Cell culturing film substrate, method for producing film substrate with cell sheet, cell culture kit, film substrate with cell sheet, and method for producing cell culturing film substrate

Abstract

A cell culturing film substrate according to the present invention has a culture surface on at least one surface of a film substrate, and has the following structure (a) and / or structure (b). (a) is a structure having, in at least a portion of both surfaces, an engraved mark including a front-rear asymmetrical shape. (b) is a structure having one or more among a notch part or a protrusion part formed in at least a portion of the outer periphery of the film substrate, or a cutout part penetrating the front and rear surfaces of the film substrate.

Description

Cell culture film substrate, method for manufacturing cell culture film substrate, cell culture kit, cell culture film substrate, and method for manufacturing cell culture film substrate

[0001] The present invention relates to a film substrate for cell culture, a method for producing a film substrate with a cell sheet, a cell culture kit, a film substrate with a cell sheet, and a method for producing a film substrate for cell culture.

[0002] Various developments have been made regarding film substrates used in cell culture. As an example of this type of technology, the technology described in Patent Document 1 is known. Patent Document 1 describes a cell culture method for producing a cell sheet on the temperature-responsive polymer layer of a film fixed to the bottom surface of a cell culture vessel, using a film comprising a resin layer bonded to the bottom surface of the vessel and a temperature-responsive polymer layer provided on the resin layer (Claim 1, paragraph 0026, etc., of Patent Document 1).

[0003] Japanese Patent Publication No. 2016-013111

[0004] Our inventors' investigations revealed that in the cell culture process, it can be difficult to determine which side of the film substrate is the culture surface, making it challenging to distinguish between the front and back. Furthermore, we found that if the film substrate placed inside a culture vessel lacks a handle, it can be difficult to remove, making the film substrate difficult to handle. Therefore, we found that there is room for improvement in the handling characteristics of substrate films used in cell culture, including visibility for distinguishing between the front and back and ease of handling.

[0005] The present inventors have achieved the present invention by obtaining the following findings (a) and (b), thereby realizing a cell culture film substrate with excellent handling properties. (a) By forming an imprint having an asymmetrical shape on the front and back of the film substrate on its surface, it becomes easy to distinguish between the front and back, that is, the visibility of the substrate is improved and its handling properties can be enhanced. (b) By forming a notch or protrusion on at least a part of the outer circumference of the film substrate, or by forming a cut that penetrates both the front and back surfaces of the film substrate, the gripping structure enhances operability and improves its handling properties.

[0006] According to one aspect of the present invention, the following cell culture film substrate, a method for producing a film substrate with a cell sheet, a cell culture kit, a film substrate with a cell sheet, and a method for producing a film substrate for cell culture are provided.

[0007] 1. A cell culture film substrate having a culture surface on at least one surface of the film substrate, comprising either one or both of the following structures: (a) a structure having an imprint including an asymmetrical shape on at least a portion of both surfaces; (b) a structure having one or more of notches or protrusions formed on at least a portion of the outer circumference of the film substrate, or notches penetrating the front and back surfaces of the film substrate. 2. The cell culture film substrate according to 1, wherein the imprint includes laser imprinting. 3. The cell culture film substrate according to 1 or 2, wherein the imprint consists of one or more letters, symbols, or patterns. 4. The cell culture film substrate according to any one of 1 to 3, wherein the imprint is located near the outer circumference of the film substrate. 5. 1 to 4. A cell culture film substrate according to any one of the above, wherein a groove is formed between the marking and the center of the culture surface, and a part of the surface is recessed. 6. A cell culture film substrate according to any one of 1 to 5, wherein at least a part of the outer peripheral surface of the film substrate, the notch, and the protrusion is a laser-processed surface. 7. A cell culture film substrate according to any one of 1 to 6, wherein a cell culture film substrate has a combination of two or more of the notch, the protrusion, and the cutout. 8. A cell culture film substrate according to any one of 1 to 7, wherein the protrusion has an asymmetrical shape on the front and back, or the notch has an asymmetrical shape on the front and back, or two or more selected from the group consisting of the protrusion and the notch are provided in asymmetrical positions on the front and back. 9. 1 to 8. A cell culture film substrate according to any one of the above, wherein the cell culture film substrate has a groove between the protruding portion and the center of the culture surface, the groove being a part of the surface that is recessed.10. A cell culture film substrate according to any one of 1 to 9, wherein the outer edge of the film substrate, as viewed from the surface, has a circular shape, and there is at least one protrusion between two notches formed by a part of the circular outer edge being missing. 11. A cell culture film substrate according to any one of 1 to 10, wherein the cell culture film substrate has an embossed surface, a haze of 35% or less, and a total light transmittance of 40% or more. 12. A cell culture film substrate according to any one of 1 to 11, wherein the thickness of the cell culture film substrate is 5 μm or more and 100 μm or less. 13. A cell culture film substrate according to any one of 1 to 12, wherein the cell culture film substrate contains an organic polymer material. 14. 1 to 13. A cell culture film substrate according to any one of the above, wherein no layer containing a temperature-responsive polymer is formed on the surface on which the culture surface is formed. 15. A method for manufacturing a cell sheet-attached film substrate, comprising: an installation step of placing a cell culture film substrate according to any one of 1 to 14 into a culture vessel; a culture step of placing a culture medium into the culture vessel, seeding cells, and culturing a cell sheet on the cell culture film substrate; and an removal step of removing the cell sheet-attached film substrate from the culture vessel. 16. A method for manufacturing a cell sheet-attached film substrate according to 15, wherein the installation step includes a step of placing a ring member on the film substrate of the cell culture film substrate in the culture vessel, and the removal step includes a step of removing the ring member from the film substrate. 17. A method for manufacturing a cell sheet-attached film substrate according to 15, wherein in the installation step, the ring member has at least one of a notch, a protrusion, and a cut. A method for manufacturing a cell sheet-attached film substrate, wherein the cell culture film substrate described in any one of the above, and the removal step includes a step of holding the portion of the film substrate near the notch, the protruding portion, or the cut portion.18. A cell culture kit comprising a culture vessel and a cell culture film substrate, wherein the cell culture film substrate is a cell culture film substrate according to any one of 1. to 14. 19. A cell culture kit according to 18., further comprising a ring member. 20. A cell culture kit according to 19., wherein the cell culture film substrate is a cell culture film substrate according to any one of 1. to 14. having a protrusion formed on at least a part of the outer circumference of the film substrate, the outer edge of the film substrate as viewed from the surface is circular, and there is at least one protrusion formed by being sandwiched between two notches formed by a part of the circular outer edge being missing, and the relationship L1 ≥ L2 is satisfied when the maximum width of the bottom surface of the culture vessel is L1 and the maximum width of the film substrate including the protrusion is L2. 21. 19. A cell culture kit as described in 22. 19. The cell culture film substrate is the cell culture film substrate as described in any one of 1 to 14, having a protrusion formed on at least a part of the outer circumference of the film substrate, the outer edge of the film substrate as viewed from the surface is circular, and has at least one protrusion protruding from the circular outer edge, and satisfies the relationship L3 ≤ L1 < L2' when L1 is the maximum width of the bottom surface of the culture container, L2' is the maximum width of the film substrate including the protrusion, and L3 is the maximum width of the film substrate excluding the protrusion. A cell culture kit as described in 22. 19. The cell culture film substrate is the cell culture film substrate as described in any one of 1 to 14, having a notch formed on at least a part of the outer circumference of the film substrate, and the notch width of the notch in the radial direction of the circular film substrate is less than or equal to the ring width of the ring member. A cell culture kit as described above, wherein the width of the notch in the radial direction of the circular film substrate is 5 mm or less.24. A film substrate with a cell sheet, comprising a cell culture film substrate according to any one of 1 to 14, and a cell sheet provided on the culture surface of the cell culture film substrate. 25. A method for manufacturing a cell culture film substrate, comprising: a surface treatment step of applying plasma treatment to at least one surface of a resin sheet to form a culture surface; a cutting step of cutting out one or more film substrates having the culture surface from the resin sheet; and a step of forming either one or both of the following structures (a) or (b): (a) A structure having an imprint including an asymmetrical shape on at least a portion of both of the surfaces; (b) A structure having one or more notches or protrusions formed on at least a portion of the outer circumference of the film substrate, or cuts that penetrate the front and back surfaces of the film substrate. 26. A method for manufacturing a cell culture film substrate according to 25, wherein the step of forming the structure is carried out by laser treatment. 27. 26. A method for manufacturing a cell culture film substrate as described in 26., wherein the step of forming the structure, including the formation of the markings, is performed before the cutting step. 28. A method for manufacturing a cell culture film substrate as described in 26. or 27., wherein the step of forming the structure, including the formation of at least one of the notches or the protrusions, is performed within the same step as the cutting step. 29. A method for manufacturing a cell culture film substrate as described in any one of 26. to 28., wherein the step of forming the structure, including the formation of the markings, includes a step of removing a part of the surface of the film substrate in the depth direction by laser treatment to form a groove between the markings and the culture surface.

[0008] The present invention provides a cell culture film substrate with excellent handling properties, a method for producing a cell sheet-attached film substrate, a cell culture kit, a cell sheet-attached film substrate, and a method for producing a cell culture film substrate.

[0009] This is a schematic top view showing an example of a film substrate for cell culture. This is a schematic top view showing an example of a film substrate for cell culture. (A) is a schematic top view showing an example of a film substrate for cell culture, and (B) is a cross-sectional view of the cell culture film substrate of (A) along line I-I'. This is a top view showing an example of a modified cell culture film substrate. This is a schematic cross-sectional view showing an example of a culture kit used in a method for manufacturing a film substrate with a cell sheet. This is a top view showing an example of a modified cell culture film substrate.

[0010] Embodiments of the present invention will be described below with reference to the drawings. In all drawings, similar components are denoted by the same reference numerals, and their descriptions are omitted as appropriate. Also, the drawings are schematic diagrams and do not correspond to the actual dimensional ratios.

[0011] <Cell Culture Film Substrate> The outline of the cell culture film substrate of this embodiment will be described below.

[0012] A cell culture film substrate is a film-like support member used for culturing cells on the surface of a film substrate. The cell culture film substrate is a film substrate having a culture surface on at least one of its surfaces and having one or both of the following structures: (a) a structure having markings including an asymmetrical shape on at least a portion of both surfaces; (b) a structure having one or more of notches or protrusions formed on at least a portion of the outer circumference of the film substrate, or cuts that penetrate the front and back surfaces of the film substrate.

[0013] The cell culture film substrate of the first embodiment has only (a) above, and has an imprint on at least a portion of both surfaces, and at least a portion of the imprint has an asymmetrical shape on the front and back.

