second locking portion 132a are examples of locking mechanisms for removably locking the culture container 110 and the lid 130 together. The first locking portion 113a and the second locking portion 132a do not both have to be helical protrusions. One of the first locking portion 113a and the second locking portion 132a may include a helical groove. This also allows for a screw structure to removably lock the culture container 110 and the lid 130 together. Moreover, the structure of the locking mechanism for removably locking the culture container 110 and the lid 130 together is not limited to a screw structure. The locking mechanism may, for example, have a structure in which a projection or groove and a claw engage to removably lock the culture container 110 and the lid 130 together.

[0048] (Regarding the culture sheet 150) The culture sheet 150 is a sheet that serves as a scaffold for culturing cells. When culturing cells, the culture sheet 150 is placed, for example, on the bottom 112 (more specifically, on the top surface 112a) and placed in the culture space 111.

[0049] The culture sheet 150 is a circular sheet having two main surfaces. One main surface is the surface that generally contacts the bottom 112 (more specifically, the top surface 112a) when it is placed in the culture space 111. The other main surface is the surface used for culturing cells (the culture surface). When cells are cultured, the cell sheet formed by the culture adheres to the culture surface.

[0050] The area of ​​the culture sheet 150 is not particularly limited, but it is desirable that it be smaller than the bottom 112 (more specifically, the top surface 112a) of the culture vessel 110 and larger than the cell sheet produced by the culture. For example, 0.1 cm ２ More than 1000cm ２ The following applies. More preferably, the lower limit is 0.2 cm. ２ More preferably 0.25 cm ２ The above is particularly preferably 0.3 cm ２That concludes the explanation. On the other hand, a more preferable upper limit would be 900 cm. ２ Further preferably 800 cm ２ The following is particularly preferred: 500 cm ２ The following applies. Note that the shape of the culture sheet 150 is not limited to a circle, but may be an appropriate shape such as an ellipse, rectangle, or polygon.

[0051] The thickness of the culture sheet 150 is, for example, 5 μm or more and 250 μm or less, preferably more than 10 μm and less than 30 μm. More preferably, the thickness of the culture sheet 150 is 25 μm or less. However, the thickness of the culture sheet 150 is not limited to those exemplified herein.

[0052] Both main surfaces of the culture sheet 150 may be smooth. "Smooth" means, for example, that there are no irregularities with a height of 1 μm or more, and that there are no irregularities that control the direction of cell growth, or both. Furthermore, the culture surface, which is one of the main surfaces for culturing cells, may be subjected to a surface treatment to maintain its smoothness. An example of such a surface treatment is plasma hydrophilization treatment. Note that the structure of the main surfaces of the culture sheet 150 is not limited to those exemplified here.

[0053] The material of the culture sheet 150 may include, for example, one or more of the following: polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyacetal (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), fibroin, cellulose, regenerated cellulose, cyclic olefin polymer, gelatin, collagen, etc.

[0054] (Regarding the ring-shaped member 170) The ring-shaped member 170 is removably housed in the culture space 111. When the ring-shaped member 170 is housed in the culture space 111, for example, the weight of the ring-shaped member 170 presses down on the culture sheet 150 placed on the upper surface 112a from above.

[0055] The ring-shaped member 170 is generally ring-shaped with open tops and bottoms, and is, for example, cylindrical or annular. However, the shape of the ring-shaped member 170 is not limited to cylindrical or annular; for example, when viewed from above, it may be a ring-shaped object of an appropriate predetermined shape such as an ellipse, rectangle, or polygon. Furthermore, the ring-shaped member 170 does not need to be a complete cylinder or annular shape; it may be a cylinder or annular shape with a portion missing, or it may be U-shaped.

[0056] The ring-shaped member 170 is housed in the culture container 110 with a gap between it and the inner surface of the culture container 110. For example, the outer surface of the ring-shaped member 170 (in this embodiment, the outer peripheral surface) is sized to fit onto the inner surface of the side wall portion 113 (in this embodiment, the inner peripheral surface). That is, in this embodiment, the inner diameter of the side wall portion 113 is larger than the outer diameter of the ring-shaped member 170.

[0057] The ring-shaped member 170 may be housed in the culture container 110 with an overall gap between its outer surface and the inner surface of the culture container 110. However, the outer surface of the ring-shaped member 170 and the inner surface of the side wall portion 113 may include a portion where one contacts the other. This allows the ring-shaped member 170 to be positioned so that it does not move within the culture space 111 when it is housed in the culture space 111.

