Cell culture kit and method for producing cell culture

Abstract

This cell culture kit (100) is provided with a culture vessel (110), a spacer (130), and a lid (150). The culture vessel (110) has therein a culture space (111) for culturing cells, and includes an opening (114a) that communicates with the culture space (111). The spacer (130) is a cylinder with an open top and bottom, and is removably stored in the culture space (111). The lid (150) can be placed on the spacer (130). The spacer (130) is taller than the culture space (111).

Description

Cell culture kit and method for producing cell culture

[0001] The present invention relates to a cell culture kit and a method for producing a cell culture.

[0002] Patent Document 1 discloses a cell culture vessel used for cell culture. The cell culture vessel described in Patent Document 1 includes a lid and a vessel body. The vessel body described in Patent Document 1 includes a side wall and a bottom, and is formed with an opening that opens upward. The opening is covered and sealed by attaching a lid. Patent Document 1 also describes that a through-hole (opening) for perfusion may be provided in the side wall of the vessel body.

[0003] International Publication No. 2006 / 123570

[0004] In the cell culture vessel described in Patent Document 1, as described above, when the lid is attached to the vessel body, the opening is sealed. This may result in a lack of gas suitable for cell culture, making it difficult to culture the cells well. Furthermore, if cells are cultured with the lid removed or if through-holes are provided in the sidewall of the vessel body, contaminants may enter the vessel body through the opening or through-hole, potentially contaminating the cells being cultured. As such, it may be difficult to culture cells well using the cell culture vessel described in Patent Document 1.

[0005] One of the objectives of the present application is to enable cells to be cultured well.

[0006] According to one aspect of the present invention, the following cell culture kit and method for producing cultured cells are provided.

[0007] 1. A cell culture kit comprising: a culture vessel having a culture space formed therein for culturing cells and including an opening communicating with the culture space; a cylindrical spacer that is open at the top and bottom and is removably housed in the culture space; and a lid that can be placed on the spacer, wherein the height of the spacer is greater than the height of the culture space. 2. The cell culture kit described in 1., wherein the spacer includes one or more holes that penetrate in a direction intersecting the height direction. 3. The cell culture kit described in 2., wherein the holes form notches that are open at the top. 4. The culture vessel includes a bottom and a side wall extending upward from the bottom, and the lid includes a plate-shaped lid portion and a hanging portion that extends downward from the lid portion and can be fitted into the side wall portion, and includes a locking mechanism for removably locking the culture vessel and the lid to each other, and the spacer has a height that prevents the locking mechanism from engaging when housed in the culture space. 5. The cell culture kit according to any one of 4., wherein the height of the spacer is smaller than the sum of the heights of the side wall portion and the hanging portion. 6. The cell culture kit according to 4. or 5., wherein the side wall portion and the hanging portion are each cylindrical, and the inner diameter of the hanging portion is larger than the outer diameter of the side wall portion. 7. The cell culture kit according to any one of 1. to 6., wherein the opening is provided above the culture vessel, and the lid is attached to the top of the culture vessel to cover the opening. 8. The cell culture kit according to any one of 4. to 7., further comprising a culture member that is a scaffold for culturing the cells, the culture member having a size and shape to be contained in the culture space and sandwiched between the bottom and the spacer. The cell culture kit according to 9. The cell culture kit according to any one of 4. to 8., wherein the lid and the culture vessel are in tight contact with each other when locked by the locking mechanism. 10. 10. The cell culture kit according to any one of 4. to 9., wherein the locking mechanism locks the lid and the culture vessel together with a screw structure.11. A method for producing a cell culture construct, comprising placing a lid on a spacer of a culture vessel having at least cells and a spacer disposed therein, culturing the cells in the culture vessel, removing the spacer from the culture vessel, and attaching a lid to the culture vessel to tightly seal it. 12. The method for producing a cell culture construct according to 11, wherein the cultured cells are a three-dimensional cell culture construct.

[0008] According to the present invention, cells can be cultured well.

[0009] 4 is a perspective view showing an example of each component constituting the cell culture kit according to embodiment 1. FIG. 5 is a side view showing an example of a culture vessel according to embodiment 1. FIG. 6 is a plan view showing an example of a culture vessel according to embodiment 1. FIG. 7 is a side cross-sectional view of the culture vessel taken along line A-A in FIG. 3. FIG. 8 is a diagram showing an example of the flow of a method for producing a cell culture construct according to embodiment 1. FIG. 9 is a side cross-sectional view showing an example of a state in which a spacer according to embodiment 1 is arranged in a culture vessel. FIG. 10 is a side cross-sectional view showing an example of a state in which a lid is placed on the spacer according to embodiment 1. FIG. 11 is a perspective view showing an example of a culture member according to embodiment 2. FIG. 12 is a diagram showing an example of the flow of a method for producing a cell culture construct according to embodiment 2. FIG. 13 is a side cross-sectional view showing an example of a state in which the spacer is arranged in the culture vessel by placing the spacer on the culture member according to embodiment 2. FIG. 14 is a perspective view showing another example of a spacer. FIG. 15 is a perspective view showing yet another example of a spacer.

