Cell culture kit, cell sheet production method, and culture sheet having cell sheet

Abstract

This cell culture kit (100) is provided with a culture vessel (110) and a culture sheet (150). The culture sheet (150) is a scaffold material for culturing cells. The culture container (110) has therein an accommodation space, in which the culture sheet (150) is accommodated, and includes an opening that communicates with the accommodation space. The culture container (110) includes a bottom part (112) on which the culture sheet is placed, and a side wall part (113) extending upward from the bottom part (112). The culture sheet (150) has a shape and size such that when placed on the bottom part (112) a gap is formed between the outer edge part of the culture sheet (150) and the side wall part (113).

Description

Cell culture kit, cell sheet manufacturing method, and culture sheet with cell sheet

[0001] The present disclosure relates to a cell culture kit, a cell sheet manufacturing method, and a culture sheet with a cell sheet.

[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, a vessel body, and a plate-like substrate. The lid covers an opening of the vessel body. The vessel body contains culture material such as cells. The plate-like substrate covers a through-hole formed in the bottom of the vessel body.

[0003] International Publication No. 2006 / 123570

[0004] In general cell culture, for example, a culture sheet (e.g., the plate-like substrate described in Patent Document 1) that serves as a scaffold for culturing cells, cells, and a culture solution are introduced into a culture vessel (e.g., the vessel body described in Patent Document 1), and the cells are cultured. As a result, a cell sheet containing the cultured cells is formed in close contact with the culture sheet. After cell culture, the culture solution that is no longer needed is removed from the culture vessel using a suction tool (e.g., a pipette, etc.).

[0005] In the cell culture vessel described in Patent Document 1, when an instrument comes into contact with the culture sheet when removing unnecessary culture medium from the storage container, the culture sheet may be distorted, which may result in damage to the cell sheet cultured on the culture sheet, such as deformation or distortion. Furthermore, when a suction tool comes into contact with the cell sheet, damage such as tearing or loss of the cell sheet may occur.

[0006] Furthermore, in the cell culture vessel described in Patent Document 1, there is a risk of damaging the cell sheet formed by cell culture when the culture sheet with the cell sheet attached thereto is removed from the vessel.

[0007] As described above, the cell culture vessel described in Patent Document 1 may be difficult to handle without damaging the cell sheet formed by cell culture.

[0008] One of the objectives of the present application is to reduce the possibility of damaging a cell sheet formed by cell culture.

[0009] According to one aspect of the present disclosure, the following cell culture kit, cell sheet manufacturing method, and culture sheet with cell sheet are provided.