[0014] The cell culture film substrate of the second embodiment has only (b) above, and has one or more selected from the group consisting of notches, protrusions, and incisions formed on the film substrate, the notches and protrusions being formed on at least a part of the outer circumference of the film substrate, and the incisions penetrating the front and back surfaces of the film substrate. In the second embodiment, if there is a structure for distinguishing the front and back surfaces by any one or more combinations of the notches, protrusions, and incisions, it may also have markings that do not have an asymmetrical shape between the front and back surfaces.

[0015] The cell culture film substrate of the third embodiment has (a) and (b) above, and has an imprint having an asymmetrical shape on at least a portion of both surfaces, and has one or more selected from the group consisting of notches, protrusions and notches formed on the film substrate, the notches and protrusions are formed on at least a portion of the outer circumference of the film substrate and the notches penetrate the front and back surfaces of the film substrate.

[0016] In the first and third embodiments, the markings function as visual information for distinguishing between the front and back sides of the film substrate. Furthermore, an asymmetrical shape may be created by combining the shape and position of the markings with the shape or position of any of the notches, protrusions, and cuts formed on the film substrate. Moreover, it is preferable that part or all of the markings be laser-engraved. Laser irradiation changes the surface structure and surface material of the film substrate, resulting in a difference in color or light reflection between the engraved area and its surroundings, improving the visibility of the laser engraving. As a result, it becomes possible to distinguish between the front and back sides of the film substrate even when the film substrate or the cell sheet provided on the film substrate is transparent.

[0017] In the second and third embodiments, the peripheral portion of the notch, the protruding portion, or the cut portion functions as a gripping portion when grasping the film substrate placed inside the culture vessel or at the transplantation site. Since such gripping portions are structured to be easily grasped by gripping tools such as tweezers, the operability of the film substrate can be improved.

[0018] The cell culture film substrate of this embodiment will be described in detail below.

[0019] Figures 1, 2, and 3(A) show schematic top views of an example of the cell culture film substrate 1. Figure 3(B) is a cross-sectional view taken along line I-I' of the cell culture film substrate shown in Figure 3(A).

[0020] The cell culture film substrate 1 includes a film substrate 150 having a culture surface 10 on at least one of its surfaces.

[0021] The film substrate 150 of the first and third embodiments has laser engraving (engraving 30) on at least one of its two surfaces. The engraving 30 and the culture surface 10 may be formed on the same surface, or they may be formed on different surfaces. Laser engraving does not use ink that may affect cell culture and does not generate much debris, so it may be formed on the same surface as the culture surface 10. In addition, laser engraving can suppress distortion in the film substrate 150 compared to forming surface irregularities by embossing.

[0022] The marking 30 is composed of recesses formed in the depth direction of the surface. These recesses are formed by the evaporation of the material on the surface of the film substrate 150 due to laser irradiation. As shown in Figure 3(B), when the thickness of the film substrate 150 is T and the depth of the marking 30 is D1, D1 / T is preferably 0.1 to 0.6, and more preferably 0.17 to 0.5. The depth of the marking 30 (D1) is preferably 1 μm to 7 μm, and more preferably 2 μm to 6 μm. In this specification, the depth can be measured using a microscope. Also, unless otherwise specified, "~" indicates that the upper and lower limits are included.

[0023] The surface of the film substrate 150 on the side where the culture surface 10 is formed may have a culture-forming region (a region suitable for cell culture) over its entire surface, or it may have both a culture-forming region and a non-culture-forming region (a region unsuitable for cell culture). In other words, in this embodiment, the culture surface 10 is defined as the surface portion that becomes the culture-forming region, and does not include the surface portion that becomes the non-culture-forming region. Furthermore, as will be explained in detail later, it is preferable that the surface portion that becomes the culture-forming region is subjected to plasma treatment. It is preferable that the markings 30 are formed only in the non-culture-forming region, that is, not formed on the culture surface 10. However, the markings 30 may be formed only in the culture-forming region, or they may be formed spanning both the culture-forming region and the non-culture-forming region. The non-culture-forming region may be formed near the outer circumference 20 of the surface of the film substrate 150 including the culture surface 10, excluding the center, or it may be formed so as to surround the outer edge of the culture-forming region (culture surface 10) located in the center. Furthermore, by adopting a configuration in which the imprint 30 is formed on the outside of the culture surface 10 when the surface of the film substrate 150 is viewed from above, the occurrence of distortion and wrinkles on the culture surface 10 can be further suppressed. This makes it possible to realize a film substrate suitable for culturing cell sheets.

[0024] The marking 30 may be located near the outer circumference 20 of the film substrate 150. For example, the marking 30 may be located within 5 mm or within 3 mm of the outer circumference 20. Specifically, it is preferable that part or all of the marking 30 be formed in a non-culture-forming area on the surface of the film substrate 150, but part of it may be formed in a culture-forming area. The marking 30 may also be formed on the surface (back surface) opposite to the culture surface 10. In that case, the marking 30 may be located near the outer circumference 20 on the back surface of the film substrate 150, or in an area other than the vicinity of the outer circumference 20.

[0025] The marking 30 may include one or more marks selected from the group consisting of letters, symbols, and patterns. Letters include languages, numbers, special characters, etc. Symbols include codes (barcodes, QR codes (registered trademarks), etc.), general symbols, mathematical symbols, etc. Patterns include geometric patterns, pictures, abstract patterns, etc. The information contained in the marking 30 makes it possible to manage the unique information of the film substrate 150, including historical information such as lot numbers. Furthermore, since the marking 30 is provided on the film substrate 150, the marking 30, including the code, can be read by a reading machine whether the film substrate 150 is dry or wet with a culture medium, etc.

[0026] The markings 30 may include those that indicate an asymmetrical shape on the front and back with a single mark, or they may include those that indicate an asymmetrical shape on the front and back with two or more marks.

[0027] The width of the markings 30 in the direction from the center of the film substrate 150 to the outer circumference 20 (for example, the height of the characters if the markings are characters) is, for example, 1 to 5 mm, preferably 1.5 to 4 mm, and more preferably 2 to 3 mm.

[0028] Furthermore, as shown in Figures 3(A) and 3(B), the film substrate 150 may have a groove 60 between the imprint 30 and the center of the culture surface 10, where a portion of the surface is recessed. The groove 60 can suppress the transmission of wrinkles and distortions in the film substrate 150 that occur during the manufacture of the imprint 30 to the culture surface 10. The groove 60 may also be formed by laser irradiation, similar to the imprint 30.

[0029] When viewed from the direction normal to the surface, the groove 60 may be formed as continuous or discontinuous. For example, when viewed from the direction normal to the surface, if the marking 30 is formed extending in an arc shape on the surface of a circular film substrate 150, the groove 60 may have an arc shape, and preferably a circular shape.

[0030] When the thickness of the film substrate 150 is T and the depth of the groove 60 is D2, D2 / T is preferably 0.1 to 0.6, and more preferably 0.17 to 0.5. Also, the depth of the groove 60 (D2) is preferably 1 μm to 7 μm, and more preferably 2 μm to 6 μm. The depth of the groove 60 (D2) may be the same as the depth of the engraving 30 (D1), or it may be greater than the depth of the engraving 30.

[0031] The film substrate 150 of the second and third embodiments may have a notch 40, a protrusion 50, or both a notch 40 and a protrusion 52 in at least a portion of the outer circumference 20.

[0032] The outer edge of the film substrate 150 may be partially or entirely straight or curved when viewed from the direction normal to the surface, and is preferably circular. As shown in Figure 1, the notch 40 has a structure in which a part of the outer edge of the film substrate 150 is cut inward toward the center.

[0033] As shown in Figure 1 or Figure 2, the protrusions 50 and 52 have a structure in which a part of the outer edge of the film substrate 150 protrudes outward from the center. In the film substrate 150, an outer edge frame is formed by extending the outer edge of the outer circumference 20 where the protrusions 50 are not formed. The protrusion 50 shown in Figure 2 has a portion (external tag) that protrudes in a region outside the outer edge frame. On the other hand, the protrusion 52 shown in Figure 1 has a portion (internal tag) that protrudes in a region inside the outer edge frame. Both the protrusion 50 (external tag) and the protrusion 52 (internal tag) are composed of a part of the structure of the film substrate 150 and may constitute a part of the surface of the film substrate 150. That is, the protrusions 50 and 52 may be formed continuously from the culture surface 10 on the surface of the film substrate 150, and other regions (e.g., non-culture regions) or other structures (grooves 60, 62, etc.) may be formed between the culture surface 10 and the protrusions 50 and 52. The protruding portion 50 (outer tag) is bendable relative to the film substrate 150 and can move in the front-to-back direction starting from the connection point with the film substrate 150. The film substrate 150 and the protruding portion 50 may be formed by cutting from the same resin film. In this case, the protruding portion 50 is an extended portion of the substrate that protrudes radially from the outer circumference 20 of the film substrate 150. Note that the protruding portion 50 does not constitute a projection that is fixed in an upward (vertical direction of the surface) position from the edge of the outer circumference 20 of the film substrate 150.

[0034] When the outer edge of the film substrate 150 is viewed from the direction normal to the surface, it may have one or more protrusions 52 (internal tags) on a part of the circular outer edge. For example, as shown in Figure 1, the protrusions 52 may be formed sandwiched between two notches 40.

[0035] As shown in Figures 3(A) and 3(B), an engraving 32 may be formed on part or all of the surface of at least one of the protruding portions 50 and 52. For example, the engraving 32 may include a code, as shown in Figure 3(A), but is not limited to this, and may be formed to include various kinds of information.

[0036] As shown in Figures 3(A) and 3(B), the film substrate 150 may have a groove 62 between the protrusion 50 and the center of the culture surface 10, where a part of the surface is recessed. The groove 62 may be formed on at least a part of the outer edge frame, or it may be formed along a line connecting the base end opposite to the tip of the protrusion 50. If the film substrate 150 has a groove 60, the groove 62 is formed outside the groove 60. When the thickness of the film substrate 150 is T and the depth of the groove 62 is D3, D3 / T is preferably 0.1 to 0.6, and more preferably 0.17 to 0.5. The depth of the groove 62 (D3) is preferably 1 μm to 7 μm, and more preferably 2 μm to 6 μm. The depth of the groove 62 (D3) may be the same as the depth of the groove 60 (D2), or it may be greater than the depth of the groove 60.

[0037] At least part or all of the side surfaces of the outer circumference 20 of the film substrate 150, the side surfaces of the outer circumference 20 of the notch 40, and the side surfaces of the outer circumference 20 of the protrusions 50 and 52 may be laser-processed surfaces. Even if the film substrate 150 is a thin film, the shape of the outer circumference 20, the notch 40, and the protrusions 50 and 52 can be improved by laser irradiation.