[0010] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all drawings, similar components are designated by similar reference numerals, and descriptions thereof will be omitted where appropriate. The drawings are schematic diagrams and do not correspond to actual dimensional proportions. Terms indicating directions such as up, down, left, and right are used for the purpose of explanation and are not intended to limit the present invention.

[0011] 1 is a perspective view showing an example of each member constituting a cell culture kit 100 according to embodiment 1. The cell culture kit 100 includes a culture vessel 110, a spacer 130, and a lid 150.

[0012] (Regarding the culture vessel 110) Fig. 2 is a side view showing an example of the culture vessel 110 according to this embodiment. Fig. 3 is a plan view showing an example of the culture vessel 110 according to this embodiment. Fig. 4 is a side cross-sectional view of the culture vessel 110 taken along line AA shown in Fig. 3.

[0013] The culture vessel 110 has a culture space 111 formed therein for culturing cells in a culture solution, and includes an opening 114 a communicating with the culture space 111 .

[0014] The culture vessel 110 according to this embodiment is cylindrical and has a bottom, and includes a bottom 112 , a sidewall 113 , and an opening 114 .

[0015] The shape of the culture vessel 110 is not limited to a cylindrical shape with a circular bottom 112 and side wall 113 when viewed from above (e.g., in the normal direction of the bottom 112). For example, the culture vessel 110 may be a cylindrical shape with a bottom, with the bottom 112 and side wall 113 having a predetermined shape such as an ellipse, rectangle, or polygon when viewed from above, or may have another shape.

[0016] The bottom portion 112 is flat and includes an upper surface 112a and a lower surface 112b.

[0017] The upper surface 112a is the inner surface of the bottom part 112 and forms the culture space 111. The upper surface 112a according to this embodiment is used as a scaffold for culturing cells.

[0018] The lower surface 112b is the outer surface of the bottom portion 112. For example, when the culture vessel 110 is placed on a support portion such as a table or an incubator, the lower surface 112b comes into contact with or faces the support portion.

[0019] In the culture vessel 110 according to this embodiment, legs are provided that extend downward from the lower surface 112b and have an outline that is approximately circular when viewed from above. Therefore, the lower surface 112b is placed opposite the mounting portion with a gap corresponding to the length of the legs. The shape of the legs is not limited to this and may be a polygonal shape such as a hexagon. Alternatively, the legs may not be provided, in which case the lower surface 112b may contact the mounting portion, for example.

[0020] 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 (inner peripheral surface in this embodiment) of the side wall portion 113, together with the upper surface 112a, forms the culture space 111. The side wall portion 113 has a first locking portion 113a at the top of its outer surface (outer peripheral surface in this embodiment). The first locking portion 113a in this embodiment is a spiral ridge.

[0021] 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. Furthermore, the opening 114a according to this embodiment is provided above the culture vessel 110.

[0022] The culture vessel 110 may be generally called, for example, a dish, Petri dish, tissue culture dish, multi-dish, flask, tissue culture flask, microplate, microwell plate, multi-plate, multi-well plate, chamber slide, Petri dish, tube, tray, culture bag, roller bottle, etc. Note that the general names of the culture vessel 110 are not limited to those exemplified here.

[0023] The material of the culture vessel 110 may be, for example, resin, glass, metal, etc. The resin may contain one or more of, for example, polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyethylene terephthalate, cellulose, silicone, nylon 6,6, cyclic olefin polymer, polycarbonate, etc. The metal may be stainless steel, aluminum, etc. The culture vessel 110 may be manufactured so as to prevent leakage of the culture solution, and may be formed as a single unit, for example. Note that the material and manufacturing method of the culture vessel 110 are not limited to those exemplified here.

[0024] The area of ​​the bottom 112 of the culture vessel 110 is not particularly limited, but any commercially available culture vessel can be used for culture without any problems. For example, ２ More than 1000cm ２ The lower limit is more preferably 0.35 cm ２More preferably, 1.0 cm or more ２ More than 1.9 cm, particularly preferably ２ On the other hand, the upper limit is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ The following is the result.

[0025] (Regarding the spacer 130) The spacer 130 is a member that is removably housed in the culture space 111. The spacer 130 includes openings 130a and 130b at the top and bottom, respectively. For example, the spacer 130 has a tubular shape that is open at the top and bottom, such as a cylindrical shape or an annular shape.

[0026] However, the shape of the spacer 130 is not limited to these. The spacer 130 may be, for example, a cylindrical shape having an appropriate predetermined shape such as an oval, a rectangle, or a polygon when viewed from above.

[0027] In detail, for example, the outer surface (in this embodiment, the outer peripheral surface) of the spacer 130 is sized to fit into the inner surface (in this embodiment, the inner peripheral surface) of the side wall portion 113. That is, in this embodiment, the inner diameter of the side wall portion 113 is larger than the outer diameter of the spacer 130. Note that when the spacer 130 is housed in the culture space 111, the outer surface of the spacer 130 and the inner surface of the side wall portion 113 may have portions where one contacts the other so that the spacer 130 does not move within the culture space 111.