[0010] 1. A cell culture kit comprising: a culture sheet which is a scaffold material for culturing cells; and a culture vessel having an internal storage space for accommodating the culture sheet and including an opening communicating with the storage space, wherein the culture vessel includes a bottom on which the culture sheet is placed and sidewalls extending upward from the bottom, and the culture sheet has a shape and size that creates a gap between the outer edge of the culture sheet and the sidewalls when placed on the bottom. 2. The cell culture kit described in 1., wherein the culture vessel further includes protrusions that protrude inward from the sidewalls and connect to the upper surface of the bottom, and the culture sheet has a shape similar to the upper surface including the portions connected to the protrusions and is no larger than the size that contacts the tips of the protrusions. 3. The cell culture kit described in 2., wherein there are multiple protrusions, and the culture sheet has a shape similar to the upper surface including the portions connected to the protrusions and is no larger than the size that contacts the tips of the multiple protrusions. 4. The cell culture kit according to 2. or 3., wherein the culture vessel is cylindrical with a bottom, and a plurality of the protrusions are provided at equal angular intervals, and each of the protrusions has a width along the circumferential direction of the side wall portion. 5. The cell culture kit according to any one of 2. to 4., wherein the upper ends of the protrusions are inclined downward and inward. 6. The cell culture kit according to any one of 1. to 5., wherein the culture sheet has a shape in which a notch is provided in the shape of the upper surface of the bottom portion, or is smaller in size than the upper surface of the bottom portion. 7. The cell culture kit according to any one of 1. to 6., further comprising a cylindrical or annular holding member that is open at the top and bottom, for holding down the culture sheet placed on the bottom portion. 8. 8. The cell culture kit according to any one of 1. to 7., further comprising a lid for closing the opening, wherein the culture vessel and the lid each include a first locking portion and a second locking portion for removably locking them to each other, and wherein the culture vessel and the lid come into tight contact with each other when the first locking portion and the second locking portion are locked together.9. A cell sheet manufacturing method comprising: using a suction tool to suck out culture medium used for culturing the cells from a cylindrical culture vessel with a bottom, on the bottom of which a culture sheet having a cell sheet containing cells attached thereto is placed, wherein sucking out the culture medium comprises positioning a tip of the suction tool above a gap formed at the bottom of the culture vessel between the culture sheet and a side wall of the culture vessel and sucking out the culture medium. 10. A cell sheet manufacturing method further comprising placing the culture sheet on the bottom of the culture vessel; placing a cylindrical or annular pressing member that is open at the top and bottom from above on the edge of the culture sheet placed on the bottom; and arranging the cells and the culture medium in a storage space formed inside the culture vessel, wherein the culture sheet with the cell sheet attached thereto includes a gap between the outer edges of the culture sheet and the outer edges of the cell sheet, and sucking out the culture medium comprises positioning the tip of the suction tool above at least one of the gap formed in the bottom and the gap formed in the culture sheet with the cell sheet attached. 11. A cell sheet-attached culture sheet comprising: a cell sheet containing cells; and a culture sheet having the cell sheet attached to one main surface, wherein at least a portion of an outer edge of the culture sheet is separated from an outer edge of the cell sheet on the one main surface. 12. A cell sheet-attached culture sheet according to 11., wherein the thickness of the culture sheet is 5 μm or more and 250 μm or less. 13. A cell sheet-attached culture sheet according to 11. or 12., wherein the material of the culture sheet includes polyether ether ketone and polyethylene terephthalate.

[0011] According to the present disclosure, it becomes possible to easily remove unnecessary culture medium from the culture vessel without damaging the cell sheet.

[0012] 1 is a perspective view showing an example of each component constituting a cell culture kit according to embodiment 1. FIG. 2 is a side view showing an example of a culture vessel according to this embodiment. FIG. 3 is a plan view showing an example of a culture vessel according to this embodiment. FIG. 4 is a side cross-sectional view of the culture vessel taken along line A-A in FIG. 3. FIG. 5 is a plan view showing an example of a culture vessel with a culture sheet according to embodiment 1 placed on the bottom. FIG. 6 is a diagram showing an example of the flow of a method for manufacturing a cell sheet according to embodiment 1. FIG. 7 is a side cross-sectional view showing an example of a culture vessel after step 3 is completed. FIG. 8 is a perspective view showing an example of a culture sheet with a cell sheet according to embodiment 1. FIG. 9 is a plan view showing an example of a culture sheet according to embodiment 2. FIG. 10 is a plan view showing the state in which the culture sheet according to embodiment 2 is placed in the culture vessel. FIG. 11 is a plan view showing an example of a culture sheet according to modified example 1. FIG. 12 is a plan view showing the state in which the culture sheet according to modified example 1 is placed in the culture vessel.

[0013] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. In all drawings, similar components are denoted 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 disclosure.

[0014] 1 is a perspective view showing an example of each component constituting a cell culture kit 100 according to embodiment 1. The cell culture kit 100 includes a culture vessel 110, a lid 130, a culture sheet 150, and a retainer member 170.

[0015] (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.

[0016] The culture vessel 110 has a storage space 111 formed therein for culturing cells in a culture solution, and includes an opening 114 a communicating with the storage space 111 .

[0017] The culture vessel 110 according to this embodiment is cylindrical and has a bottom, and includes a bottom 112, a sidewall 113, an opening 114, and a plurality of protrusions 115. Although four protrusions 115 are provided in Figures 1 and 3, the number of protrusions 115 is not particularly limited and can be any number between 1 and 12, with three, four, five, six, seven, or eight being preferred.

[0018] 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.

[0019] The bottom portion 112 is flat and includes an upper surface 112a and a lower surface 112b.