[0038] Furthermore, the film substrate 150 of the second and third embodiments may have a notch 70 that penetrates both the front and back surfaces, as shown in Figure 2. The film substrate 150 can be manipulated by inserting tweezers or the like into the notch 70. It is also possible to manipulate the notch 70 by looping a thread around it. The shape of the notch 70 when viewed from the direction normal to the culture surface 10 can be, for example, a cross, a V-shape, a U-shape, an arc, or an I-shape, but is not limited thereto. The size of the notch 70 is not particularly limited, but for example, it can be 1 to 5 mm or 2 to 4 mm on each side.

[0039] The film substrate 150 may have two or more notches 40, two or more protrusions 50, 52, two or more notches 70, or two or more different notches selected from the group consisting of "notches 40", "protrusions 50, 52", and "notches 70" (specifically, including combinations of "notches 40" and "protrusions 50, 52", "notches 40" and "notches 70", "protrusions 50, 52" and "notches 70", and "notches 40", "protrusions 50, 52", and "notches 70"). In the film substrate 150, two or more notches selected from the group consisting of "notches 40", "protrusions 50, 52", and "notches 70" may be provided in asymmetrical positions on the front and back.

[0040] An example of the cell culture film substrate 1 of this embodiment is shown in Figure 4, but is not limited thereto. The film substrate 150 may have, for example, an imprint 30, a notch 40, a protrusion 50, a protrusion 52, and a cut 70, each individually, or it may have combinations such as imprint 30 and a protrusion 50, imprint 30 and a protrusion 52, a protrusion 50 and a cut 70, a protrusion 52 and a cut 70, imprint 30, a protrusion 50 and a cut 70, imprint 30, a protrusion 52 and a cut 70, and so on. Another example of the cell culture film substrate 1 will be described with reference to Figure 6. The film substrate 150 shown in Figure 6 has a protrusion 50 (external tag), and further has a groove 62 between the protrusion 50 and the culture surface 10, in which a part of the surface is recessed. It is preferable that this groove 62 is provided at a position formed along the outer edge of the film substrate 150, and if the outer edge is circular, it is more preferable that it is formed in an arc shape. With this configuration, the protruding portion 50 can be easily bent at the position of the groove portion 62, and distortion and wrinkles on the surface of the culture surface 10 can be further suppressed when bending. As another modification, in the above configuration, the groove portion 62 may have one of the following structures. That is, from the viewpoint of strength and ease of bending, it may have a structure in which a plurality of through holes that penetrate both the front and back are arranged along the outer edge, or it may have a structure in which non-penetrating recesses are arranged continuously or discontinuously along the outer edge. As another modification, in the above configuration, an imprint 30 may be further formed on the protruding portion 50 (external tag). By moving the location of the imprint 30 to the protruding portion 50 (external tag), the transmission of distortion and wrinkles to the surface of the culture surface 10 can be suppressed. Furthermore, the occurrence of distortion and wrinkles on the surface of the culture surface 10 can be suppressed even more than when the imprint 30 is formed near the outer circumference 20. As another modification, in the above configuration, a structure for distinguishing the front and back may be formed on a part of the protruding portion 50 (external tag). Specifically, a shape in which a part of the protruding portion 50 is missing may be adopted. This makes it easy to distinguish between the front and back sides of the film substrate. In this case, it is not necessarily required to form the markings 30 on the protruding portion 50. The grooves 62 and markings 30 may be formed on the same surface as the culture surface 10, or on the surface (back side) opposite to the culture surface 10.Furthermore, the width (w1) of the portion where the protruding portion 50 (external tag) is joined to the outer edge of the film substrate 150 is, for example, 2 to 20 mm, preferably 2 to 15 mm, and more preferably 3 to 10 mm. In addition, the width (w2) of the protruding portion 50 (external tag) that extends beyond the outer edge of the film substrate 150, that is, the maximum distance from the line connecting both ends of the joining portion between the protruding portion 50 and the outer edge to the outer edge of the protruding portion 50, is, for example, 3 to 30 mm, preferably 4 to 20 mm, and more preferably 5 to 15 mm. The area (s) of the protruding portion 50 (external tag) is, for example, 9 to 900 mm². ２ Preferably 20 to 500 mm ２ , more preferably 30 to 90 mm ２ That is the case.

[0041] The film substrate 150 may contain organic polymer materials and may be configured not to contain biological membranes such as collagen film or amniotic membrane. When a biological membrane is used as the substrate, it tends not to be possible to immediately detach the cell sheet formed on the biological membrane. In contrast, with a film substrate 150 containing organic polymer materials, the cell sheet formed on the culture surface 10 can be immediately detached from the transplant site after being attached. The time from attachment to detachment may be 10 minutes or less.

[0042] The materials constituting the film substrate 150 (hereinafter also referred to as resin materials) include, specifically, polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), and ethylene tetraphosphate. Examples of organic polymer materials include 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 polymers, gelatin, and collagen. In particular, examples of synthetic polymer materials used as resin materials include polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), 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), and cyclic olefin polymers.An example of the film substrate 150 may be a film mainly composed of at least one of these resin materials, preferably a polyetheretherketone film, polyethylene terephthalate film, or polystyrene film, more preferably a polyetheretherketone film. The film substrate 150 preferably contains one or more resin layers made of the above resin materials. The two or more resin layers may contain different types of materials. The film substrate 150 may be composed of a resin substrate containing resin layers made of the above materials, or it may be composed of a laminate substrate containing a resin layer made of the above materials and a resin layer and / or an inorganic layer made of other materials. However, the resin layers do not have to include layers formed by chemical vapor deposition such as parylene or wet coating layers. Furthermore, the inorganic layer in the laminate substrate preferably does not contain glass, but may contain metal foil. In addition, it is preferable that the film substrate 150 does not contain nonwoven fabric and fiber substrate on the culture surface 10 side. When considering all aspects from the viewpoints of low coefficient of thermal expansion, solvent resistance, and heat resistance, the film substrate 150 containing PEEK is preferable to the case containing PET. Furthermore, it is preferable that the film substrate 150 is composed of a material with a specific gravity higher than that of the culture medium.

[0043] The film substrate 150 may be transparent. Transparency means that the haze is 30% or less. Even with a transparent film substrate 150, the visibility of the markings 30 is excellent because it has a contrast ratio. Furthermore, if the surface of the film substrate 150 is embossed, it is preferable that the haze is 35% or less, preferably 30% or less, more preferably 25% or less, and the total light transmittance is 40% or more, preferably 60% or more, more preferably 80% or more. According to the inventors' findings, cell observation is possible even with a large haze value when there is no embossing, but when embossing is present on the surface, it has been found that the haze needs to be below the above upper limit value from the viewpoint of cell observation. In the case of PEEK film, it is preferable not only to control the haze by thickness but also to reduce the haze by lowering the crystallinity. That is, in addition to the above conditions, it is more preferable to use a low-crystallinity PEEK film.

[0044] It is preferable that at least the culture-forming region (culture surface 10) of the film substrate 150, including the culture surface 10, is subjected to a hydrophilic treatment. This improves cell adhesion. Examples of this hydrophilic treatment include UV ozone treatment and plasma treatment. Among these, it is preferable to use a film substrate 150 in which the culture surface 10 has been plasma-treated. It is also preferable that the surface opposite to the side on which the culture surface 10 is formed is subjected to the above-mentioned hydrophilic treatment, specifically plasma treatment. By hydrophilizing both surfaces, the compatibility (wettability) with the culture medium is improved. As a result, when the film substrate 150 is immersed in the culture medium, the floating of the film can be suppressed, and the operability of the substrate can be further improved. In this specification, a hydrophilic surface means a surface in which the upper limit of the water droplet contact angle θ measured by the θ / 2 method is 70° or less. On the other hand, the lower limit of the water droplet contact angle θ of the hydrophilic surface is preferably 3° or more, but is not limited thereto. The water droplet contact angle θ is measured by placing 2 μl of pure water on the substrate at 25°C and measuring the angle between the water droplet and the substrate using a contact angle meter in accordance with the θ / 2 method in accordance with ISO 19403-2:2017. Furthermore, the lower limit of the surface roughness Ra of the hydrophilized culture surface is 0.3 nm or more, preferably 0.5 nm or more, and the upper limit is 100 nm or less, preferably 10 nm or less, more preferably 2 nm or less. Here, surface roughness Ra refers to the arithmetic mean roughness in a square region with sides of 100 nm, using surface shape data measured by an atomic force microscope (AFM).

[0045] The shape of the film substrate 150 in the direction normal to the surface is preferably circular, but it may be other shapes, such as regular polygons like squares, regular pentagons, regular hexagons, and regular octagons, or ovals, rectangles, etc.

[0046] The thickness of the film substrate 150 is not particularly limited, but is preferably 5 μm or more and 100 μm or less. The lower limit of the substrate thickness is more preferably 6 μm or more, even more preferably 8 μm or more, 10 μm or more, or 12 μm or more. On the other hand, the upper limit of the substrate 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 substrate thickness of the film substrate 150 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. Furthermore, by setting the thickness to be above the lower limit, it is possible to prevent the film substrate 150 from tearing or curling during transport, thereby improving handling. By setting the thickness to be below the upper limit, it is possible to improve conformability to the affected area during transplantation. Note that the film substrate 150 in a cross-sectional view in the thickness direction may have a flat, sheet-like surface overall.

[0047] Furthermore, from the viewpoint of functioning as a self-supporting membrane during culture, the lower limit of the film substrate thickness 150 is preferably greater than 10 μm, more preferably 11 μm or more, and even more preferably 12 μm or more. This makes it possible to manufacture cell sheets 180 without fixing or adhering the film substrate 150 to the inside of the culture vessel 110 during the cell culture process by using a film substrate 150 with a substrate thickness greater than 10 μm. A self-supporting membrane is preferably a membrane in which the planar state of the culture surface 10 of the film substrate 150 is maintained even in the culture medium, and more preferably the planar state is maintained even after the cell sheet 180 is formed on the culture surface 10. Because the planar state of the culture surface 10 is maintained, the non-curling film substrate 150 can enhance the cell culture characteristics in the culture medium without adhering. In addition, this self-supporting membrane may also be able to maintain its sheet shape to a certain extent when one end is grasped and lifted with forceps. When viewed from the direction normal to the surface, it is preferable that the film substrate 150 does not have openings that penetrate the front and back surfaces in the region where the culture surface 10 is formed, and when placed in the culture container 110, it is preferable that it does not have openings that expose the bottom surface of the culture container 110. In other words, it is preferable that the film substrate 150 is not an annular structure and that the region where the culture surface 10 is formed is solid.