[0058] When the ring-shaped member 170 is placed on the culture sheet 150 which is placed inside the culture container 110, it sandwiches at least a portion of the culture sheet 150 between itself and the bottom of the culture container 110. In other words, when the bottom surface of the ring-shaped member 170 is housed in the culture space 111, at least a portion of it overlaps with the culture sheet 150 which is placed on the upper surface 112a.

[0059] For example, since the culture sheet 150 and the ring-shaped member 170 are housed in the culture space 111, the diameter of the culture sheet 150 and the outer diameter of the ring-shaped member 170 may be smaller than the inner diameter of the side wall portion 113. More specifically, if the difference between the diameter of the culture sheet 150 and the outer diameter of the ring-shaped member 170 and the inner diameter of the side wall portion 113 is small, and the culture sheet 150 and the ring-shaped member 170 fit loosely into the side wall portion 113, then the culture sheet 150 and the ring-shaped member 170 will not be able to move much on the upper surface 112a. In this case, it is desirable that the inner diameter of the ring-shaped member 170 is smaller than the diameter of the culture sheet 150. This allows the culture sheet 150 to be roughly fixed on the upper surface 112a by pressing down on it from above with the weight of the ring-shaped member 170.

[0060] For example, if there is a large difference between the diameter of the culture sheet 150 and the outer diameter of the ring-shaped member 170 and the inner diameter of the side wall portion 113, then each can move on the upper surface 112a. It is desirable that the culture sheet 150 and the ring-shaped member 170 are sized such that when they move on the upper surface 112a, they always include a portion that overlaps with each other when viewed from above. This allows the culture sheet 150 to be generally fixed on the upper surface 112a by the weight of the ring-shaped member 170 pressing down on the culture sheet 150 from above, even if either the culture sheet 150 or the ring-shaped member 170 moves on the upper surface 112a.

[0061] The height of the ring-shaped member 170 may be, for example, less than or equal to the height of the culture space 111. The culture space 111 is a sealed space formed inside, for example, when a lid 130 is attached to a culture container 110. This allows the culture space 111 to be sealed by attaching the lid 130 to the culture container 110 while the ring-shaped member 170 is housed in the culture space 111. The degree of sealing may be, for example, airtight or liquid-tight.

[0062] The ring-shaped member 170 may include, for example, a holding portion 172 that is held for removal of the ring-shaped member 170. This allows the ring-shaped member 170 to be easily removed from the culture space 111.

[0063] More specifically, the ring-shaped member 170 may include a ring-shaped main body portion 171 that is open at the top and bottom, and a holding portion 172 provided on the main body portion 171.

[0064] The part to be held 172 may, for example, form a projection. The part to be held 172 may, for example, be a projection that protrudes upward from the main body 171.

[0065] The holding portion 172 may include opposing surfaces 172a, 172b, 172c, and 172d for gripping and holding using an instrument such as tweezers. The opposing surfaces 172a and 172b may be composed of opposing surfaces along the circumferential direction of the ring-shaped member 170 (main body portion 171). The opposing surfaces 172c and 172d may be composed of opposing surfaces along the radial direction of the ring-shaped member 170 (main body portion 171).

[0066] Note that the configuration of the held portion 172 is not limited to that exemplified here.

[0067] The specific gravity of the ring-shaped member 170 is greater than 1.0. It is desirable that the specific gravity of the ring-shaped member 170 be 1.05 or higher so that it sinks stably in the culture medium.

[0068] The material of the ring-shaped member 170 may include one or more of the following: resin, glass, ceramics, metal, etc. From the viewpoint of moldability and productivity, resin is preferred for the ring-shaped member 170. The resin may include, for example, one or more of the following: PEEK (polyether ether ketone), PET (polyethylene terephthalate), PBT (polybutylene terephthalate), PS (polystyrene), PC (polycarbonate), mPPE (modified polyphenylene ether), PPS (polyphenylene sulfide), PSU (polysulfone), PAR (polyarylate), LCP (liquid crystal polymer), ABS (acrylonitrile butadiene styrene), PES (polyethersulfone), POM (polyacetal), PI (polyimide), PA (polyamide, including nylon 6 and nylon 66), PGA (polyglycolic acid), PLA (polylactic acid), AS resin (acrylonitrile styrene copolymer resin), MS resin (methyl methacrylate styrene copolymer resin), PMMA (methacrylic resin), acrylic resin, polyurethane resin, fluororesin (PTFE (polytetrafluoroethylene), PVDF (polyvinylidene fluoride), etc.), polyphenyl ether resin, polyetherimide resin, vinyl chloride resin, etc. The glass may be soda-lime glass, borosilicate glass, quartz glass, etc. The metal may include one or more of the following: stainless steel, aluminum, etc. Furthermore, the ring-shaped member 170 may contain various additives, with the materials exemplified herein as the main material.