[0020] The upper surface 112a forms the storage space 111. The culture sheet 150 is placed on the upper surface 112a. That is, the bottom 112 is the portion where the culture sheet 150 is placed on the upper surface 112a.

[0021] 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.

[0022] 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 storage 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.

[0023] 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 accommodation space 131. That is, the culture vessel 110 includes the opening 114a that communicates with the accommodation space 131.

[0024] Each of the protrusions 115 protrudes inward from the side wall 113 (i.e., toward the storage space 111). Each of the protrusions 115 is connected to the upper surface 112a of the bottom 112. Each of the protrusions 115 has a width along the circumferential direction of the side wall 113. The upper end of each of the protrusions 115 is inclined downward and inward. The amount by which the protrusions 115 protrude inward from the side wall 113 (the thickness of the protrusions 115 in FIG. 3 ) is preferably 0.5 mm or more, and more preferably 0.8 mm or more. With such a protrusion amount, it is possible to easily remove the culture medium using a suction tool, for example, by providing a gap T as described below.

[0025] In this embodiment, four protrusions 115 are provided at equal angular intervals when the side wall portion 113 is viewed from above.

[0026] 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.

[0027] 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.

[0028] 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 ２The lower limit is more preferably 0.35 cm. ２ More 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.

[0029] (Regarding the lid 130) The lid 130 is a member for closing the opening 114a. The lid 130 includes a second locking portion 132a for removably locking the incubation vessel 110 and the lid 130 by engaging with the first locking portion 113a.

[0030] The lid 130 according to this embodiment includes a lid portion 131 and a hanging portion 132. Note that the lid 130 in FIG.

[0031] The lid portion 131 has a flat plate shape. More specifically, the lid portion 131 has a circular flat plate shape.

[0032] Hanging portion 132 extends downward from lid portion 131. Specifically, hanging portion 132 extends downward from lid portion 131 when lid 130 is attached to culture vessel 110. More specifically, hanging portion 132 extends downward from the outer edge of lid portion 131.

[0033] The hanging part 132 includes a second locking part 132a at an upper part of the inner surface (inner circumferential surface in this embodiment). The second locking part 132a according to this embodiment is a spiral ridge.

[0034] The inner surface of hanging portion 132 is sized to fit loosely onto the outer surface of sidewall portion 113. When lid 130 is attached to incubation vessel 110, first locking portion 113a and second locking portion 132a fit together using a screw structure. Therefore, incubation vessel 110 and lid 130 can be removably locked to each other by first locking portion 113a and second locking portion 132a. That is, incubation vessel 110 and lid 130 each include first locking portion 113a and second locking portion 132a for locking them to each other.

[0035] It is desirable that the culture vessel 110 and the lid 130 be in close contact with each other when the first locking portion 113 a and the second locking portion 132 a are locked together, thereby making it difficult for the culture medium and the like contained in the storage space 111 to leak out.

[0036] The material of the lid 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. The lid 130 may be manufactured so as to prevent leakage of the culture medium when engaged with the culture vessel 110, and may be manufactured, for example, as a single unit, or may be configured as a single unit by combining multiple parts. Note that the material and manufacturing method of the lid 130 are not limited to those exemplified here.

[0037] The shape of the lid 130 is not limited to that described here. Furthermore, the first locking portion 113a and the second locking portion 132a need not be spiral protrusions, as long as they can removably lock the culture vessel 110 and the lid 130 to each other. One of the first locking portion 113a and the second locking portion 132a may include a spiral groove. This also allows the culture vessel 110 and the lid 130 to be removably locked to each other using a screw structure. Furthermore, the structure for removably locking the culture vessel 110 and the lid 130 to each other is not limited to a screw structure. This structure may be, for example, a structure in which a protrusion or groove and a claw engage with each other to removably lock the culture vessel 110 and the lid 130 to each other.

[0038] (Regarding the culture sheet 150) The culture sheet 150 is a scaffold for culturing cells. When culturing cells, the culture sheet 150 is placed on, for example, the bottom 112 (more specifically, the upper surface 112a) and accommodated in the accommodation space 111.