[0048] The area of the film substrate 150 is not particularly limited, but for example, it is 0.3 cm ２ or more and 1000 cm ２ or less. As the lower limit value of the area of the film substrate 150, it may be 0.6 cm ２ or more, 1.6 cm ２ or more and 3 cm ２ or more, 4 cm ２ or more, 5 cm ２ or more, 6 cm ２ or more, 7 cm ２ or more, 8 cm ２ or more or 10 cm ２ or more. On the other hand, as the upper limit value of the area of the film substrate 150, it is more preferably 900 cm ２ or less, still more preferably 800 cm ２ or less, particularly preferably 500 cm ２ or less, and it may be 100 cm ２ or less, 50 cm ２ or less or 20 cm ２ or less. For example, the area of the film substrate 150 is 0.6 cm ２ or more and 900 cm ２ or less, 1.6 cm ２ or more and 800 cm ２ or less, 3 cm ２ or more and 500 cm ２ or less, 6 cm ２ or more and 100 cm ２ or less, 8 cm ２ or more and 50 cm ２ or less or 10 cm ２ or more and 20 cm ２ or less.

[0049] The film substrate 150 is such that, when the culture surface 10 of the film substrate 150 is measured with a laser microscope, there are three or fewer holes in a 100 μm square area, each with a diameter of 1 μm or more and 100 μm or less, and a depth of 0.5 μm or more and 100 μm or less. The diameter of the holes is, for example, 1 μm or more and 100 μm or less, preferably 2 μm or more and 50 μm or less, and more preferably 3 μm or more and 30 μm or less. The depth of the holes is, for example, 0.5 μm or more and 100 μm or less, preferably 1 μm or more and 50 μm or less, and more preferably 2 μm or more and 20 μm or less. The ranges of hole diameter and hole depth include, for example, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 0.5 μm or more and 100 μm or less, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 1 μm or more and 50 μm or less, a hole diameter of 1 μm or more and 100 μm or less and a hole depth of 2 μm or more and 20 μm or less, a hole diameter of 2 μm or more and 50 μm or less and a hole depth of 0.5 μm or more and 100 μm or less, and the hole diameter The pores are 2 μm to 50 μm in diameter and 1 μm to 50 μm in depth, 2 μm to 50 μm in diameter and 2 μm to 20 μm in depth, 3 μm to 30 μm in diameter and 0.5 μm to 100 μm in depth, 3 μm to 30 μm in diameter and 1 μm to 50 μm in depth, and 3 μm to 30 μm in diameter and 2 μm to 20 μm in depth. The upper limit of the porosity of the film substrate 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 film substrate 150 is not particularly limited, but may be 0% or more. By ensuring that the number of holes in the culture surface 10 is three or less, and / or by keeping the porosity of the film substrate 150 below the upper limit, the adhesion force with the cell sheet 180 can be made appropriate. The presence or absence of holes may be measured on the surface of the resin layer formed on the culture surface 10 side of the film substrate 150. The porosity is calculated from the theoretical density and the measured density. Specifically, it is calculated using the formula: porosity = {1 - (actual density / theoretical density)} × 100.

[0050] The film substrate 150 does not necessarily need to have a layer containing the temperature-responsive polymer formed on the surface on which the culture surface 10 is formed. This prevents a decrease in adhesion between the cell sheet and the film substrate 150 in low-temperature environments such as cryopreservation processes. The temperature-responsive polymer is a material that exhibits cell adhesion at the temperature at which cells are cultured, and then exhibits cell non-adhesion by changing the temperature, making it possible to easily peel off the cell sheet. It is preferable that the temperature range in which the temperature-responsive polymer exhibits cell adhesion is 10°C to 45°C, particularly 33°C to 40°C, as this allows for stable cell culture. It is also preferable that the temperature range in which the temperature-responsive polymer exhibits cell non-adhesion is 1°C to 36°C, particularly 4°C to 32°C, as this reduces the damage to the peeling of the cell sheet. Examples of temperature-responsive polymers that can be used as materials for temperature-responsive polymers include 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, among which PNIPAAm, poly-N-n-propylmethacrylamide, and poly-N,N-diethylacrylamide are preferred. The bottom surface of the culture vessel 110 may also be configured so as not to contain temperature-responsive polymers.

[0051] Next, an example of a method for producing cell culture film substrates will be described.

[0052] An example of a method for manufacturing a cell culture film substrate according to the first embodiment includes: a surface treatment step of applying plasma treatment to at least one surface of a resin sheet to form a culture surface 10; a cutting step of cutting out one or more film substrates 150 having the culture surface 10 from the resin sheet; and a step of forming either one or both of the following structures (a) or (b): (a) a structure having an imprint 30 including an asymmetrical shape on at least a portion of both surfaces; (b) a structure having one or more of notches 40 or protrusions 50, 52 formed on at least a portion of the outer circumference of the film substrate 150, or cuts 70 penetrating the front and back surfaces of the film substrate 150.

[0053] The resin sheet can be made of the same material and thickness as the film substrate 150 described above. The resin sheet may be a roll of film or a sheet of film obtained by cutting a roll. Plasma treatment of the resin sheet may be performed only on the surface that will become the culture surface 10. After treating the said surface, plasma treatment may also be performed on the opposite surface (back side). Furthermore, instead of plasma treatment, the aforementioned hydrophilization treatment may be performed. The cutting process may be performed by cutting with a blade or die-cutting, but is preferably performed by laser treatment. Specifically, the outer circumference 20 of the film substrate 150 may be cut from the resin sheet by laser treatment. Laser treatment improves the processability of the film substrate 150, which is a thin film of 100 μm or less, compared to cutting with a blade or die-cutting. Similarly, the process of forming the structure may be performed by laser treatment. That is, at least one of the markings 30, notches 40, protrusions 50, 52, or cuts 70 can be formed by laser treatment.

[0054] In the first embodiment, the structure formation step, including the formation of the imprint 30, may be performed before the cutting step. For example, if laser processing is used for both the formation of the imprint 30 and the cutting step, it is preferable to cut along the outer edge of the film substrate 150 after forming the imprint 30. When the resin sheet is thin, if the imprint is formed by embossing, distortion may occur in the film, making it difficult to form a homogeneous cell sheet. In contrast, laser processing allows for the formation of the imprint 30 even on a film substrate 150 with a thin film thickness of 100 μm or less. The contrast ratio of the imprint 30 can be controlled by the laser irradiation intensity, the number of irradiations, etc.

[0055] In the second embodiment, the structural formation step, which includes forming the notch 40 or at least one of the protrusions 50, 52, may be carried out in the same step as the cutting step.

[0056] For example, in the process of forming the notches 40 or the protrusions 50, 52 and the cutting process, the outer circumference 20 of the notches 40 and the protrusions 50, 52 can be cut out simultaneously with the outer circumference 20 of the film substrate 150. In this case, the resin sheet may be cut by laser processing. Furthermore, at the corners of the outer circumference 20 of the notches 40 and the protrusions 50, 52 formed by laser processing, higher processing accuracy can be obtained than when using blades or die-cutting. Moreover, since there are molten portions formed by laser irradiation at these corners, it is possible to suppress the propagation of cracks (slits) inward from these portions, which can easily cause the film substrate 150 to tear. In the cutting process of the first embodiment, laser processing may be performed to remove a portion of the film substrate 150 surface in the depth direction, forming a groove 60 between the marking 30 and the center of the culture surface 10. Also, in at least one of the cutting processes of the first and second embodiments, laser processing may be performed to remove a portion of the film substrate 150 surface in the depth direction, forming a groove 62 between the protrusions 50 and the culture surface 10. Furthermore, in the cutting process in the first and second embodiments, it is preferable to cut the film substrate 150 into multiple pieces from the resin sheet. This increases productivity.

[0057] Furthermore, in the third embodiment, the method for manufacturing a cell culture film substrate involves forming (a') an imprint 30 that does not have an asymmetrical shape on at least a portion of both surfaces, and the structure of (b) in the structure formation step of the first embodiment. That is, in the third embodiment, the asymmetrical shape may be formed by the imprint 30, or by a combination of the imprint 30 and at least one of the "notch 40", "protrusions 50, 52", and "cutout 70". Also, in the first and third embodiments, with respect to the structure of (b), either the "protrusions 50, 52" have an asymmetrical shape, or the "notch 40" has an asymmetrical shape, or two or more selected from the group consisting of "protrusions 50, 52" and "notch 40" (two protrusions may be selected) are provided in positions that are asymmetrical. Note that "protrusions 50, 52" have an asymmetrical shape means that a structure such as a chip may be formed on a part of the protruding portion.

[0058] The cell culture film substrate of this embodiment may have a surface protective material coated on the culture surface 10. Examples of surface protective materials include metal foil, resin sheets, fiber sheets, etc. The cell culture film substrate may also be sterilized. Examples of sterilization methods include electron beam sterilization, gamma ray sterilization, EOG sterilization, and autoclaving. The sterilization treatment may be performed on a package containing the cell culture film substrate or on a packing body containing the cell culture film substrate. The sterilization treatment may also be performed on the cell culture film substrate with the surface protective material. The package can be any package that contains the cell culture film substrate or the cell culture film substrate with the surface protective material. The bag may be a heat-sealed bag or a bag with a zipper, but specifically, two-sided bags, three-sided sealed bags, three-sided zippered bags, stand-up pouches, etc., can be used. The sealing method should be selected to suit the sealing performance required for the packaging. The packing body includes a packaging box that contains the package. The packaging box may be a paper box such as a corrugated cardboard box.

[0059] <Cell Sheet Attached Film Substrate> Next, the cell sheet attached film substrate and its manufacturing method will be described.

[0060] The cell sheet-attached film substrate of this embodiment includes the cell culture film substrate described above and a cell sheet provided on the culture surface of the cell culture film substrate.

[0061] Figure 5 is a schematic cross-sectional view showing an example of a culture kit used in the manufacturing method of a cell sheet-attached film substrate.

[0062] An example of a method for manufacturing a cell sheet-attached film substrate according to this embodiment includes: an installation step of placing the above-mentioned cell culture film substrate into a culture vessel 110; a culture step of placing a culture medium P into the culture vessel 110, seeding cells, and culturing the cell sheet 180 on the film substrate 150 of the cell culture film substrate; and an removal step of removing the cell sheet-attached film substrate from inside the culture vessel 110.

[0063] The installation step may include the step of placing the ring member 170 on the film substrate 150 inside the culture vessel 110. The removal step may also include the step of removing the ring member 170 from the film substrate 150.

[0064] Furthermore, when a cell culture film substrate having at least one of a notch 40, protrusions 50, 52, and a notch 70 is used in the installation process, the removal process may include a step of holding the portion of the film substrate 150 near the notch 40, the protrusions 50, 52, or the notch 70.