[0028] The height of the spacer 130 is greater than the height of the culture space 111 .

[0029] The spacer 130 also includes two holes 131 that penetrate in a direction intersecting the height direction. Each of the holes 131 forms a hole that penetrates in the thickness direction of the spacer 130. Each of the holes 131 according to this embodiment forms a notch that is open at the top end of the spacer 130.

[0030] The number of hole 131 may be one or three or more. The hole 131 may be at least one of a notch that is open upward at the upper end of the spacer 130 and a notch that is open downward at the lower end of the spacer 130.

[0031] The material of the spacer 130 may be, for example, resin, glass, metal, etc. The resin may include, for example, one or more of polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyethylene terephthalate, cellulose, silicone, nylon 6,6, cyclic olefin polymer, polycarbonate, etc. The metal may be stainless steel, aluminum, etc.

[0032] (Regarding the Lid 150) The lid 150 covers the opening 114a by being attached to the culture vessel 110. In this embodiment, the lid 150 is attached above the culture vessel 110 to cover the opening 114a.

[0033] The lid 150 is a member that can be placed on the spacer 130. When the lid 150 is placed on the spacer 130, it covers the opening 130a above the spacer 130.

[0034] The lid 150 includes a second locking portion 152a that engages with the first locking portion 113a to removably lock the incubation vessel 110 and the lid 150 together.

[0035] The lid 150 according to this embodiment includes a lid portion 151 and a hanging portion 152. Note that the lid 150 in FIG.

[0036] The lid portion 151 has a flat plate shape. More specifically, the lid portion 151 has a circular flat plate shape.

[0037] Hanging portion 152 extends downward from lid portion 151. Specifically, hanging portion 152 extends downward from lid portion 151 when lid 150 is attached to culture vessel 110. More specifically, hanging portion 152 extends downward from the outer edge of lid portion 151.

[0038] The hanging part 152 includes a second locking part 152a at the upper part of the inner surface (inner circumferential surface in this embodiment). The second locking part 152a according to this embodiment is a spiral ridge.

[0039] The inner surface of hanging portion 152 is sized to fit loosely onto the outer surface of side wall portion 113. In the present embodiment, the inner diameter of hanging portion 152 is larger than the outer diameter of side wall portion 113. That is, hanging portion 152 can be fitted into side wall portion 113. Also, as described above, the outer surface (in the present embodiment, the outer peripheral surface) of spacer 130 is sized to fit onto the inner surface (in the present embodiment, the inner peripheral surface) of side wall portion 113. Therefore, the inner surface of hanging portion 152 is larger than the outer surface of spacer 130, and by placing lid 150 on spacer 130, opening 130a above spacer 130 can be covered.

[0040] When the lid 150 is attached to the incubation vessel 110, the first locking portion 113a and the second locking portion 152a fit together using a screw structure. Therefore, the incubation vessel 110 and the lid 150 can be removably locked to each other by the first locking portion 113a and the second locking portion 152a. That is, the incubation vessel 110 and the lid 150 each include the first locking portion 113a and the second locking portion 152a for locking them to each other.

[0041] It is desirable that the culture vessel 110 and the lid 150 closely contact each other when the first locking portion 113a and the second locking portion 152a are locked. In this embodiment, the upper end of the side wall portion 113 and the lower surface of the lid portion 151 are both flat, so the culture vessel 110 and the lid 150 can closely contact each other when they are fastened and locked with a screw structure. This makes it difficult for the culture medium and the like contained in the culture space 111 to leak to the outside.

[0042] The material of the lid 150 may be, for example, resin, glass, metal, etc. The resin may include, for example, one or more of polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyethylene terephthalate, cellulose, silicone, nylon 6,6, cyclic olefin polymer, polycarbonate, etc. The metal may be stainless steel, aluminum, etc. The lid 150 may be manufactured so as to prevent leakage of the culture solution when engaged with the culture vessel 110, and may be formed integrally, for example. Note that the material and manufacturing method of the lid 150 are not limited to those exemplified here.

[0043] The shape of the lid 150 is not limited to that described here. For example, the lid portion 151 may be a flat plate having an appropriate shape such as a rectangular shape, or may be a plate having an appropriate shape.

[0044] Furthermore, the first locking portion 113a and the second locking portion 152a are an example of a locking mechanism for detachably locking the culture vessel 110 and the lid 150 to each other. The first locking portion 113a and the second locking portion 152a do not both have to be spiral protrusions. One of the first locking portion 113a and the second locking portion 152a may include a spiral groove. This also allows the culture vessel 110 and the lid 150 to be detachably locked to each other using a screw structure. Furthermore, the structure by which the locking mechanism detachably locks the culture vessel 110 and the lid 150 to each other is not limited to a screw structure. The locking mechanism may be, for example, a structure in which a protrusion or groove and a claw engage with each other to detachably lock the culture vessel 110 and the lid 150 to each other.