[0039] The culture sheet 150 is a sheet having two main surfaces. One main surface is in general contact with the bottom 112 (specifically, the upper surface 112a) when accommodated in the accommodation space 111. The other main surface is the main surface (culture surface) used for cell culture. When cells are cultured on the culture surface, a cell sheet formed thereby adheres to the culture surface.

[0040] The area of ​​the culture sheet 150 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 It may be the following:

[0041] The lower limit of the area of ​​the culture sheet 150 is more preferably 0.6 cm ２ More preferably, 1.6 cm 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 sheet 150 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 It may be the following:

[0042] For example, the area of ​​the culture sheet 150 is 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 cm２ 20cm or more ２ It may be the following:

[0043] The thickness of the culture sheet 150 may be, for example, 5 μm or more and 250 μm or less. The lower limit of the thickness of the culture sheet 150 is more preferably 6 μm or more, 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 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 sheet 150 may be 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. The thickness of the culture sheet 150 is not limited to those exemplified here.

[0044] Both main surfaces of the culture sheet 150 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 control the direction of growth of the cultured cells, 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 smoothness. An example of such a surface treatment is plasma hydrophilization. The structure of the main surface of the culture sheet 150 is not limited to the example shown here.

[0045] When placed on the bottom 112, the culture sheet 150 has a shape and size that creates a gap S between the outer edge of the culture sheet 150 and the side wall 113 (more specifically, the inner surface of the side wall 113), as shown in the plan view of Figure 5.

[0046] The culture sheet 150 has, for example, a shape similar to the upper surface 112a including the portion where the protrusion 115 is connected. In FIG. 1, the upper surface 112a including the portion where the protrusion 115 is connected is circular because the culture vessel 110 is cylindrical as described above. The culture sheet 150 is, for example, no larger than the size that contacts each tip of the protrusion 115. Note that when there is one protrusion, the culture sheet 150 may be, for example, no larger than the size that contacts the tip of the protrusion 115.

[0047] The shape of the culture sheet 150 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 sheet 150 may not have an opening that penetrates through the front and back surfaces. When placed on the culture vessel 110 and viewed from the surface normal direction of the culture sheet 150, it is preferable that the culture sheet 150 does not have an opening that exposes the bottom surface of the culture vessel 110.

[0048] The material of the culture sheet 150 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, etc. The culture sheet 150 may be made of a film containing at least one of these materials as a main component. The culture sheet 150 is preferably made of a material that is transparent, has a specific gravity greater than 1.0, and is excellent in various durability, mechanical strength, and processability. The main component of the culture sheet 150 is preferably a polyether ether ketone film, a polyethylene terephthalate film, or polystyrene, and more preferably a polyether ether ketone film.

[0049] The surface roughness Ra of the culture surface of the culture sheet 150 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, using surface shape data measured with an atomic force microscope (AFM).

[0050] When the culture surface of the culture sheet 150 is measured with a laser microscope, it is preferable that there are three or fewer holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm within a 100 μm square area, and more preferably zero. The diameter of these holes is, for example, 1 μm to 100 μm, preferably 2 μm to 50 μm, 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, more preferably 2 μm to 20 μm.

[0051] The combination of the ranges of the hole diameter and hole depth on the culture surface of the culture sheet 150 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. m or less, 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, 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.

[0052] The upper limit of the porosity of the culture sheet 150 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 sheet 150 is not particularly limited, but may be 0% or more.

[0053] By limiting the number of holes on the culture surface of the culture sheet 150 to three or less and / or by limiting the porosity of the culture sheet 150 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.

[0054] The presence or absence of holes on the culture surface of the culture sheet 150 may be measured on the surface of the resin layer formed on the culture surface side of the culture sheet 150 .

[0055] 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.

[0056] The culture surface of the culture sheet 150 may be configured not to contain a temperature-responsive polymer, which can prevent a decrease in adhesion between the cell sheet and the culture sheet 150 in a low-temperature environment such as during a cryopreservation process.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] (Regarding the holding member 170) The holding member 170 is a member for holding down the culture sheet 150 placed on the bottom 112 (specifically, the upper surface 112a). The holding member 170 has a shape that is open at the top and bottom, and is, for example, cylindrical or annular.