[0065] The cells are clinically useful cells for treating or preventing symptoms related to cell, tissue, or organ defects and dysfunction, or cultureable cells for use in non-clinical trials, etc., and are not particularly limited as long as they are cells isolated from a living organism. Examples of cells include living tissue cells, mesenchymal stem cells that have the ability to differentiate into cells belonging to mesenchymal tissues, pluripotent stem cells that have the ability to differentiate into various living tissues, and differentiation-inducible stem cells and progenitor cells. The cells may be adherent cells or suspension cells.

[0066] Specific examples of living tissue cells include, for example, fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiomyocytes, myoblasts, osteocytes, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, renal cells, gingival cells, periosteal cells, skin cells, and endothelial cells. Specific examples of mesenchymal stem cells include, for example, 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. Specific examples of pluripotent stem cells include, for example, induced pluripotent stem cells, embryonic stem cells, nuclear transfer embryonic stem cells, embryonic tumor cells, and embryonic germ cells. These cells may be cultured individually or in combination of two or more types. These cells should be appropriately selected from known types depending on the intended use of the cells.

[0067] The origin of the cells is not particularly limited, but examples include mammals, birds, amphibians, fish, insects, plants, and microorganisms. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and chickens.

[0068] The cell sheet 180 has a sheet structure in which cells are physically and functionally connected to each other via adhesion molecules, extracellular matrix, etc. The cell sheet 180 may be a monolayer structure composed of one cell layer, or a layered structure composed of two or more cell layers. There are no particular limitations on the layered structure, but examples include multilayer structures of two, three, four, five layers, etc.

[0069] If the cell sheet 180 has a multilayer structure, the multilayer structure may be obtained when cultured on the film substrate 150, or it can be obtained by stacking single-layer cell sheets. In particular, by preparing multiple cell sheet-attached film substrates of the present invention, stacking one cell sheet on top of another, and peeling off the culture carrier substrate from the other cell sheet, a cell sheet with a multilayer structure can be obtained.

[0070] The thickness of the cell sheet 180 is not particularly limited, but for example, it is between 0.001 mm and 2.0 mm. The lower limit of the thickness of the cell sheet 180 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 180 is more preferably 1.5 mm or less, even more preferably 1.2 mm or less, and particularly preferably 1.0 mm or less. The thickness of the cell sheet 180 is, 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 1.5 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, and 0.05 mm to 1.0 mm. By setting the thickness of the cell sheet 180 within the above range, a high cell activity rate within the cell sheet 180 and excellent shape retention capabilities advantageous for cell transplantation can be achieved.

[0071] The area of ​​the cell sheet 180 is not particularly limited, but for example, 0.3 cm² ２ More than 1000cm ２ The following applies: The lower limit for the area of ​​cell sheet 180 is 0.6 cm². ２ Above 1.6 cm ２ More than 3cm ２ More than 4cm ２ Above, 5cm ２ Above 6 cm ２ More than 7cm ２ Above 8cm ２ or more or 10 cm ２ It may be greater than or equal to this. On the other hand, the upper limit of the area of ​​the cell sheet 180 is more preferably 900 cm². ２ Further preferably 800 cm ２ The following is particularly preferred: 500 cm ２ The following is the case, 100 cm ２ Below, 50cm２ Below or 20 cm ２ The following may also be the case. For example, the area of ​​the cell sheet 180 is 0.6 cm². 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 Below or 10 cm ２ 20cm or more ２ The following applies: Conventionally, when transplanting cell sheets alone, the strength of the cell sheets is low, and in the case of large areas, there is a high possibility of damage during transport. However, in this disclosure, since the cell sheet 180 is supported by a culture carrier substrate, it is possible to prevent the cell sheet from tearing during transplantation, and the size of the cell sheet 180 is 8 cm. ２ The size can be made even larger. Alternatively, a large cell sheet can be prepared and then adjusted to the appropriate size for the affected area. In the case of a film substrate 150 with a cell sheet 180, it is preferable that the entire lower surface of the cell sheet 180 is positioned to overlap the surface (culture surface 10) of the film substrate 150.

[0072] Cell culture is not particularly limited, and conventional methods used in the fields of medicine, pharmaceuticals, quasi-drugs, cosmetics, food, veterinary medicine, and basic technologies such as regenerative medicine and biotechnology can be used.

[0073] The culture conditions are not particularly limited as long as they allow the cultured cells to be brought to the desired state. Typical culture conditions include, for example, 37°C and 5% CO2 using a prepared basal medium. ２under such an environment. The cell culture period is not particularly limited as long as the cultured cells reach the target state. For example, the cell culture period can be within 28 days, within 21 days, within 14 days, within 7 days, within 5 days, or within 3 days. When culturing for a long period, the medium can be changed. The frequency and method of medium change are not particularly limited. Generally, it is preferable to change the medium every 1 to 7 days. Particularly preferably, it is every 1 to 5 days. At this time, the entire amount of the medium can be changed, or a part of the medium can be left and new medium can be added.

[0074] The density of the cells to be cultured is not particularly limited as long as it is suitable for the cells to be cultured, the culture vessel, and the use of the cultured cells. For example, it is ２ cells / cm ２ or more and 1×10 ９ cells / cm ２ or less. As the lower limit value of the cell density, more preferably, it is 1×10 ３ cells / cm ２ or more, still more preferably 5×10 ３ cells / cm ２ or more, and particularly preferably 5×10 ４ cells / cm ２ or more. On the other hand, as the upper limit value of the cell density, more preferably, it is 1×10 ８ cells / cm ２ or less, still more preferably 5×10 ７ cells / cm ２ or less, and particularly preferably 1×10 ７ cells / cm ２ or less. The density of the cells to be cultured is, for example, 5×10 ２ cells / cm ２ or more and 1×10 ９ cells / cm ２ or less, 5×10 ２ cells / cm ２ or more and 1×10 ８ cells / cm ２ or less, 5×10 ２ cells / cm ２ or more and 5×10 ７ cells / cm ２ or less, 5×10 ２ cells / cm ２ or more and 1×10 ７ cells / cm２ The following is 1×10 ３ cells / cm ２ or more, 1×10 ９ cells / cm ２ or less, 1×10 ３ cells / cm ２ or more, 1×10 ８ cells / cm ２ or less, 1×10 ３ cells / cm ２ or more, 5×10 ７ cells / cm ２ or less, 1×10 ３ cells / cm ２ or more, 1×10 ７ cells / cm ２ or less, 5×10 ３ cells / cm ２ or more, 1×10 ９ cells / cm ２ or less, 5×10 ３ cells / cm ２ or more, 1×10 ８ cells / cm ２ or less, 5×10 ３ cells / cm ２ or more, 5×10 ７ cells / cm ２ or less, 5×10 ３ cells / cm ２ or more, 1×10 ７ cells / cm ２ or less, 5×10 ４ cells / cm ２ or more, 1×10 ９ cells / cm ２ or less, 5×10 ４ cells / cm ２ or more, 1×10 ８ cells / cm ２ or less, 5×10 ４ cells / cm ２ or more, 5×10 ７ cells / cm ２ or less, 5×10 ４ cells / cm ２ or more, 1×10 ７ cells / cm ２ is as follows.

[0075] The culture medium is not particularly limited as long as it is suitable for the cells being cultured. Examples of culture medium components include sugars, amino acids, vitamins, inorganic salts, trace metals, and additives. These culture medium components may be formulated individually or in combination of two or more. These culture medium components should be appropriately selected from known components depending on the cells being cultured.

[0076] Specific examples of sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; cyclodextrins; and oligosaccharides.

[0077] Specific examples of amino acids include L-glutamic acid, L-glutamine, L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, and L-hydroxyproline.

[0078] Specific examples of vitamins include, for instance, L-sodium ascorbate, L-ascorbic acid diphosphate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0079] Specific examples of inorganic salts include sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, potassium bicarbonate, calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, calcium carbonate, magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, and magnesium carbonate. Specific examples of trace metals include iron sulfate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate.

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

[0081] Examples of culture media containing the above-mentioned culture medium components include AIM V medium, HFDM-1 medium, equilibrium buffers such as Dulbecco's phosphate-buffered saline (D-PBS) and Hanks equilibrium salt solution (HBSS), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), α-MEM (Minimum Essential Medium alpha Modification), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), and Ham's F-10. medium, Ham's F-12 medium, Ham's F-12K medium, RPMI medium 1640, M-199 medium, L-15 medium, McCoy's 5A Medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's MB752 / 1 medium, CMRL-1066 Examples of basal culture media include medium, Williams' medium E, Brinster's BMOC-3 Medium, and E8 medium. These basal culture media may be used individually or in combination of two or more. Furthermore, the basal culture media may be modified by adding, removing, increasing, or decreasing the amount of media components depending on the type and state of the cells. These basal culture media should be appropriately selected from known media depending on the cells being cultured.

[0082] <Cell Culture Kits> Next, we will explain cell culture kits.

[0083] An example of a cell culture kit according to this embodiment includes a culture vessel 110 and the cell culture film substrate 1 described above.

[0084] The culture vessel 110 is a container for culturing cells in a culture medium, and is not particularly limited as long as it is suitable for the type of cells to be cultured and for the intended use. Examples of culture vessels 110 include dishes, petri dishes, tissue culture dishes, multi-dishes, flasks, tissue culture flasks, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles. The general names for culture vessels 110 are not limited to those exemplified here.

[0085] The material of the culture vessel 110 is not particularly limited as long as it does not allow the culture medium to pass through, but for example, it may be resin, glass, metal, etc. Examples of resins include polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), and tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer. The culture vessel may contain one or more of the following: PFA, acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyacetal (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), cyclic olefin polymer, AS resin (acrylonitrile-styrene copolymer), MS resin (methyl methacrylate-styrene copolymer), methacrylic resin (PMMA), acrylic resin, polyurethane resin, polytetrafluoroethylene (PTFE), etc. The glass may be soda glass, borosilicate glass, quartz glass, etc. The metal may be stainless steel, aluminum, etc. The culture vessel 110 only needs to be manufactured so that the culture medium does not leak, and may be integrally formed, for example. The materials and manufacturing methods of the culture vessel 110 are not limited to those exemplified herein.

[0086] The area of ​​the culture vessel 110 is not particularly limited, but culture can be performed without problems using commercially available culture vessels. For example, 0.3 cm² ２ More than 1000cm ２ The following applies. More preferably, the lower limit is 0.35 cm. ２ More preferably 1.0 cm ２ The above is particularly preferably 1.9 cm ２ That concludes the explanation. On the other hand, a more preferable upper limit would be 900 cm. ２Further preferably, 800 cm ２ The following is particularly preferred: 500 cm ２ The following applies:

[0087] The cell culture kit may also include a ring member 170. The ring member 170 is removably housed in the internal space of the culture vessel 110. The ring member 170 can press down on and fix the film substrate 150 placed inside the culture vessel 110.