[0045] (Example of a method for producing a cell culture construct using the cell culture kit 100) Hereinafter, an example of a method for producing a cell culture construct using the cell culture kit 100 will be described. The cell culture construct contains cultured cells. The cell culture construct is, for example, a three-dimensional cell culture construct such as a cell sheet, a spheroid, or an organoid, but is not limited to these.

[0046] FIG. 5 is a diagram showing an example of the flow of a method for producing a cell culture construct.

[0047] (Step 1) The spacer 130 is placed in the culture vessel 110. In detail, for example, the spacer 130 is placed on the bottom 112 (specifically, the upper surface 112 a) with the hole 131 facing upward, thereby placing the spacer 130 in the culture space 111.

[0048] 6 is a side cross-sectional view showing an example of a state in which the spacer 130 is placed in the culture vessel 110. As described above, the height of the spacer 130 is greater than the height of the culture space 111. Therefore, when the spacer 130 is accommodated in the culture space 111, it protrudes above the culture vessel 110.

[0049] (Step 2) Cells are introduced into the culture space 111.

[0050] The cell culture method may be adhesion cell culture, in which cells are seeded on the bottom 112 (specifically, the upper surface 112a). Alternatively, a culture medium may be introduced into the culture space 111. The culture medium may be liquid or solid. The cell culture method may be suspension cell culture, in which cells are suspended in a culture medium.

[0051] Note that step 1 may be performed after step 2 is performed.

[0052] (Step 3) The lid 150 is placed on the spacer 130. In detail, for example, the lid 150 is placed on the spacer 130 of the culture vessel 110 having at least cells and the spacer 130 arranged inside (culture space 111). For example, when a culture medium is used, the lid 150 is placed on the spacer 130 of the culture vessel 110 having cells, the culture medium, and the spacer 130 arranged inside.

[0053] FIG. 7 is a side cross-sectional view showing an example of the state in which the lid 150 is placed on the spacer 130. As described above, the inner surface of the hanging portion 152 is larger than the outer surface of the spacer 130. Therefore, for example, the hanging portion 152 can be directed downward, the spacer 130 can be positioned within the hanging portion 152, and the lid 150 can be placed on the spacer 130. This allows the upper opening 130a of the spacer 130 to be covered by the lid portion 151. This makes it difficult for substances that contaminate cells in the culture space 111 to enter the culture space 111, thereby reducing the possibility of cell contamination. Furthermore, since evaporation and drying of the culture medium can be reduced, a good culture environment can be maintained for longer than if the lid 150 is not provided. This enables cells to be cultured well.

[0054] On the other hand, the spacer 130 creates a gap between the vicinity of the upper part of the culture vessel 110 and the lid 150, through which gases such as oxygen can flow. This allows gases suitable for cell culture to flow into the culture space 111. In particular, in this embodiment, the spacer 130 is provided with the holes 131, which allows gases suitable for cell culture to flow more smoothly into the culture space 111. This makes it possible to culture cells well.

[0055] Here, it is desirable that the spacer 130 has a height that prevents the locking mechanism (e.g., the first locking portion 113a and the second locking portion 152a) from engaging with each other when the spacer 130 is housed in the culture space 111. This makes it possible to more reliably provide a gap between the vicinity of the upper portion of the culture vessel 110 and the lid 150. This allows for better cell culture.

[0056] Furthermore, it is desirable that the height h1 of the spacer 130 be smaller than the sum of the heights h2 and h3 of the sidewall 113 (culture space 111) and the hanging portion 152 (the inner surface of the hanging portion 152) (i.e., height h1 of the spacer 130 < height h2 of the sidewall 113 + height h3 of the hanging portion 152). This creates an overlap between the hanging portion 152 and the sidewall 113 when viewed from the side, making it more difficult for substances that contaminate cells in the culture space 111 to enter the culture space 111, further reducing the possibility of cell contamination. Furthermore, since evaporation and drying of the culture medium can be reduced, a better culture environment can be maintained for longer than in a case where the lid 150 is not provided. This allows for better cell culture.

[0057] (Step 4) The cells are cultured in the culture space 111. The culture vessel 110 may be placed in a constant temperature incubator, and the cells are cultured in an appropriate environment. When the cell culture is completed, a cell culture mass is formed in the culture space 111.

[0058] (Step 5) The lid 150 is removed from the spacer 130, and the spacer 130 is taken out from the culture vessel 110 (specifically, the culture space 111).

[0059] After the spacers 130 are removed, the cell culture mass containing the cultured cells remains attached to the bottom 112 (specifically, the upper surface 112a).

[0060] (Step 6) The lid 150 is attached to the culture vessel 110, completing the production of the cell culture. At this time, the culture vessel 110 and the lid 150 may be locked with the first locking portion 113a and the second locking portion 152a. The culture vessel 110 and the lid 150 may be tightly attached to seal the culture space 111. The cell culture may then be stored using the culture vessel 110 with the lid 150 attached. For storage, a cryopreservation solution may be poured into the culture space 111 before the lid 150 is attached, and the cell culture may be cryopreserved. Alternatively, the culture medium may be removed and replaced with the cryopreservation solution.