[0061] However, the shape of the holding member 170 is not limited to these. The holding member 170 may have a cylindrical or annular shape when viewed from above, for example, an elliptical, rectangular, polygonal, or other appropriate predetermined shape.

[0062] The holding member 170 is placed, for example, from above on the culture sheet 150 placed on the bottom 112. In detail, for example, it is placed from above on the edge of the culture sheet 150 placed on the bottom 112. This makes it possible to hold down the culture sheet 150.

[0063] Therefore, the outer surface (in this embodiment, the outer peripheral surface) of the pressing member 170 may be, for example, small enough to contact the tips of the protrusions 115. Also, for example, the bottom of the pressing member 170 may be large enough to contact at least a portion of the outer edge of the culture sheet 150, preferably the entire outer edge of the culture sheet 150. That is, for example, the outer edge of the bottom of the pressing member 170 may be large enough to contact the outer edge of the culture sheet 150 or larger, and the inner edge of the bottom of the pressing member 170 may be small enough to contact the outer edge of the culture sheet 150.

[0064] The material of the retaining member 170 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, gelatin, collagen, etc. The metal may be stainless steel, aluminum, etc.

[0065] (Example of a method for producing a cell sheet using the cell culture kit 100) Hereinafter, an example of a method for producing a cell sheet containing cells using the cell culture kit 100 will be described.

[0066] FIG. 6 is a diagram showing an example of the flow of the cell sheet manufacturing method.

[0067] (Step 1) The culture sheet 150 is placed on the bottom 112 (specifically, the upper surface 112 a ) of the culture container 110 . As a result, the culture sheet 150 is accommodated in the accommodation space 111 .

[0068] As described above, the culture sheet 150 has a shape similar to that of the upper surface 112a including the portion where the protrusions 115 are connected, and is smaller than the size that contacts each tip of the protrusions 115. This allows the culture sheet 150 to be placed inside the protrusions 115 and placed on the bottom 112.

[0069] As described above, the upper end of each of the protrusions 115 is inclined downward and inward. This inclination makes it difficult for the culture sheet 150 to get caught on the tip of the protrusion 115, making it easier to place the culture sheet 150 inside the protrusion 115.

[0070] (Step 2) The holding member 170 is placed from above on the edge of the culture sheet 150 placed on the bottom 112. Specifically, the holding member 170 is placed from above on the edge of the culture surface (one main surface) of the culture sheet 150. As a result, the holding member 170 fits loosely into the four protrusions 115 and holds down the culture sheet 150 from above.

[0071] (Step 3) The culture medium P and cells are introduced into the storage space 111.

[0072] 7 is a side cross-sectional view showing an example of the culture vessel 110 after completion of step 3. Since the culture sheet 150 is held down from above by the holding member 170, the culture sheet 150 is less likely to float in the culture solution P, and the culture sheet 150 can be reliably immersed in the culture solution P.

[0073] (Step 4) The lid 130 is attached to the culture vessel 110, and cells are cultured in an appropriate environment such as a constant temperature incubator. At this time, the culture vessel 110 and the lid 130 may be locked together by the first locking portion 113a and the second locking portion 132a.

[0074] (Step 5) When the cell culture is completed, the lid 130 is removed from the culture vessel 110, and the retainer 170 is taken out from the culture vessel 110 (specifically, the accommodation space 111).

[0075] At this time, a cell sheet containing cultured cells is attached to the culture sheet 150 placed on the bottom 112 (specifically, the upper surface 112a). That is, a culture sheet 200 with a cell sheet attached thereto is produced.

[0076] FIG. 8 is a perspective view showing an example of a cell-sheet-attached culture sheet 200 (hereinafter also referred to as "cell-attached culture sheet 200") according to this embodiment. The cell-sheet-attached culture sheet 200 includes a cell sheet 201 containing cells and a culture sheet 150 with the cell sheet 201 attached to a culture surface (one main surface) Q. The cell sheet 201 is formed by cultured cells being physically and functionally connected to each other via adhesion molecules, extracellular matrix, and the like to form a sheet structure. During cell culture, the restraining member 170 rests on the edge of the culture sheet 150 (culture surface Q), so that at least a portion of the outer edge of the culture sheet 150 and the outer edge of the cell sheet 201 are separated on the culture surface Q. That is, the culture surface Q includes a gap T between the outer edge of the culture sheet 150 and the outer edge of the cell sheet 201.