[0069] (Example of a method for producing cell cultures using cell culture kit 100) From here, an example of a method for producing and using cell cultures using cell culture kit 100 will be described.

[0070] Figure 3 shows an example of a flow chart for manufacturing cell cultures.

[0071] (Step 1) The culture sheet 150 is placed inside the culture container 110. More specifically, for example, the culture sheet 150 is placed with the culture surface facing upwards at the bottom 112 (more specifically, the top surface 112a). This places the culture sheet 150 within the culture space 111.

[0072] (Step 2) The ring-shaped member 170 is placed on the culture sheet 150. More specifically, for example, the ring-shaped member 170 is placed on the culture sheet 150 such that it overlaps at least a portion of the edge of the culture sheet 150 which is placed on the bottom 112. As a result, the culture sheet 150 is pressed down from above by the weight of the ring-shaped member 170.

[0073] (Step 3) The culture medium P is introduced into the culture space 111.

[0074] The culture medium P is not particularly limited as long as it is suitable for the cells being cultured. Examples of culture medium components include sugars, amino acids, vitamins, inorganic salts, trace metals, and additives. These culture medium components may be formulated individually or in combination of two or more. These culture medium components should be appropriately selected from known components depending on the cells being cultured.

[0075] Specific examples of sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; cyclodextrins; and oligosaccharides.

[0076] Specific examples of amino acids include L-glutamic acid, L-glutamine, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, and L-hydroxyproline.

[0077] Specific examples of vitamins include, for instance, L-sodium ascorbate, L-ascorbic acid diphosphate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0078] Specific examples of inorganic salts include, for instance, sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium bicarbonate, calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, calcium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, and magnesium carbonate.

[0079] Specific examples of trace metals include, for instance, iron sulfate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate.

[0080] Specific examples of additives include serums such as bovine serum, horse serum, and human serum; growth factors such as FGF2, EGF, HGF, VEGF, and PDGF; proteins such as albumin; antioxidants such as glutathione, ascorbic acid, and ascorbic acid derivatives; antibiotics such as penicillin and streptomycin; pH adjusters such as HEPES; organic acids such as lactic acid and propionic acid; lipids such as cholesterol; fatty acids such as linolenic acid; amines such as ethanolamine and putrescine; reducing agents such as mercaptoethanol and 3-mercapto-1,2-propanediol; thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, and pullulan; and pH indicators such as phenol red.

[0081] Examples of culture medium P containing the above-mentioned culture medium components include AIM V medium, HFDM-1 medium, equilibrium buffers such as Dulbecco's phosphate-buffered saline (D-PBS) and Hanks equilibrium salt solution (HBSS), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), α-MEM (Minimum Essential Medium alpha Modification), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), and Ham's F-10. medium, Ham's F-12 medium, Ham's F-12K medium, RPMI medium 1640, M-199 medium, L-15 medium, McCoy's 5A Medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's MB752 / 1 medium, CMRL-1066 Examples of basic culture media include medium, Williams' medium E, Brinster's BMOC-3 Medium, and E8 medium.

[0082] Furthermore, these basic culture media may be used individually or in combination of two or more. Additionally, the basic culture media may be modified by adding, removing, increasing, or decreasing the amount of culture media components depending on the type and condition of the cells. These basic culture media should be appropriately selected from known options depending on the cells being cultured.

[0083] (Step 4) Cells are introduced into the culture space 111.

[0084] Figure 4 is a side cross-sectional view showing an example of the culture container 110 after steps 1 to 4 have been completed. Since the culture sheet 150 is held down from above by the ring-shaped member 170, the culture sheet 150 is less likely to float in the culture medium P, and the culture sheet 150 can be reliably immersed in the culture medium P.

[0085] The order of the steps—arranging the culture sheet 150 (step 1), arranging the ring-shaped member 170 (step 2), introducing the culture medium P (step 3), and introducing the cells (step 4)—may be changed as appropriate. However, the arrangement of the ring-shaped member 170 (step 2) is performed after the arrangement of the culture sheet 150 (step 1). The introduction of the cells (step 4) is performed after the introduction of the culture medium P (step 3). Figure 4 shows an example in which an amount of culture medium P is introduced that completely immerses the ring-shaped member 170, but the amount of culture medium P introduced may be an amount that only partially immerses the ring-shaped member 170.

[0086] (Step 5) The lid 130 is placed on top of the culture container 110. For example, the lid 130 is placed on top of the culture container 110 so that the upper end of the culture container 110 fits into the hanging part 132. At this time, the culture container 110 and the lid 130 do not have to be completely locked together by the first locking part 113a and the second locking part 132a. In other words, the culture space 111 does not have to be airtight.