[0061] Alternatively, an appropriate process such as centrifugation may be performed on the cell culture while it is housed in the culture space 111. After this process, the lid 150 may be removed from the culture vessel 110, and the medium may be removed from the culture space 111 by a technique such as suction. Thereafter, the lid 150 may be reattached to the culture vessel 110, and the cell culture may be stored using the culture vessel 110 with the lid 150 attached.

[0062] After the spacer 130 is removed (step 5), the lid 150 does not have to be attached. For example, the cells together with the medium may be removed from the culture space 111 by a method such as suction, and an appropriate process such as centrifugation may be performed.

[0063] According to this embodiment, during cell culture, the spacer 130 can be placed inside the culture vessel 110 and the lid 150 can be placed on the spacer 130. This reduces the possibility of contamination of the cells being cultured, thereby enabling the cells to be cultured well.

[0064] Furthermore, the height of the spacer 130 is greater than the height of the culture space 111. Therefore, the spacer 130 forms a gap between the lid 150 placed on top of it and the culture vessel 110. Because gas such as air can circulate through this gap, it is possible to improve the breathability for culturing cells compared to placing the lid 150 directly on the culture vessel 110. Therefore, it becomes possible to culture cells well.

[0065] Furthermore, after cell culture, the lid 150 can be locked to the culture vessel 110 by removing the spacer 130. Therefore, the culture vessel 110 with the lid 150 attached can be used for processes such as centrifugation. Furthermore, storing the culture vessel 110 with the lid 150 attached reduces the possibility of the cell culture being contaminated by the lid 150 being removed, etc. Therefore, the culture vessel 110 can be suitably used for processing and storing the cell culture.

[0066] [Embodiment 2] The cell culture kit may further include a culture member 270. The culture member 270 is a scaffold for culturing cells. When culturing cells, the culture member 270 is placed, for example, on the bottom 112 (specifically, the upper surface 112a) and accommodated in the culture space 111. Therefore, the culture member 270 has a size that can be accommodated in the culture space 111.

[0067] 8 is a perspective view showing an example of a culture member 270. The culture member 270 shown in the figure is a sheet having two main surfaces. Specifically, the culture member 270 is a circular sheet that is smaller than the bottom portion 112 (specifically, the upper surface 112a).

[0068] The shape of the culture member 270 when viewed from the surface normal direction may be any suitable shape, such as a rectangle, a regular polygon such as a square, a regular pentagon, a regular hexagon, or a regular octagon, or an oval. When viewed from the surface normal direction, the culture member 270 may not have an opening that penetrates through the front and back surfaces. When placed in the culture vessel 110 and viewed from the surface normal direction of the culture member 270, it is preferable that the culture member 270 does not have an opening that exposes the bottom surface of the culture vessel 110.

[0069] One of the main surfaces of the culture member 270 is the main surface that is in general contact with the bottom 112 (specifically, the upper surface 112a) when the culture member 270 is housed in the culture space 111. The other main surface is the main surface (culture surface) used for cell culture. When cells are cultured, a cell culture formed thereby adheres to the culture surface. In other words, the culture member 270 is also a member that supports a cell sheet containing cells cultured thereon.

[0070] The area of ​​the culture member 270 is not particularly limited, but is preferably smaller than the bottom of the culture vessel and the same as or larger than the cell sheet. ２ More than 1000cm ２ The lower limit of the area of ​​the culture member 270 is preferably 0.6 cm ２ More preferably, 1.6 cm or more ２ More than 8 cm, especially preferred ２ More than 3 cm ２ More than 4cm ２ Above, 5cm ２ More than 6cm ２ More than 7cm ２ or more or 10 cm ２ On the other hand, the upper limit of the area of ​​the culture member 270 is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably, 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm ２ For example, the area of ​​the culture member 270 may be 0.6 cm ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ The following is the result.

[0071] The thickness of the culture member 270 is, for example, 5 μm or more and 250 μm or less. The lower limit of the thickness of the culture member 270 is more preferably 6 μm or more, and even more preferably 8 μm or more, 10 μm or more, 11 μm or more, or 12 μm or more. On the other hand, the upper limit of the thickness of the culture member 270 is more preferably 50 μm or less, 30 μm or less, 25 μm or less, and even more preferably 20 μm or less. For example, the thickness of the culture member 270 is 6 μm or more and 50 μm or less, 8 μm or more and 30 μm or less, or 10 μm or more and 20 μm or less. Note that the thickness of the culture member 270 is not limited to those exemplified here.

[0072] Both main surfaces of the culture member 270 may be smooth. "Smooth" means, for example, that there are no irregularities of 1 μm or more in height, or that there are no irregularities that would restrict the direction of cell growth. Furthermore, the culture surface, which is one of the main surfaces for culturing cells, may be subjected to a surface treatment to maintain smoothness. An example of such a surface treatment is plasma hydrophilization.