[0077] The culture sheet 200 with a cell sheet illustrated in Figure 8 is an example manufactured using a presser member 170 having a bottom that contacts the entire outer edge of the culture sheet 150. Therefore, at least a portion of the outer edge of the culture sheet 150 and the outer edge of the cell sheet 201 are separated on the culture surface Q. For example, as illustrated in Figure 8, the outer edge of the culture sheet 150 and the outer edge of the cell sheet 201 may be separated entirely on the culture surface Q. Note that the outer edges of the culture sheet 150 and the outer edges of the cell sheet 201 may be partially separated from each other on the culture surface Q, for example, by a method in which the culture sheet 200 with a cell sheet is manufactured using a presser member 170 having a bottom that contacts a portion of the outer edge of the culture sheet 150.

[0078] (Cell Sheet) The cell sheet 201 has a sheet structure in which cells are physically and functionally connected to one another via adhesion molecules, extracellular matrix, and the like.

[0079] The cell sheet 201 may have a single-layer structure consisting of one cell layer, or may have 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.

[0080] When the cell sheet 201 has a multilayer structure, the multilayer structure may be obtained when cultured on the culture sheet 150, or it may be obtained by stacking cell sheets with a single layer structure. In particular, a cell sheet with a multilayer structure can be obtained by preparing a plurality of the cell sheet-attached culture sheets 200 of the present disclosure, overlaying one cell sheet on another, and peeling the culture sheet from the other cell sheet.

[0081] The thickness of the cell sheet 201 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.

[0082] The thickness of the cell sheet 201 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 201 within the above ranges, high cell activity within the cell sheet 201 and excellent shape retention ability, which are advantageous for cell transplantation, can be achieved.

[0083] The area of ​​the cell sheet 201 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 201 is more preferably 0.6 cm ２ More preferably, 1.6 cm 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 201 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 201 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 ２More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ It may be the following:

[0084] Generally, when a cell sheet is transplanted alone, the strength of the cell sheet is low, and therefore there is a high possibility that the cell sheet will break during transportation if it is large in area. In contrast, in the present disclosure, the cell sheet 201 is supported by the culture sheet, which prevents the cell sheet from breaking during transplantation. Therefore, the size of the cell sheet 201 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.

[0085] In the cell-attached culture sheet 200, it is preferable that the entire lower surface of the cell sheet 201 is arranged to overlap the surface (culture surface) of the culture sheet 150.

[0086] (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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] (Step 6) The culture solution P used for culturing the cells is sucked out from the culture vessel 110 (specifically, the storage space 111) using a suction tool, and the production of the cell sheet is completed.

[0093] The suction tool is, for example, a pipette, but is not limited to this.

[0094] As described above, the culture sheet 150 has a shape and size that creates a gap S between the outer edge of the culture sheet 150 and the side wall 113 (specifically, the inner surface of the side wall 113) when placed on the bottom 112. Therefore, a gap S is formed between the culture sheet 150 and the side wall 113 at the bottom 112 (specifically, the upper surface 112a) of the culture container 110. Furthermore, in the cell-attached culture sheet 200, a gap T is formed between the outer edge of the culture sheet 150 and the outer edge of the cell sheet 201.

[0095] Aspirating the culture solution P includes positioning the tip of an aspirator above at least one of the gap S and the gap T and aspirating the culture solution P.

[0096] This makes it easy to suck out the culture solution P without contacting the suction tool with the culture sheet 150 or the cell sheet 201. This reduces the possibility of damaging the cell sheet 201 formed by cell culture.

[0097] By positioning the tip of the suction tool above the gap S and suctioning the culture solution P, it becomes easier to suction out the culture solution P without bringing the suction tool into contact with the culture sheet 150 or the cell sheet 201. This makes it possible to further reduce the possibility of damaging the cell sheet 201 formed by cell culture.

[0098] The cell-attached culture sheet 200 may be stored or transported using the culture vessel 110. At this time, the lid 130 may be attached to the culture vessel 110 while the cell-attached culture sheet 200 is housed in the housing space 111. This makes it possible to reduce the possibility of the cell sheet 201 being contaminated during storage or transport of the cell-attached culture sheet 200.