[0087] (Step 6) The introduced cells are cultured.

[0088] For example, the culture vessel 110 with the lid 130 on top is managed in a suitable environment, such as inside a constant-temperature incubator, and cells are cultured in the culture vessel 110. By not sealing the culture space 111 in step 5, the air necessary for cell culture can be circulated between the culture space 111 and the outside, allowing cells to be cultured in a suitable environment.

[0089] Once the cell culture is complete, as shown in Figure 5, the culture sheet 150 is covered with a cell culture containing the cultured cells. In other words, a "cell culture contained in a container" is produced. The "cell culture contained in a container" comprises a culture vessel 110, a lid 130, a culture sheet Q to which the cell culture is attached, and a ring-shaped member 170. The ring-shaped member 170 presses down from above on at least a portion of the culture sheet Q that does not have a cell culture attached.

[0090] (Step 7) Cool the cell culture.

[0091] For example, in cell cultures contained in a container, the cell cultures are immersed in a culture medium P, but this culture medium P may be replaced with a cryopreservation solution in the cooling step (step 7). It is preferable that the culture sheet Q to which the cell cultures are attached is cooled in the cryopreservation solution using a non-through-flow cooling device. By cooling the cell cultures on a suitable culture sheet using a non-through-flow cooling device, it is possible to cool the cell cultures, which are the object to be cooled, to a uniform temperature, thereby reducing damage to the cells and suppressing the decrease in cell activity.

[0092] In the cooling process, the cooling rate between 0 and -5°C is, for example, 0.1°C / min to 15°C / min, preferably 0.25°C / min to 12.5°C / min, and more preferably 0.5°C / min to 10°C / min. Setting the rate above the lower limit reduces the contact time between unnecessary liquid cryoprotective material and cells. Setting the rate below the upper limit suppresses the formation of intracellular ice crystals.

[0093] (Regarding cryopreservation solutions) The cryopreservation solution is not particularly limited as long as it is suitable for cryopreservation of cells, but it may contain culture medium components, cryoprotectants, etc. Regarding the culture medium components such as sugars, amino acids, vitamins, inorganic salts, trace metals, and additives, it is sufficient if they satisfy the above-mentioned explanation regarding the culture medium. Furthermore, it is preferable that the cryopreservation solution has a coagulation initiation temperature in the range of -15°C to -5°C.

[0094] Cryoprotective agents are substances used to reduce cell damage caused by freezing and thawing during cryopreservation. Examples of cryoprotective agents include cell-impermeable cryoprotective agents and cell-permeable cryoprotective agents. Specific examples of cell-impermeable cryoprotective agents include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine. Specific examples of cell-permeable cryoprotective agents include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, and propanediol. These cryoprotective agents may be formulated individually or in combination of two or more. The appropriate cryoprotective agents should be selected from known ones depending on the type of cell, the composition of the cryopreservation solution, etc.

[0095] Examples of commercially available cryopreservation solutions that do not contain DMSO include: Stem Cell Banker® DMSO-Free GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Banbanker® DMSO-Free (CG Lymphotec Co., Ltd.), Cryoscarless® DMSO-Free (Bioverde Co., Ltd.), Stem Cell Keep (Bioverde Co., Ltd.), CryoNovo® X12 (Akron BioProducts LCC Co., Ltd.), CryoNovo® P24 (Akron BioProducts LCC Co., Ltd.), DMSO-Free Cell Cryopreservation Solution for Human ES / iPS Cells (Reprocell Co., Ltd.), Cell Reserver One (Nacalai Tesque Co., Ltd.), ThelioKeep® (Bioverde Co., Ltd.), Cellvation® (Protide Co., Ltd.) Examples include Pharmaceuticals, ReproCryo RM (ReproCell), and SOFORO Cryo (Saraya). Examples of commercially available cryopreservation solutions containing DMSO include Stem Cell Banker® GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Stem Cell Banker® EX GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Banbankar® hRM (CG Lymphotek), Banbankar® (CG Lymphotek), iStock (CG Lymphotek), CryoStor CS5 (Charles River Laboratories Cell Solutions, Inc.), and CryoStor CS10 (Charles River Laboratories Cell Solutions, Inc.).

[0096] (Step 8) The cell cultures are frozen. This completes the method for producing the cell cultures.

[0097] The freezing temperature is not particularly limited as long as it can freeze the cultured cells. For example, the freezing temperature is between -196°C and -25°C. The lower limit is more preferably -180°C or higher, even more preferably -160°C or higher, and particularly preferably -150°C or higher. On the other hand, the upper limit is more preferably -25°C or lower, even more preferably -30°C or lower, and particularly preferably -35°C or lower.