[0088] The ring member 170 is, for example, cylindrical or annular. However, the shape of the ring member 170 is not limited to cylindrical or annular; for example, when viewed from above, it may be a ring shape with an appropriate predetermined shape such as an ellipse, rectangle, or polygon. Furthermore, the ring member 170 does not need to be a perfect cylinder or annular shape; it may be a cylinder or annular shape with a portion missing, or it may be U-shaped.

[0089] The specific gravity of the ring member 170 is greater than 1.0. It is desirable that the specific gravity of the ring member 170 be 1.05 or higher so that it sinks stably in the culture medium.

[0090] The material of the ring member 170 may include one or more of the following: resin, glass, ceramics, metal, etc. From the viewpoint of moldability and productivity, resin is preferred for the ring member 170. The resin may include, for example, one or more of the following: PEEK (polyether ether ketone), PET (polyethylene terephthalate), PBT (polybutylene terephthalate), PS (polystyrene), PC (polycarbonate), mPPE (modified polyphenylene ether), PPS (polyphenylene sulfide), PSU (polysulfone), PAR (polyarylate), LCP (liquid crystal polymer), ABS (acrylonitrile butadiene styrene), PES (polyethersulfone), POM (polyacetal), PI (polyimide), PA (polyamide, including nylon 6 and nylon 66), PGA (polyglycolic acid), PLA (polylactic acid), AS resin (acrylonitrile styrene copolymer resin), MS resin (methyl methacrylate styrene copolymer resin), PMMA (methacrylic resin), acrylic resin, polyurethane resin, fluororesin (PTFE (polytetrafluoroethylene), PVDF (polyvinylidene fluoride), etc.), polyphenyl ether resin, polyetherimide resin, vinyl chloride resin, etc. The glass may be soda glass, borosilicate glass, quartz glass, etc. The metal may include one or more of the following: stainless steel, aluminum, etc. Furthermore, the retaining member 170 may contain various additives in addition to the materials exemplified herein as its main material.

[0091] The cell culture film substrate 1 includes a film substrate 150 on which protrusions 52 are formed on at least a part of the outer circumference 20, and the outer edge of the film substrate 150, when viewed from the direction normal to the surface, has a circular shape, and may be used which has at least one protrusion 52 (internal tag) formed by being sandwiched between two notches 40, which are formed by a part of the circular outer edge being missing. In this case, it is preferable that the cell culture kit satisfies L1 ≥ L2, where L1 is the maximum width of the bottom surface of the culture vessel 110 and L2 is the maximum width of the film substrate 150 including the protrusions 52. L1-L2 is, for example, 0.1 to 4.5 mm, preferably 0.3 to 3 mm, and more preferably 0.5 to 1.5 mm.

[0092] The cell culture film substrate 1 includes a film substrate 150 on which the protrusions 50 are formed on at least a portion of the outer circumference 20, and the outer edge of the film substrate 150, when viewed from the direction normal to the surface, has a circular shape, and may be used that has at least one protrusion 50 (external tag) protruding from the circular outer edge. In this case, the cell culture kit preferably satisfies L3 ≤ L1 < L2', where L1 is the maximum width of the bottom surface of the culture vessel 110, L2' is the maximum width of the film substrate 150 including the protrusions 50, and L3 is the maximum width of the film substrate 150 not including the protrusions 50. L2' - L1 is, for example, 3 to 30 mm, preferably 4 to 20 mm, more preferably 5 to 15 mm. L2' - L3 is, for example, 3 to 30 mm, preferably 4 to 20 mm, more preferably 5 to 15 mm. This further improves the operability of the protrusions 50.

[0093] The cell culture film substrate 1 may include a film substrate 150 in which notches 40 are formed on at least a portion of the outer circumference 20. In this case, it is preferable that the cell culture kit satisfies the condition that the width of the notches 40 in the radial direction of the circular film substrate 150 is less than or equal to the ring width of the ring member 170. When the film substrate 150 and the ring member 170 are placed inside the culture vessel 110, it is preferable that the notches 40 overlap the ring member 170.

[0094] The notch width of the radial notch 40 in the circular film substrate 150 is, for example, 1 to 5 mm, preferably 1.5 to 4 mm, and more preferably 2 to 3 mm. Here, the notch width refers to the maximum distance (depth) from the circular outer edge frame formed by stretching along the outer edge of the film substrate 150, as shown in Figure 1, to the tip of the radial notch 40.

[0095] <Storage> One example of the cryopreservation method of this embodiment includes a cooling step of cooling the cell sheet-attached film substrate in a cryopreservation solution. The cryopreservation of the cell sheet-attached film substrate may also be carried out inside the culture vessel 110.

[0096] Cryopreservation solution is a solution used to reduce cell damage during cryopreservation. While the cryopreservation solution is not particularly limited as long as it is suitable for cell cryopreservation, it may contain culture medium components, cryoprotectants, etc. Regarding the culture medium components, such as sugars, amino acids, vitamins, inorganic salts, trace metals, and additives, it is sufficient if they satisfy the description in the (culture medium) section above. Furthermore, it is preferable that the cryopreservation solution has a freezing initiation temperature in the range of -15°C to -5°C.

[0097] Cryoprotective agents are substances used to reduce cell damage caused by freezing and thawing during cryopreservation. Examples of cryoprotective agents include cell-impermeable cryoprotective agents and cell-permeable cryoprotective agents. Specific examples of cell-impermeable cryoprotective agents include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine. Specific examples of cell-permeable cryoprotective agents include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, and propanediol. These cryoprotective agents may be formulated individually or in combination of two or more. The appropriate cryoprotective agents should be selected from known ones depending on the type of cell, the composition of the cryopreservation solution, and other factors.

[0098] Examples of commercially available cryopreservation solutions that do not contain DMSO include: Stem Cell Banker® DMSO-Free GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Banbanker® DMSO-Free (GC Lymphotec Co., Ltd.), Cryoscares® DMSO-Free (Bioverde Co., Ltd.), Stem Cell Keep (Bioverde Co., Ltd.), CryoNovo® X12 (Akron BioProducts LCC Co., Ltd.), CryoNovo® P24 (Akron BioProducts LCC Co., Ltd.), DMSO-Free Cell Cryopreservation Solution for Human ES / iPS Cells (Reprocell Co., Ltd.), Cell Reserver One (Nacalai Tesque Co., Ltd.), ThelioKeep® (Bioverde Co., Ltd.), Cellvation® (Protide Co., Ltd.) Examples include Pharmaceuticals, ReproCryo RM (ReproCell), and SOFORO Cryo (Saraya). Examples of commercially available cryopreservation solutions containing DMSO include Stem Cell Banker® GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Stem Cell Banker® EX GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Banbankar® hRM (GC Lymphotek), Banbankar® (GC Lymphotek), iStock (GC Lymphotek), CryoStor CS5 (Charles River Laboratories Cell Solutions, Inc.), and CryoStor CS10 (Charles River Laboratories Cell Solutions, Inc.).

[0099] In the cooling process, it is preferable to cool the cell sheet-attached film substrate in a cryopreservation solution using a non-through-flow cooling device. A non-through-flow cooling device allows the cell sheet, which is the object to be cooled, to be cooled at a uniform temperature, thus minimizing damage to the cells and suppressing the decrease in cell activity.

[0100] In the cooling process, the cooling rate between 0 and -5°C is, for example, 0.1°C / min to 15°C / min, preferably 0.25°C / min to 12.5°C / min, and more preferably 0.5°C / min to 10°C / min. Setting the rate above the lower limit reduces the contact time between unnecessary liquid cryoprotective material and cells. Setting the rate below the upper limit suppresses the formation of intracellular ice crystals.

[0101] In the cooling process, the freezing temperature is not particularly limited as long as it can freeze the cultured cells and cryopreservation solution. For example, the freezing temperature is between -196°C and -25°C. For example, the lower limit is between -150°C, -160°C, -180°C, and -196°C. On the other hand, for example, the upper limit is between -25°C, -30°C, and -35°C. The temperature range for freezing is, for example, -196°C to -25°C, -196°C to -30°C, -196°C to -35°C, -180°C to -25°C, -180°C to -30°C, -180°C to -35°C, -160°C to -25°C, -160°C to -30°C, -160°C to -35°C, -150°C to -25°C, -150°C to -30°C, and -150°C to -35°C.

[0102] The cooling device used for the freezing process is not particularly limited, and examples include rapid freezing devices and cryogenic refrigeration devices. As for the cooling device, a freezing device that does not come into contact with the heat transfer means and freezes by blowing cold air onto the culture vessel from multiple directions, preferably all directions, rather than from one direction, is preferable from the viewpoint of freezing cells at a uniform temperature and increasing the viability of cells after thawing. Specifically, examples of freezing devices that freeze by blowing cold air onto the culture vessel include cooling devices that cool the object to be cooled by circulating cold air with a cooling fan, such as the non-through-flow type cooling device equipped with a cooling fan disclosed in Japanese Patent Application Publication No. 2005-127666. The above "non-through-flow type" means a method in which most of the through-air from the object to be cooled does not pass through the cooler.

[0103] The cryopreservation method preferably includes a cryopreservation step in which, after a cooling step, the cell sheet-attached film substrate is stored at, for example, -196°C to -60°C, preferably -196°C to -65°C, and more preferably -196°C to -80°C. This allows the cell sheet to be stably maintained for a long period of time. The temperature in the cooling step may be the set temperature of the cooling device.

[0104] Cryopreservation is not particularly limited as long as the cells can be safely frozen and stored. Methods of cryopreservation include, for example, contact with the liquid or gas phase of a coolant, and the use of an ultra-low temperature freezer. From a temperature standpoint, the preferred method of cryopreservation is contact with the liquid or gas phase of a coolant. Examples of coolants include liquid nitrogen, liquid ethane, liquid propane, liquid helium, and dry ice.

[0105] The cryopreservation temperature is not particularly limited as long as it allows for stable cryopreservation of the cells. For example, the cryopreservation temperature may be within the ranges of -196°C to -60°C, -196°C to -134°C, or -134°C to -60°C. The cryopreservation temperature may also be the surface temperature of the cell sheet 180 to be frozen. The surface temperature can be measured, for example, using a K-type thermocouple.

[0106] The frozen cell sheet-attached film substrate comprises a film substrate 150 and a cell sheet 180 formed on the surface (culture surface 10) of the film substrate 150. In the cell sheet-attached film substrate, the film substrate 150 and the cell sheet 180 are in a cryopreserved state.

[0107] In the above-described frozen product, the film substrate 150 and the cell sheet 180 are stored frozen at, for example, the above-described freezing temperature, specifically preferably -60°C or lower, more preferably -135°C or lower. Alternatively, the frozen product may be stored frozen in the range of -134°C to -60°C. Furthermore, the frozen product may be stored frozen at -196°C or higher.