[0073] The shape and structure of the culture member 270 are not limited to those described here.

[0074] The material of the culture member 270 may include one or more of, for example, polyetheretherketone (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, and the like. The culture member 270 may be made of a film containing at least one of these materials as a main component. The culture member 270 is preferably made of a transparent material with a specific gravity greater than 1.0 and excellent durability, mechanical strength, and processability. The main component of the culture member 270 is preferably a polyether ether ketone film, a polyethylene terephthalate film, or polystyrene, and more preferably a polyether ether ketone film.

[0075] The surface roughness Ra of the culture surface of the culture member 270 has a lower limit of, for example, 0.3 nm or more, preferably 0.5 nm or more, and an upper limit of, for example, 100 nm or less, preferably 10 nm or less, more preferably 2 nm or less. Note that the surface roughness Ra here refers to the arithmetic mean roughness in a square area with sides of 100 nm, measured using surface shape data measured with an atomic force microscope (AFM).

[0076] When the culture surface of the culture member 270 is measured with a laser microscope, it is preferable that there are three or fewer holes, and more preferably zero holes, each having a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm within a 100 μm square area. The diameter of these holes is, for example, 1 μm to 100 μm, preferably 2 μm to 50 μm, and more preferably 3 μm to 30 μm. The depth of these holes is, for example, 0.5 μm to 100 μm, preferably 1 μm to 50 μm, and more preferably 2 μm to 20 μm.

[0077] The combination of the ranges of the hole diameter and hole depth on the culture surface of the culture member 270 is, for example, a hole diameter of 1 μm to 100 μm and a hole depth of 0.5 μm to 100 μm, a hole diameter of 1 μm to 100 μm and a hole depth of 1 μm to 50 μm, a hole diameter of 1 μm to 100 μm and a hole depth of 2 μm to 20 μm, or a hole diameter of 2 μm to 50 μm and a hole depth of 0.5 μm to 100 μm. The hole diameter is 2 μm or more and 50 μm or less and the hole depth is 1 μm or more and 50 μm or less, the hole diameter is 2 μm or more and 50 μm or less and the hole depth is 2 μm or more and 20 μm or less, the hole diameter is 3 μm or more and 30 μm or less and the hole depth is 0.5 μm or more and 100 μm or less, the hole diameter is 3 μm or more and 30 μm or less and the hole depth is 1 μm or more and 50 μm or less, and the hole diameter is 3 μm or more and 30 μm or less and the hole depth is 2 μm or more and 20 μm or less.

[0078] The upper limit of the porosity of the culture member 270 is, for example, 15% or less, preferably 10% or less, and more preferably 5% or less. On the other hand, the lower limit of the porosity of the culture member 270 is not particularly limited, but may be 0% or more.

[0079] By limiting the number of holes on the culture surface of the culture member 270 to three or less and / or by limiting the porosity of the culture member 270 to the above upper limit or less, the adhesive strength with the cell sheet can be made appropriate. Here, "A and / or B" means that either "A and B" or "A or B" is acceptable.

[0080] The presence or absence of holes on the culture surface of the culture member 270 may be measured on the surface of the resin layer formed on the culture surface side of the culture member 270 .

[0081] The porosity is calculated from the theoretical density and the actually measured density. Specifically, for example, the porosity may be calculated by the formula: porosity={1−(actual density / theoretical density)}×100.

[0082] The culture surface of the culture member 270 may be configured not to contain a temperature-responsive polymer, which can prevent a decrease in adhesion between the cell sheet and the culture member 270 in a low-temperature environment such as during a cryopreservation process.

[0083] A temperature-responsive polymer is a material that exhibits cell adhesiveness at the temperature used for cell culture, and exhibits cell non-adhesiveness by changing the temperature from that temperature, making it possible to easily detach a cell sheet. The temperature range in which the temperature-responsive polymer exhibits cell adhesiveness is preferably 10°C to 45°C, particularly 33°C to 40°C, because this allows stable cell culture. Furthermore, the temperature range in which the temperature-responsive polymer exhibits cell non-adhesiveness is preferably 1°C to 36°C, particularly 4°C to 32°C, because this reduces damage to the detachment of the cell sheet.

[0084] The material constituting the temperature-responsive polymer may be, for example, a temperature-responsive polymer such as poly-N-isopropylacrylamide (PNIPAAm), poly-N-n-propylacrylamide, poly-N-n-propylmethacrylamide, poly-N-ethoxyethylacrylamide, poly-N-tetrahydrofurfurylacrylamide, poly-N-tetrahydrofurfurylmethacrylamide, and poly-N,N-diethylacrylamide. Of these, PNIPAAm, poly-N-n-propylmethacrylamide, and poly-N,N-diethylacrylamide are preferred as the material constituting the temperature-responsive polymer.

[0085] The bottom 112 (specifically, the upper surface 112a) of the culture vessel 110 may also be configured so as not to contain a temperature-responsive polymer.