[0098] The cooling device used for the freezing process is not particularly limited, and examples include rapid freezing devices and cryogenic refrigeration devices. As for the cooling device, a freezing device that does not come into contact with the heat transfer means and freezes by blowing cold air onto the culture vessel from multiple directions, preferably all directions, rather than from one direction, is preferable from the viewpoint of freezing cells at a uniform temperature and increasing the viability of cells after thawing. Specifically, examples of freezing devices that freeze by blowing cold air onto the culture vessel include cooling devices that cool the object to be cooled by circulating cold air with a cooling fan, such as the non-through-flow type cooling device equipped with a cooling fan disclosed in Japanese Patent Application Publication No. 2005-127666. The above "non-through-flow type" means a method in which most of the through-air from the object to be cooled does not pass through the cooler.

[0099] As a result, the cell cultures are frozen while still attached to the culture sheet 150, while remaining in the culture vessel 110 with the lid 130 attached. The cell cultures can then be stored (cryopreserved) and transported in their frozen state. The location where such storage takes place may be, for example, the manufacturer that produced the "cell cultures contained in the containers," or it may be a hospital or other facility where they are transported, but it is not limited to these locations.

[0100] The method of cryopreservation and transport is not particularly limited as long as it can stably cryopreserve the cells, but examples include contact of the coolant with the liquid or gas phase, and the use of an ultra-low temperature freezer. From a temperature perspective, the preferred method of cryopreservation and transport is contact of the coolant with the liquid or gas phase. Examples of coolants include liquid nitrogen, liquid ethane, liquid propane, liquid helium, and dry ice.

[0101] The temperature for cryopreservation and transport is not particularly limited as long as it allows for stable cryopreservation of the cells, but it can be within any of the following ranges, for example, -196 to -60°C, -196 to -134°C, or -134 to -60°C. This allows for the stable maintenance of cell sheets over a long period of time. The surface temperature of the cell culture to be frozen may be used as the temperature for cryopreservation and transport. The surface temperature can be measured, for example, using a K-type thermocouple.

[0102] As described above, in the cell culture kit 100, the height of the ring-shaped member 170 is less than or equal to the height of the culture space 111. Therefore, with the ring-shaped member 170 and the culture sheet Q to which the cell cultures are attached contained, the lid 130 can be attached to the culture container 110 (locked by the first locking part 113a and the second locking part 132a) to seal the culture space 111. This makes it possible to use the cell cultures contained in the container as they are and prevent foreign matter or microorganisms from entering the culture space 111 from the outside during work outside the sterile space (such as storage and transportation), thereby reducing the possibility of contamination of the cell cultures.

[0103] Furthermore, with the cell culture kit 100, there is no need to remove the ring-shaped member 170 for storage or transport of the cell cultures. Therefore, it is possible to prevent foam from being generated when removing the ring-shaped member 170 and contaminating the culture environment in which the cell culture kit 100 is placed. Cell culture is sometimes performed in a sterile environment, and if foam is generated and the culture medium and cells are scattered, it will contaminate the sterile environment. However, with the cell culture kit 100 of this disclosure, it is possible to proceed to the next process such as storage or transport without removing the ring-shaped member 170 after culturing, thus preventing contamination of the sterile environment.

[0104] The ring-shaped member 170 may be removed from the culture space 111 before freezing the culture medium P and cell cultures for storage, transport, etc.

[0105] Figure 6 shows an example of the flow of how to use cell cultures. The use of cell cultures begins, for example, when a cell culture kit 100 containing cell cultures is transported to a site where cell cultures are used, such as a medical facility.

[0106] (Step 11) The cell culture is thawed.

[0107] For example, cell cultures are thawed according to the instructions of a physician or other medical professional when they are used in surgery. Note that the timing and location of thawing are not limited to those exemplified here.

[0108] The thawing method is not particularly limited, and conventional methods used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as in basic technical fields such as regenerative medicine and biotechnology, can be used. Examples of thawing methods include using a water bath, bead bath, incubator, or hot plate, immersing in a thawing solution at a temperature higher than the freezing point, or leaving the product undisturbed in an environment at a temperature higher than the freezing point. The ambient temperature in contact with the frozen product during thawing is not particularly limited, as long as it is higher than the freezing point and 45°C or lower. Thawing temperatures above 45°C are undesirable because they may cause thermal damage to the cells. Alternatively, the product may be temporarily stored in an environment below its freezing point during thawing. For example, a frozen product stored at -80°C can be exposed to an ambient temperature of -30°C and then thawed at an ambient temperature above its freezing point.