[0108] Frozen culture carrier substrates with cell sheets, frozen inside culture vessels or cryopreservation containers, can be packaged in packaging material while still frozen in the culture vessel or cryopreservation container, or after being removed from the container. Sealed packaging prevents microbial contamination of the cell sheets and allows for storage and distribution without damaging them. For example, a packaged frozen product can be obtained by packaging a container containing the cell sheet culture carrier substrate and cryopreservation solution in a film-like packaging material, sealing it, and then freezing it. Preferred packaging materials include aluminum, polyethylene terephthalate, ionomer, polyethylene, polyvinylidene chloride, polyvinyl alcohol, polypropylene, polyester, polycarbonate, polyacrylonitrile, ethylene vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-methacrylic acid copolymer, polyimide, perfluoroalkoxy fluororesins, fluoropolymers such as tetrafluoroethylene-hexafluoropropylene copolymer (FEP), nylon, cellophane, and paper. These may be used individually or in combination of two or more.

[0109] The freezing method of this embodiment may further include a thawing step of thawing the cell sheet-attached film substrate.

[0110] The thawing method is not particularly limited, and conventional methods used in the fields of medicine, pharmaceuticals, quasi-drugs, cosmetics, food, veterinary medicine, and basic technology fields such as regenerative medicine and biotechnology can be used. Examples of thawing methods include using a water bath, incubator, hot plate, or defroster, immersing in a thawing solution at a temperature higher than the freezing point, or leaving the product undisturbed in an environment at a temperature higher than the freezing point. The ambient temperature in contact with the frozen product during thawing is not particularly limited, as long as it is higher than the freezing point and below 50°C. The upper limit of the ambient temperature is, for example, 49°C or lower, preferably 45°C or lower, and more preferably 40°C or lower. On the other hand, the lower limit of the ambient temperature is, for example, 0°C or higher, preferably 5°C or higher, more preferably 10°C, and even more preferably 15°C or higher. The range of ambient temperatures in contact with the frozen material during thawing is, for example, 0°C to 49°C, 0°C to 45°C, 0°C to 40°C, 5°C to 49°C, 5°C to 45°C, 5°C to 40°C, 10°C to 49°C, 10°C to 45°C, 10°C to 40°C, 15°C to 49°C, 15°C to 45°C, and 15°C to 40°C. However, thawing at temperatures above 50°C is undesirable as it may cause thermal damage to the cells. Furthermore, the frozen material may be temporarily stored in an environment below its freezing point during thawing. For example, a frozen material stored at -80°C can be exposed to an ambient temperature of -30°C and then thawed at an ambient temperature above its freezing point. The time required to thaw the frozen material is not particularly limited, as long as no freezing damage occurs to the cells. Normally, thawing can be done without problems for more than 10 seconds and up to 60 minutes. The lower limit of the time required to thaw frozen material is sufficient to be more than 10 seconds, preferably 20 seconds or more, more preferably 30 seconds or more, and even more preferably 1 minute or more. The upper limit is sufficient to be 60 minutes or less, preferably 50 minutes or less, more preferably 40 minutes or less, and even more preferably 30 minutes or less.For example, the thawing time is between 10 seconds and 60 minutes, between 10 seconds and 50 minutes, between 10 seconds and 40 minutes, between 10 seconds and 30 minutes, between 20 seconds and 60 minutes, between 20 seconds and 50 minutes, between 20 seconds and 40 minutes, between 20 seconds and 30 minutes, between 30 seconds and 60 minutes, between 30 seconds and 50 minutes, between 30 seconds and 40 minutes, between 30 seconds and 30 minutes, between 1 minute and 60 minutes, between 1 minute and 50 minutes, between 1 minute and 40 minutes, and between 1 minute and 30 minutes. If thawing is too fast, thermal shock due to the temperature difference may cause cracks in the frozen material and damage the cell sheet. If thawing is too slow, water molecules will recrystallize under sub-zero conditions, causing larger ice crystals and serious damage to the cells, which is undesirable. During the above thawing time, the above ambient temperature may be set to be constant, or it may be set to fluctuate, such as gradually increasing.

[0111] The thawing medium is not particularly limited, as long as it does not damage the cultured cells. Examples of components that can be contained in the thawing medium include sucrose, glucose, maltose, trehalose, and fructose. The thawing medium may also contain the components listed in the (culture medium) section above.

[0112] The temperature of the melt is not particularly limited as long as it is higher than the freezing point. For example, the temperature of the melt is between 0°C and 45°C. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower. Examples of melt temperatures include 0°C to 45°C, 0°C to 40°C, 0°C to 39°C, 0°C to 38°C, 4°C to 45°C, 4°C to 40°C, 4°C to 39°C, 4°C to 38°C, 25°C to 45°C, 25°C to 40°C, 25°C to 39°C, 25°C to 38°C, 28°C to 45°C, 28°C to 40°C, 28°C to 39°C, and 28°C to 38°C.

[0113] The thawed cell sheet 180 and film substrate 150 may be washed with a cell washing solution immediately after thawing, if necessary. The cell washing solution is not particularly limited and may contain the components described in the (culture medium) section above. The temperature of the cell washing solution is not particularly limited. For example, the temperature of the cell washing solution is 0°C to 45°C. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower. The number of times the cultured cells are washed is not particularly limited and may be one or more times (for example, two, three, four, five times, etc.).

[0114] In this disclosure, since the culture surface 10 does not contain a temperature-responsive polymer, it is possible to suppress the peeling of the cell sheet 180 from the film substrate 150 in low-temperature environments such as the cryopreservation process, and a cell sheet-attached film substrate can be obtained that maintains the adhesion between the cell sheet and the film even after the cryopreservation and thawing processes.

[0115] <Transplantation> The transplantation method of this embodiment includes a transplantation step of attaching a cell sheet 180 formed on the culture surface 10 of a film substrate 150 to the transplant site, and then peeling the film substrate 150 from the cell sheet 180.

[0116] The transplant site may be at least a part of the body of a recipient organism such as a mammal, bird, amphibian, fish, insect, plant, or microorganism. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cattle, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and chickens. However, the transplant site may exclude the internal and external parts of the human body. Examples of transplant sites other than the recipient organism include other cell sheets, medical devices, and tissues or organs separated from the recipient organism. Examples of tissues or organs include skin, oral tissue, esophagus, trachea, bronchi, lungs, lung lobes, stomach, duodenum, pancreas, spleen, small intestine, large intestine, muscle tissue, and bone (excluding tissues or organs that were taken from the recipient organism and then returned to the same organism for therapeutic purposes).

[0117] Cell transplantation therapy aims to suppress and prevent the onset and recurrence of symptoms associated with cell, tissue, and organ defects, dysfunction, and impairment. Examples of diseases treated with cell transplantation therapy include spinal cord injury, knee joint cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell deficiency, aplastic anemia, severe lower limb ischemia, intractable skin ulcers, prevention of postoperative complications (e.g., anastomotic leakage of various organs, bronchial stump fistula, pancreatic fistula, bile leakage), and burns. The cells used in cell transplantation therapy may be autologous cells, allogeneic non-autologous cells, or xenologous cells. From the perspective of clinical application and safety, autologous cells are preferred, while non-autologous cells are preferred from the perspective of clinical application and productivity.