[0086] (Cell Sheet) A cell sheet has a sheet structure in which cells are physically and functionally connected to one another via adhesion molecules, extracellular matrix, and the like.

[0087] The cell sheet may have a single-layer structure consisting of one cell layer, or a laminated structure consisting of two or more cell layers. The laminated structure is not particularly limited, but examples include multi-layer structures such as two-layer, three-layer, four-layer, and five-layer structures.

[0088] When a cell sheet has a multilayer structure, the multilayer structure may be obtained when cultured on the culture member 270, or it may be obtained by stacking cell sheets having a single layer structure. In particular, a cell sheet having a multilayer structure can be obtained by preparing a plurality of culture members with cell sheets of the present disclosure, overlaying one cell sheet on another cell sheet, and peeling the culture member from the other cell sheet.

[0089] The thickness of the cell sheet is not particularly limited, but may be, for example, 0.001 mm or more and 2.0 mm or less. The lower limit of the thickness of the cell sheet is more preferably 0.01 mm or more, even more preferably 0.03 mm or more, and particularly preferably 0.05 mm or more. On the other hand, the upper limit of the thickness of the cell sheet is more preferably 1.5 mm or less, even more preferably 1.2 mm or less, and particularly preferably 1.0 mm or less.

[0090] The thickness of the cell sheet may be, for example, 0.001 mm to 2.0 mm, 0.001 mm to 1.5 mm, 0.001 mm to 1.2 mm, 0.001 mm to 1.0 mm, 0.01 mm to 2.0 mm, 0.01 mm to 1.5 mm, 0.01 mm to 1.2 mm, 0.01 mm to 1.0 mm, 0.03 mm to 2.0 mm, 0.03 mm to 1.2 mm, 0.03 mm to 1.0 mm, 0.05 mm to 2.0 mm, 0.05 mm to 1.5 mm, 0.05 mm to 1.2 mm, or 0.05 mm to 1.0 mm. By keeping the thickness of the cell sheet within the above ranges, high cell activity within the cell sheet and excellent shape retention ability, which is advantageous for cell transplantation, can be achieved.

[0091] The area of ​​the cell sheet is not particularly limited, but may be, for example, 0.3 cm ２ More than 1000cm ２ The lower limit of the area of ​​the cell sheet is more preferably 0.6 cm ２ More preferably, 1.6 cm or more ２ More than 8 cm, especially preferred ２ More than 3 cm ２ More than 4cm ２ Above, 5cm ２ More than 6cm ２ More than 7cm ２ or more or 10 cm ２ On the other hand, the upper limit of the area of ​​the cell sheet is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm ２ For example, the area of ​​the cell sheet may be 0.6 cm or less. ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ The following is the result.

[0092] Generally, when a cell sheet is transplanted alone, the strength of the cell sheet is low, and therefore, if the cell sheet has a large area, it is likely to break during transportation. In contrast, in the present disclosure, when the cell sheet is supported by a culture member, it is possible to prevent the cell sheet from breaking during transplantation, and therefore the size of the cell sheet can be reduced to 8 cm. ２ It is also possible to prepare a large cell sheet and adjust it to the size of the affected area as needed.

[0093] In the culture member with the cell sheet, it is preferable that the entire lower surface of the cell sheet is arranged to overlap the surface (culture surface) of the culture member 270 .

[0094] (Cells) The cells are not particularly limited as long as they are clinically useful cells for treating or preventing symptoms associated with cell, tissue, or organ deficiency, dysfunction, or dysfunction, or culturable cells for use in non-clinical trials, and are cells isolated from a living body.

[0095] Examples of cells include biological tissue cells, mesenchymal stem cells capable of differentiating into cells belonging to mesenchymal tissue, pluripotent stem cells capable of differentiating into various biological tissues, stem cells and progenitor cells that can be induced to differentiate, etc. The cells may be adherent cells or suspension cells.

[0096] Specific examples of biological tissue cells include fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiac muscle cells, myoblasts, bone cells, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, kidney cells, gingival cells, periosteal cells, skin cells, and endothelial cells.

[0097] Specific examples of mesenchymal stem cells include adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells.

[0098] 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 alone 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.

[0099] The origin of the cells is not particularly limited, and examples thereof include mammals, birds, amphibians, fish, insects, plants, microorganisms, etc. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, chickens, etc.

[0100] Figure 9 is a diagram showing another example of the flow of the method for producing a cell culture construct. The method for producing a cell culture construct shown in this figure includes steps 21 and 22 instead of steps 1 and 2 in the method for producing a cell culture construct shown in Figure 6. The subsequent steps 2 to 6 may be the same as those shown in Figure 6.

[0101] (Step 21) The culture member 270 is placed on the bottom 112 (specifically, the upper surface 112a) of the culture vessel 110. As a result, the culture member 270 is accommodated in the culture space 111.

[0102] (Step 22) The spacer 130 is placed in the culture vessel 110. In detail, for example, the spacer 130 is placed on the culture member 270 placed on the bottom 112 (specifically, the upper surface 112a) with the hole 131 facing upward, thereby placing the spacer 130 in the culture space 111. Note that step 22 may be performed after step 2 is performed.