[0109] The thawing medium is not particularly limited, as long as it does not damage the cultured cells. Examples of components that can be contained in the thawing medium include sucrose, glucose, maltose, trehalose, and fructose. The thawing medium may also contain the components listed in the above section (culture medium).

[0110] The temperature of the molten material is not particularly limited, as long as it is higher than the freezing point. For example, the temperature of the molten material is between 0°C and 45°C. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower.

[0111] (Step 12) The ring-shaped member 170 is removed.

[0112] For example, after removing the lid 130 from the culture container 110, the ring-shaped member 170 is removed from the culture space 111 through the opening 114a. At this time, the part to be held 172 can be held by gripping it with an instrument such as tweezers. Therefore, it becomes possible to easily hold the ring-shaped member 170 and remove it from the culture space 111.

[0113] In detail, for example, in settings where cell cultures are used, such as medical facilities, the ring-shaped member 170 is removed from the culture space 111. In such cases, it is particularly desirable that the ring-shaped member 170 can be easily removed from the culture space 111. As described above, by including the holding portion 172, it is possible to easily remove the ring-shaped member 170 from the culture space 111. Therefore, it is possible to minimize the decrease in convenience when using cell cultures.

[0114] (Step 13) The culture sheet Q with the cell culture attached is removed.

[0115] In detail, for example, using an instrument such as tweezers, the portion of the culture sheet 150 to which the cell culture is attached is held in place, while the culture sheet Q to which the cell culture is attached is held in place. The culture sheet Q to which the cell culture is attached is removed from the culture space 111 through the opening 114a.

[0116] When the culture sheet Q to which the cell cultures are attached is removed from the culture space 111, it may be washed with a cell washing solution as needed. The cell washing solution is not particularly limited and may contain the components described in the (culture medium) section above. The temperature of the cell washing solution is not particularly limited. For example, the temperature of the cell washing solution is 0°C or higher and 45°C or lower. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower. Note that the culture sheet Q to which the cell cultures are attached does not need to be washed.

[0117] (Step 14) The cell culture is peeled off the culture sheet 150. This completes the use of the cell culture. For example, the cell culture is attached to a transplant site such as a wound or affected area inside or outside the human body, and then peeled off from the culture sheet 150. The transplant site may be other than inside or outside the human body. Examples of transplant sites other than the recipient include other cell sheets, medical devices, and tissues or organs separated from the recipient. Examples of tissues or organs include skin, oral tissue, esophagus, trachea, bronchi, lungs, lung lobes, stomach, duodenum, pancreas, spleen, small intestine, large intestine, muscle tissue, bone, etc. (However, this excludes tissues that are returned to the same person for treatment as those collected from the recipient).

[0118] Embodiment 1 of the present disclosure has been described above.

[0119] (Function and Effects) According to this embodiment, the cell culture kit 100 comprises a culture container 110, a lid 130, and a ring-shaped member 170. The culture container 110 has a culture space 111 formed inside for culturing cells and includes an opening 114a that communicates with the culture space 111. The lid 130 is detachably attached to the culture container 110 so as to close the opening 114a. The ring-shaped member 170 is ring-shaped with open top and bottom and is detachably housed in the culture space 111. The height of the ring-shaped member 170 is less than or equal to the height of the culture space 111. The ring-shaped member 170 includes a holding portion 172 that forms a projection for holding the ring-shaped member 170 in order to remove it.

[0120] This allows the ring-shaped member 170 to be removed from the culture space 111 using the holding portion 172. Therefore, it becomes possible to easily hold and remove the ring-shaped member 170 from the culture space 111. For example, in settings where cell cultures are used, such as in medical settings, it becomes possible to easily remove the ring-shaped member 170 from the culture space 111.

[0121] Generally, for large areas (for example, an area of ​​8 cm²), ２While the cell sheets described above can be applied to affected areas of various sizes by adjusting their size as needed, many cell sheets are not very strong and may break during transport. The cell culture kit 100 allows for transport with the cell culture bodies contained within the kit. Therefore, the possibility of cell sheet damage can be reduced.

[0122] Furthermore, with the ring-shaped member 170 and the culture sheet Q to which the cell cultures are attached contained, the lid 130 can be attached to the culture container 110 to store or transport the cell cultures. This prevents the cell cultures from being exposed to the liquid surface during storage, freezing, or transport. Consequently, it becomes possible to reduce the risk of contamination of the cell cultures and drying of the cell cultures.