[0099] Furthermore, because of the gaps S and T, it is easy to hold the culture sheet 150 and remove the cell-attached culture sheet 200 from the culture container 110 (more specifically, the storage space 111) without contacting the cell sheet 201 with an instrument such as tweezers. This reduces the possibility of damaging the cell sheet 201 formed by cell culture.

[0100] [Embodiment 2] In the first embodiment, an example was described in which the culture vessel 110 includes a protrusion 115. However, the culture sheet only needs to have a shape and size that creates a gap S between the outer edge of the culture sheet and the side wall 113 (specifically, the inner surface of the side wall 113) when placed on the bottom 112. In the present embodiment, an example is described in which a gap S is created between the outer edge of the culture sheet and the side wall 113 (specifically, the inner surface of the side wall 113) in a culture vessel that does not include a protrusion 115.

[0101] Fig. 9 is a plan view showing an example of a culture sheet 250 according to this embodiment. Fig. 10 is a plan view showing a state in which the culture sheet 250 according to this embodiment is placed in a culture container 210.

[0102] The cell culture kit according to this embodiment includes a culture container 210 and a culture sheet 250 instead of the culture container 110 and the culture sheet 150. Except for these, the cell culture kit according to this embodiment may be configured similarly to the cell culture kit 100 according to the first embodiment.

[0103] The culture vessel 210 according to this embodiment may be configured similarly to the culture vessel 110 according to the first embodiment, except that it does not include the protrusion 115 .

[0104] The culture sheet 250 is a substantially circular sheet that is the same size as the bottom 112 (specifically, the upper surface 112a) or smaller than the bottom 112 (specifically, the upper surface 112a), and includes a notch 250a.

[0105] When the culture sheet 250 is placed on the bottom 112, a gap S is generated between the outer edge of the culture sheet 250 and the side wall 113 (specifically, the inner surface of the side wall 113). The gap S is generated in a portion corresponding to the notch 250a.

[0106] Such a culture sheet 250 is an example of a culture sheet having a shape in which a notch is provided in the shape of the upper surface 112a of the bottom portion 112.

[0107] According to this embodiment, the culture solution P can also be aspirated by positioning the tip of the aspirator above at least one of the gaps S and T. This makes it easy to aspirate the culture solution P without bringing the aspirator into contact with the culture sheet 250 or the cell sheet 201. This reduces the possibility of damaging the cell sheet 201 formed by cell culture.

[0108] Furthermore, by positioning the tip of the suction tool above the gap S and suctioning the culture solution P, it becomes easier to suction out the culture solution P without bringing the suction tool into contact with the culture sheet 150 or the cell sheet 201. This makes it possible to further reduce the possibility of damaging the cell sheet 201 formed by cell culture.

[0109] Furthermore, because of the gaps S and T, it is easy to hold the culture sheet 250 and remove the cell-attached culture sheet 200 from the culture container 110 (more specifically, the storage space 111) without contacting the cell sheet 201 with an instrument such as tweezers. This reduces the possibility of damaging the cell sheet 201 formed by cell culture.

[0110] (Modification 1) Fig. 11 is a plan view showing an example of a culture sheet 350 according to Modification 1. Fig. 12 is a plan view showing the culture sheet 350 according to Modification 1 placed in the culture container 210. The culture sheet 350 can be used in place of the culture sheet 250 in the cell culture kit according to the second embodiment.

[0111] The culture sheet 350 is a polygonal sheet, and has a size smaller than the bottom 112 (specifically, the upper surface 112a).

[0112] When the culture sheet 250 is placed on the bottom 112, a gap S is generated between the outer edge of the culture sheet 250 and the side wall 113 (specifically, the inner surface of the side wall 113). The gap S is generated between each side of the culture sheet 250 and the side wall 113.

[0113] Such a culture sheet 250 is an example of a culture sheet that is smaller than the upper surface 112 a of the bottom portion 112 .

[0114] This modification also provides the same effects as those of the second embodiment.