[0103] 10 is a side cross-sectional view showing an example of a state in which the spacer 130 is placed on the culture member 270, thereby arranging the spacer 130 in the culture vessel 110. The culture member 270 is accommodated in the culture space 111 and sandwiched between the bottom 112 and the spacer 130.

[0104] In other words, the culture member 270 has a size and shape that allows it to be accommodated in the culture space 111 and sandwiched between the bottom 112 and the spacer 130. In this embodiment, for example, the outer edge of the culture member 270 is smaller than the outer edge of the bottom of the spacer 130 and larger than the inner edge of the bottom of the spacer 130.

[0105] This allows the spacer 130 to hold down the culture member 270 placed on the bottom 112 (specifically, the upper surface 112 a ).

[0106] The spacer 130 is placed, for example, from above on the culture member 270 placed on the bottom 112. More specifically, for example, the spacer 130 is placed from above on the edge of the culture member 270 placed on the bottom 112. This holds down the culture member 270, making it difficult for it to move. This allows cells to be cultured well.

[0107] Since the culture member 270 is held down from above by the spacer 130, when a culture medium is used, for example, the culture member 270 is less likely to float in the culture medium, and the culture member 270 can be reliably immersed in the culture medium. This makes it possible to culture cells satisfactorily.

[0108] 11 and 12 are diagrams showing other examples of spacers. A spacer 230 shown in FIG. 11 does not include the hole 131, and may be configured similarly to the spacer 130 except for this point. A spacer 330 shown in FIG. 12 is an example in which two hole portions 331 penetrating in a direction intersecting the height direction are provided between the upper and lower ends. These spacers 230 and 330 can be used in place of the spacer 130. This also achieves the same effects as the embodiment.

[0109] The above describes embodiments of the present invention with reference to the drawings. However, these are merely examples of the present invention and may be modified as appropriate. For example, in the flowcharts used in the above description, multiple steps (processes) are described in order, but the order in which these steps are executed is not limited to the order described. The order of the steps shown in the drawings may be changed as long as it does not interfere with the content. For example, it goes without saying that applicable general technology may be used to realize the functions and processes exemplified in the embodiments. For example, the embodiments and modified examples may be combined as long as the content is not contradictory.

[0110] This application claims priority based on Japanese Patent Application No. 2024-049020, filed March 26, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0111] 100 Cell culture kit 110 Culture container 111 Culture space 112 Bottom 112a Top surface 112b Bottom surface 113 Side wall 113a First locking portion 114 Opening 114a Opening 130, 230, 330 Spacer 130a, 130b Opening 131, 331 Hole 150 lid 151 lid part 152 hanging part 152a second locking part 270 culture member

Claims

1. A cell culture kit comprising: a culture vessel having a culture space formed therein for culturing cells and including an opening communicating with the culture space; a cylindrical spacer that is open at the top and bottom and is removably housed in the culture space; and a lid that can be placed on the spacer, wherein the height of the spacer is greater than the height of the culture space.

2. The cell culture kit according to claim 1, wherein the spacer includes one or more holes penetrating in a direction intersecting the height direction.

3. The cell culture kit according to claim 2, wherein the hole forms a notch that is open at the top.

4. A cell culture kit according to any one of claims 1 to 3, wherein the culture vessel includes a bottom and a side wall extending upward from the bottom, the lid includes a plate-shaped lid portion and a hanging portion extending downward from the lid portion and capable of being fitted into the side wall portion, and includes a locking mechanism for removably locking the culture vessel and the lid to each other, and the spacer has a height that prevents the locking mechanism from engaging when the spacer is accommodated in the culture space.

5. The cell culture kit according to claim 4, wherein the height of the spacer is smaller than the sum of the heights of the side wall portion and the hanging portion.

6. The cell culture kit according to claim 5, wherein the side wall portion and the hanging portion are each cylindrical, and the inner diameter of the hanging portion is larger than the outer diameter of the side wall portion.

7. A cell culture kit according to any one of claims 1 to 3, wherein the opening is provided above the culture vessel, and the lid is attached to the top of the culture vessel to cover the opening.

8. The cell culture kit according to claim 4, further comprising a culture member that is a scaffold for culturing the cells, the culture member having a size and shape that allows it to be housed in the culture space and sandwiched between the bottom and the spacer.

9. The cell culture kit according to claim 4, wherein the lid and the culture vessel are tightly attached to each other when locked by the locking mechanism.

10. The cell culture kit according to claim 4, wherein the locking mechanism has a screw structure that locks the lid and the culture vessel together.

11. A method for producing a cell culture, comprising placing a lid on a spacer of a culture vessel having at least cells and a spacer disposed therein, culturing the cells in the culture vessel, removing the spacer from the culture vessel, and attaching a lid to the culture vessel to tightly seal it.

12. The method for producing a cell culture construct according to claim 11, wherein the cells to be cultured are a three-dimensional cell culture construct.

Images