[0123] Furthermore, since the ring-shaped member 170 can be easily removed at the destination, it is not necessary to remove the ring-shaped member 170 in advance under the culture environment before storage or transportation. Therefore, it is possible to prevent foam from being generated and contaminating the culture environment when removing the ring-shaped member 170.

[0124] According to this embodiment, the ring-shaped member 170 is housed in the culture container 110 with a gap between it and the inner surface of the culture container 110.

[0125] This allows the ring-shaped member 170 to be housed in the culture vessel 110 without being fixed inside. Consequently, the ring-shaped member 170 can be easily removed.

[0126] According to this embodiment, the holding portion 172 is a projection and includes opposing surfaces 172a, 172b, 172c, and 172d for gripping and holding with a tool.

[0127] This allows the ring-shaped member 170 to be removed from the culture space 111 by gripping the part to be held 172. Therefore, the ring-shaped member 170 can be easily removed.

[0128] According to this embodiment, the opposing surfaces 172a and 172b are composed of opposing surfaces along the circumferential direction of the ring-shaped member 170.

[0129] This allows the retained portion 172 to be easily grasped and the ring-shaped member 170 to be removed from the culture space 111. Consequently, the ring-shaped member 170 can be removed even more easily.

[0130] According to this embodiment, the specific gravity of the ring-shaped member 170 is greater than 1.0.

[0131] This allows the ring-shaped member 170 to be submerged in the culture medium, ensuring that the culture sheet 150 is securely held down from above. Consequently, it becomes possible to reliably culture cells in the culture medium and produce cell cultures.

[0132] According to this embodiment, the material of the ring-shaped member 170 includes at least one of the following: PEEK (polyether ether ketone), PET (polyethylene terephthalate), PBT (polybutylene terephthalate), PS (polystyrene), PC (polycarbonate), mPPE (modified polyphenylene ether), PPS (polyphenylene sulfide), PSU (polysulfone), PAR (polyarylate), LCP (liquid crystal polymer), ABS (acrylonitrile butadiene styrene), PES (polyethersulfone), POM (polyacetal), PI (polyimide), PA (polyamide, including nylon 66), PGA (polyglycolic acid), PLA (polylactic acid), AS resin (acrylonitrile styrene copolymer resin), MS resin (methyl methacrylate styrene copolymer resin), PMMA (methacrylic resin), acrylic resin, polyurethane resin, PTFE (polytetrafluoroethylene), PVDF (polyvinylidene fluoride), glass, ceramics, and metal.

[0133] According to this embodiment, since the specific gravity of the ring-shaped member 170 is greater than 1.0, the ring-shaped member 170 can be submerged in the culture medium and the culture sheet 150 can be securely held down from above. Therefore, it becomes possible to reliably culture cells in the culture medium and produce cell cultures.

[0134] [Embodiment 2] In Embodiment 1, an example was described in which the retained portion 172 is a projection. In this embodiment, an example is described in which the retained portion forms a recess. In this embodiment, the differences from Embodiment 1 will be mainly described.

[0135] Figure 7 shows another example of the ring-shaped member 270. The ring-shaped member 270 includes a ring-shaped main body 272 that is open at the top and bottom, similar to the main body 171 in Embodiment 1, and a holding portion 272 provided on the main body 271. The holding portion 272 forms a recess in the main body 271. More specifically, for example, the holding portion 272 may be a part that forms a recess on the inner surface (e.g., the inner circumferential surface) of the main body 271. As a result, the holding portion 272 forms a recess that protrudes toward the outer surface (e.g., the outer circumferential surface) of the main body 271. For example, the tip of tweezers or the like can be hooked into the recess of the holding portion 272 to hold the ring-shaped member 270. Therefore, the holding portion 272 makes it possible to easily remove the ring-shaped member 270 from the culture space 111. The recess may also be a through hole extending from the inner circumferential surface to the outer circumferential surface of the main body 271.

[0136] Therefore, a cell culture kit equipped with a ring-shaped member 270 instead of the ring-shaped member 170 described in Embodiment 1 will also produce the same effects as in Embodiment 1.

[0137] Although the present disclosure has been described above with reference to embodiments, the present disclosure is not limited to the embodiments described above. Various modifications to the structure and details of the present disclosure are possible, as can be understood by those skilled in the art within the scope of the present disclosure. Furthermore, each embodiment can be combined with other embodiments as appropriate.

[0138] Furthermore, although the flowchart used in the above explanation shows multiple steps (processes) in order, the execution order of the steps performed in each embodiment is not limited to the order in which they are described. In each embodiment, the order of the illustrated steps can be changed to the extent that it does not impair the content.

[0139] This application claims priority based on Japanese Patent Application No. 2024-195453, filed on 7 November 2024, and incorporates all of its disclosures herein.