[0115] The above describes embodiments of the present disclosure with reference to the drawings. However, these are merely examples of the present disclosure 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 in which they are 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 technologies 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.

[0116] This application claims priority based on Japanese Patent Application No. 2024-049019, filed March 26, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0117] 100 Cell culture kit 110, 210 Culture vessel 111 Storage space 112 Bottom 112a Upper surface 113 Side wall 113a First locking portion 114 Opening 114a Opening 115 Protrusion 130 Lid 131 Lid 132 Hanging portion 132a Second locking portion 150, 250, 350 Culture sheet 170 Retaining member 200 Culture sheet with cells 201 Cell sheet 250a Notch P Culture solution Q Culture surface S, T Gap

Claims

1. A cell culture kit comprising: a culture sheet, which is a scaffolding material for culturing cells; and a culture vessel having an internal storage space for storing the culture sheet and including an opening communicating with the storage space, wherein the culture vessel has a bottom on which the culture sheet is placed and side walls extending upward from the bottom, and the culture sheet has a shape and size that, when placed on the bottom, creates a gap between the outer edge of the culture sheet and the side walls.

2. The cell culture kit according to claim 1, wherein the culture vessel further includes a protrusion that protrudes inward from the side wall and connects to the top surface of the bottom, and the culture sheet has a shape similar to the top surface including the portion connected to the protrusion, and is smaller than or equal to the size of the tip of the protrusion.

3. The cell culture kit according to claim 2, wherein the protrusions are multiple, the culture sheet has a shape similar to the upper surface including the portion where the protrusions are connected, and is smaller than the size that contacts each tip of the multiple protrusions.

4. The cell culture kit according to claim 3, wherein the culture vessel is cylindrical with a bottom, the protrusions are provided in a number of four at equal angular intervals, and each of the protrusions has a width along the circumferential direction of the side wall portion.

5. The cell culture kit according to any one of claims 2 to 4, wherein the upper end of the protrusion is inclined downward and inward.

6. The cell culture kit according to claim 1, wherein the culture sheet has a shape in which a notch is provided in the shape of the upper surface of the bottom portion, or is smaller in size than the upper surface of the bottom portion.

7. The cell culture kit according to any one of claims 1 to 4 and 6, further comprising a cylindrical or annular holding member that is open at the top and bottom and that holds down the culture sheet placed on the bottom.

8. A cell culture kit according to any one of claims 1 to 4 and 6, further comprising a lid for closing the opening, wherein the culture vessel and the lid each include a first locking portion and a second locking portion for removably locking them to each other, and the culture vessel and the lid come into close contact with each other when the first locking portion and the second locking portion are locked together.

9. A cell sheet manufacturing method comprising using a suction tool to suck out culture medium used for culturing cells from a cylindrical culture vessel with a bottom, on the bottom of which is placed a culture sheet to which a cell sheet containing cells is attached, wherein sucking out the culture medium includes positioning the tip of the suction tool above a gap formed between the culture sheet and a side wall of the culture vessel at the bottom of the culture vessel and sucking out the culture medium.

10. The cell sheet manufacturing method according to claim 9, further comprising: placing the culture sheet on the bottom of the culture vessel; placing a cylindrical or annular restraining member that is open at the top and bottom from above on the edge of the culture sheet placed on the bottom; and arranging the cells and the culture solution in a storage space formed inside the culture vessel; the culture sheet to which the cell sheet is attached comprises a gap between the outer edges of the culture sheet and the outer edges of the cell sheet; and sucking out the culture solution comprises positioning the tip of the suction tool above at least one of the gap formed in the bottom and the gap formed in the culture sheet to which the cell sheet is attached, and sucking out the culture solution.

11. A culture sheet with a cell sheet, comprising: a cell sheet containing cells; and a culture sheet having the cell sheet attached to one main surface, wherein the outer edge of the culture sheet and the outer edge of the cell sheet are at least partially separated on the one main surface.

12. The culture sheet with a cell sheet according to claim 11, wherein the thickness of the culture sheet is 5 μm or more and 250 μm or less.

13. The culture sheet with a cell sheet according to claim 11 or 12, wherein the material of the culture sheet includes polyether ether ketone and polyethylene terephthalate.

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