[0140] 100 Cell Culture Kit 110 Culture Container 111 Culture Space 112 Bottom 112a Top 112b Bottom 113 Side Wall 113a First Stop 114 Opening 114a Opening 115 Foot 130 Cap 131 Cap 132 Drooping Part 132a Second Stop 150 Culture Sheet 170, 270 Ring-shaped Parts 171, 271 Body 172, 272 Holding Parts 172a, 172b Opposite Surface

Claims

1. A cell culture kit comprising: a culture vessel having a culture space formed inside for culturing cells and an opening communicating with the culture space; a lid that can be attached to the culture vessel so as to close the opening; a culture sheet for culturing the cells; and a ring-shaped member that is open at the top and bottom and is removably housed in the culture space, wherein the ring-shaped member has a height less than or equal to the height of the culture space and includes a holding portion that forms a projection or recess for holding the ring-shaped member for removal.

2. The cell culture kit according to claim 1, wherein the ring-shaped member is housed in the culture vessel with a gap between it and the inner surface of the culture vessel.

3. The cell culture kit according to claim 1 or 2, wherein the holding portion is the projection and includes opposing surfaces for gripping and holding with an instrument.

4. The cell culture kit according to claim 3, wherein the opposing surfaces are composed of opposing surfaces along the circumferential direction of the ring-shaped member.

5. The cell culture kit according to claim 1 or 2, wherein the specific gravity of the ring-shaped member is greater than 1.

0.

6. The material of the ring-shaped member is PEEK (polyether ether ketone), PET (polyethylene terephthalate), PBT (polybutylene terephthalate), PS (polystyrene), PC (polycarbonate), mPPE (modified polyphenylene ether), PPS (polyphenylene sulfide), PSU (polysulfone), PAR (polyarylate), LCP (liquid crystal polymer), ABS (acrylonitrile butadiene styrene), PES (polyethersulfone), POM (polyacetal), PI (polyimide), PA (polyamide, A cell culture kit according to claim 1 or 2, comprising at least one of the following materials: nylon 6, nylon 66, PGA (polyglycolic acid), PLA (polylactic acid), AS resin (acrylonitrile-styrene copolymer resin), MS resin (methyl methacrylate-styrene copolymer resin), PMMA (methacrylic resin), acrylic resin, polyurethane resin, fluororesin (PTFE (polytetrafluoroethylene), PVDF (polyvinylidene fluoride), etc.), polyphenyl ether resin, polyetherimide resin, vinyl chloride resin, glass, ceramics, and metal.

7. The cell culture kit according to claim 1 or 2, wherein when the ring-shaped member is placed on the culture sheet placed inside the culture vessel, at least a portion of the culture sheet is sandwiched between the ring-shaped member and the bottom of the culture vessel.

8. The cell culture kit according to claim 1 or 2, wherein the culture space can be sealed when the lid is attached to the culture vessel.

9. The cell culture kit according to claim 2, wherein the holding portion is the projection and includes opposing surfaces for gripping and holding with an instrument, the ring-shaped member, when placed on the culture sheet placed inside the culture container, grips at least a portion of the culture sheet between itself and the bottom of the culture container, and the culture space can be sealed when the lid is attached to the culture container.

10. A method for producing a cell culture, comprising: placing a culture sheet for culturing cells inside a culture vessel having a culture space formed inside and an opening communicating with the culture space; placing a ring-shaped member, which is open at the top and bottom and is removablely housed in the culture space, on top of the culture sheet; introducing a culture medium into the culture space; introducing cells into the culture space; placing a removable lid on top of the culture vessel so as to close the opening; and culturing the introduced cells to obtain a cell culture on the culture sheet, wherein the ring-shaped member has a height less than or equal to the height of the culture space and includes a holding portion that forms a projection or recess for holding the ring-shaped member for removal.

11. The method for producing a cell culture according to claim 10, further comprising freezing the cell culture.

12. A culture vessel comprising: an internal culture space for culturing cells and an opening communicating with the culture space; a lid that can be attached to and removed from the culture vessel to close the opening; a culture sheet disposed inside the culture vessel to which cell cultures are attached; and a ring-shaped member that is open at the top and bottom and is removably housed in the culture space, wherein the ring-shaped member has a height less than or equal to the height of the culture space and includes a holding portion that forms a projection or recess for holding the ring-shaped member for removal, and at least a portion of the culture sheet to which the cell cultures are not attached is sandwiched between the bottom of the culture vessel.

13. The cell culture is contained in the container according to claim 12, wherein the cell culture is frozen by being immersed in a cryopreservation solution.

Images