[0118] The embodiments of the present invention have been described above, but these are merely examples of the present invention, and various other configurations can be adopted. Furthermore, the present invention is not limited to the embodiments described above, and modifications, improvements, etc., within the scope that can achieve the objectives of the present invention are included in the present invention. Examples of reference embodiments are listed below. 1. A cell culture film substrate having a culture surface on at least one surface of the film substrate, wherein at least a portion of both surfaces has an imprint, and at least a portion of the imprint has an asymmetrical shape between the front and back. 2. A cell culture film substrate having a culture surface on at least one surface of the film substrate, wherein the film substrate has one or more selected from the group consisting of notches, protrusions, and incisions, wherein the notches and protrusions are formed on at least a portion of the outer circumference of the film substrate, and the incisions penetrate the front and back surfaces of the film substrate. 3. A cell culture film substrate having a culture surface on at least one surface of the film substrate, wherein at least a portion of both surfaces has an imprint, and the film substrate has one or more selected from the group consisting of notches, protrusions, and incisions, wherein the notches and protrusions are formed on at least a portion of the outer circumference of the film substrate, and the incisions penetrate the front and back surfaces of the film substrate. 4. A cell culture film substrate according to 1. or 3., wherein the imprint includes laser imprinting. 5. A cell culture film substrate according to any one of 1., 3., and 4., wherein the imprint includes one or more selected from the group consisting of letters, symbols, and patterns. 6. A cell culture film substrate according to any one of 1., 3. to 5., wherein the imprint is located near the outer circumference of the film substrate. 7. 1., 3. to 6. A cell culture film substrate according to any one of the above, wherein a groove is provided between the imprint and the center of the culture surface, the groove having a part of the surface recessed.8. A cell culture film substrate according to 2. or 3., wherein at least a portion of the outer peripheral surface of the film substrate, the notches, and the protrusions is a laser-processed surface. 9. A cell culture film substrate according to any one of 2., 3., and 8., wherein it has two or more notches, two or more protrusions, two or more cuts, or two or more different features selected from the group consisting of the notches, protrusions, and cuts. 10. A cell culture film substrate according to any one of 2., 3., 8., and 9., wherein two or more features selected from the group consisting of the notches and protrusions are provided in asymmetrical positions on the front and back. 11. 2., 3., and 8. to 10. A cell culture film substrate according to any one of the above, wherein a groove is formed between the protruding portion and the center of the culture surface, and a part of the surface is recessed. 12. A cell culture film substrate according to any one of 2., 3., and 8. to 11., wherein the outer edge of the film substrate, as viewed from the surface, is circular, and the cell culture film substrate has at least one protruding portion formed by being sandwiched between two notches, each notched by a part of the circular outer edge being missing. 13. A cell culture film substrate according to any one of 1. to 12., wherein the cell culture film substrate is transparent. 14. A cell culture film substrate according to any one of 1. to 13., wherein the thickness of the cell culture film substrate is 5 μm or more and 100 μm or less. 15. 1. to 14. A cell culture film substrate according to any one of the above, wherein the cell culture film substrate contains an organic polymer material. 16. A cell culture film substrate according to any one of 1 to 15, wherein no layer containing a temperature-responsive polymer is formed on the surface on which the culture surface is formed.17. A method for manufacturing a film substrate with a cell sheet, comprising: an installation step of placing a cell culture film substrate according to any one of 1. to 16. into a culture vessel; a culture step of placing a culture medium into the culture vessel, seeding cells, and culturing a cell sheet on the cell culture film substrate; and an removal step of removing the film substrate with the cell sheet from the culture vessel. 18. A method for manufacturing a film substrate with a cell sheet according to 17., wherein the installation step includes a step of placing a ring member on the film substrate of the cell culture film substrate in the culture vessel, and the removal step includes a step of removing the ring member from the film substrate. 19. A method for manufacturing a film substrate with a cell sheet according to 17. or 18., wherein in the installation step, a ring member having at least one of a notch, a protrusion, and a cut portion is provided. A method for manufacturing a cell sheet-attached film substrate, wherein the cell culture film substrate described in is used, and the removal step includes a step of holding the portion of the film substrate near the notch, the protruding portion, or the cut portion. 20. A cell culture kit comprising a culture vessel and a cell culture film substrate, wherein the cell culture film substrate is a cell culture film substrate described in any one of 1 to 16. 21. A cell culture kit according to 20, further comprising a ring member. 22. A cell culture kit according to 21, wherein the cell culture film substrate has a protruding portion formed on at least a part of the outer circumference of the film substrate, as described in 2 or 3. A cell culture kit comprising the cell culture film substrate described in [reference], wherein the outer edge of the film substrate, as viewed from the surface, has a circular shape, and has at least one protruding portion formed by being sandwiched between two notches, which are part of the circular outer edge, and satisfies L1 ≥ L2 when the maximum width of the bottom surface of the culture vessel is L1 and the maximum width of the film substrate including the protruding portion is L2.23. A cell culture kit according to 21., wherein the cell culture film substrate is the cell culture film substrate according to 2. or 3. having a protrusion formed on at least a part of the outer circumference of the film substrate, the outer edge of the film substrate as viewed from the surface is circular, and there is at least one protrusion protruding from the circular outer edge, and when the maximum width of the bottom surface of the culture vessel is L1, the maximum width of the film substrate including the protrusion is L2', and the maximum width of the film substrate excluding the protrusion is L3, the cell culture kit satisfies L3 ≤ L1 < L2'. 24. A cell culture kit according to 21., wherein the cell culture film substrate is the cell culture film substrate according to 2. or 3. having a notch formed on at least a part of the outer circumference of the film substrate, and the notch width of the notch in the radial direction of the circular film substrate is less than or equal to the ring width of the ring member. 25. 24. A cell culture kit as described in 26. A cell culture kit wherein the width of the notch in the radial direction of the circular film substrate is 5 mm or less. 26. A film substrate with a cell sheet, comprising: a cell culture film substrate as described in any one of 1 to 16; and a cell sheet provided on the culture surface of the cell culture film substrate. 27. A method for manufacturing a cell culture film substrate, comprising: a surface treatment step of performing plasma treatment on at least a part of the surface of a resin sheet to form a culture surface; an engraving step of forming an engraving on a part of the plasma-treated surface; and a cutting step of cutting out a film substrate from the resin sheet having the culture surface and the engraving on its surface. 28. A method for manufacturing a cell culture film substrate as described in 27, wherein in the engraving step, the engraving is formed by laser treatment. 29. A method for producing a film substrate for cell culture, comprising: a surface treatment step of performing plasma treatment on at least a portion of the surface of a resin sheet to form a culture surface; and a cutting step of cutting out a film substrate having notches and / or protrusions on at least a portion of its outer circumference from the resin sheet.30. A method for manufacturing a cell culture film substrate according to any one of 27 to 29, wherein the cutting step is performed by laser processing. 31. A method for manufacturing a cell culture film substrate according to any one of 27, 28, and 30, wherein in the cutting step, a notch and / or protrusion is formed on at least a part of the outer circumference of the film substrate. 32. A method for manufacturing a cell culture film substrate according to any one of 27 to 31, wherein in the cutting step, a notch is formed that penetrates the front and back surfaces of the film substrate. 33. A method for manufacturing a cell culture film substrate according to any one of 27, 28, 30, and 31, wherein in the cutting step, laser processing is performed to remove a part of the depth direction on the surface of the film substrate, and a groove is formed between the marking and the center of the culture surface.

[0119] The present invention will be described in detail below with reference to examples, but the present invention is not limited in any way to the descriptions of these examples.

[0120] <Manufacturing of Cell Culture Film Substrate> The manufacturing of the cell culture film substrate was carried out in two parts: the (surface treatment) process and the (cutting and engraving) process described below. (Surface Treatment) A 12 μm thick PEEK film was used as the film substrate, and plasma treatment was performed on the surface of the film substrate under the conditions shown in Table 1. The plasma treatment was performed in RIE mode. RIE stands for reactive ion etching. Specifically, the procedure was as follows: The film substrate was placed on the cathode electrode in the chamber. After depressurizing the chamber, a predetermined gas was flowed at a predetermined flow rate, and after the pressure in the chamber stabilized, high-frequency power (13.56 MHz) was applied at a predetermined power to generate plasma. After the predetermined processing time had elapsed, the application of power was stopped, the gas was stopped, the chamber was opened to the atmosphere, and the film substrate was removed. The surface that underwent plasma treatment became the culture surface. In Table 1, the discharge treatment intensity was calculated from RF (radio frequency) power × processing time / electrode area. The electrode area of ​​the electrode used was 736 cm². ２ That was the case.

[0121] The PEEK film used in the examples had a porosity of 5% or less, and when the culture surface was measured with a laser microscope, there were no 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, indicating a solid film. The porosity was calculated from "{1 - (measured density / theoretical density)} × 100". In addition, the surface roughness of the film used in the examples was rougher after plasma treatment than before plasma treatment. The Ra after the second stage of plasma treatment was 0.5 nm in Example 1. The Ra measurement was performed as follows: An AFM (Shimadzu SPM-9700) was used, and a silicon single crystal probe with a small tip diameter (radius of curvature approximately 10 nm) was used. The surface was observed in a 1 μm × 1 μm area. To calculate Ra, analysis software included with the device was used. After smoothing in the X and Y directions, surface roughness analysis was performed in a 100 nm x 100 nm area, avoiding foreign matter and surface scratches. The arithmetic mean roughness Ra is the average value of the distance from the reference line within the reference length.

[0122]

[0123] (Cutting and Engraving) As described above, cell culture film substrates for Examples 1 and 2 and Comparative Example 1 were prepared by cutting and engraving the surface-treated film substrates with a UV laser (Table 2). The UV laser device used was the MD-U1020C manufactured by Keyence Corporation.

[0124] As shown in Figure 2, the cell culture film substrate 1 of Example 1 has a circular film substrate 150 with one protrusion 50 on its outer circumference 20, and has markings 30 on the surface of the film substrate 150 facing the culture surface 10, near the outer circumference 20, with numbers from 0 to 9 and several letters. In Example 1, the maximum width (L2') of the film substrate 150 including the protrusion 50 was 43.5 mm, and the maximum width (L3) of the film substrate 150 excluding the protrusion 50 was 33.5 mm.

[0125] As shown in Figure 1, the cell culture film substrate 1 of Example 2 has a circular film substrate 150 with a protrusion 52 sandwiched between two notches 40 on its outer circumference 20, and the film substrate 150 has markings 30 near the outer circumference 20 on the culture surface 10 side, including numbers from 0 to 9 and several letters. In Example 2, the maximum width (L2) of the film substrate 150 including the protrusion 52 was 33.5 mm, and the notch width was 3 mm.

[0126] The cell culture film substrate of Comparative Example 1 is a circular film substrate with a diameter of 33.5 mm and does not have any of the notches 40, protrusions 50, or markings 30.

[0127] The resulting cell culture film substrates were evaluated for their operability when placed in culture vessels and for their ability to distinguish between the front and back sides.

[0128]

[0129] <Evaluation of Operability> Based on the following (culture conditions), a cell sheet was cultured on the culture surface of the cell culture film substrate obtained above to obtain a film substrate with a cell sheet. After placing the obtained film substrate with a cell sheet in a transparent plastic culture container with a maximum bottom width (L1) of 34.4 mm, the ease with which the film substrate could be removed with tweezers was evaluated. In Example 1, the film substrate 150 could be easily removed from the culture container by grasping the protruding portion 50 with tweezers, and in Example 2, by grasping the protruding portion 52 sandwiched between the two notches 40, thus preventing damage to the cell sheet. On the other hand, in Comparative Example 1, when the tip of the tweezers was inserted between the film substrate and the bottom of the culture container in order to grasp the film body with tweezers, the film substrate was distorted, causing damage to the cell sheet. Furthermore, in the (surface treatment) described above, plasma treatment was applied to the opposite surface (back surface) as well as the surface, and a reference example of a cell culture film substrate was prepared in the same manner as in Examples 1 and 2. When the cell culture film shown in the reference example was immersed in the liquid culture medium used under the culture conditions described below, it showed better compatibility with the liquid culture medium and further suppressed the floating of the film substrate during the initial stages of immersion, compared to a substrate treated with plasma on only one side. In cell culture films where the opposite surface (back side) was not treated with plasma, the wettability of the back side was poor, and the film would float when immersed in the liquid culture medium, often requiring immersion with tweezers or similar tools. From the standpoint of reducing damage to the film and improving workability, it is preferable to perform plasma treatment on both the front and back sides.

[0130] (Culture conditions) Frozen human fibroblasts (derived from human oral tissue) were thawed at 37°C and washed with culture medium. 5 × 10 ５ Suspend individual cells in a 5% serum-containing medium and place them in two dishes (60.1 cm²). ２ ) 2.5 x 10 ５ After sowing individual seeds, the cells were cultured for 3 days. The cultured cells were harvested, suspended in a 5% serum-containing medium, and placed in four flasks (225 cm²). ２ ) 2.5 x 10 ５After seeding each cell individually and subculturing, the cells were cultured for 4 days. The above film substrates were washed in 70% ethanol aqueous solution, phosphate buffer, and culture medium in that order, and then placed on the flat bottom of each well in a cell culture multiwell plate (6 wells). At this time, the culture surface of the film substrate was positioned facing the opening of the well plate, that is, the back surface of the film substrate was in contact with the flat bottom of the well. The cultured cells were harvested, suspended in 2% serum-containing medium, and 27.9 × 10¹⁴ cells were placed in each well of the cell culture multiwell plate with the film substrates already in place. ４ pieces / cm ２ Seeds were sown at the following density, at 37°C and 5% CO2. ２ The cells were incubated in the specified environment for one day, and a cell sheet was prepared on the culture surface of a film substrate.

[0131] <Evaluation of Front / Back Distinguishing Ease> The cell culture film substrates obtained above were observed with the naked eye before cell culture to evaluate how easily the front and back sides could be distinguished. In Examples 1 and 2, it was easy to distinguish the front and back sides of the film by the letters and numbers inscribed on it, and it was possible to set the film in the culture vessel with the culture side facing upwards. Furthermore, when transplanting cell sheets after cell culture, the presence of markings on the film substrate made it easier to determine which side of the film substrate the cell sheet was formed on, thus facilitating cell sheet transplantation. On the other hand, in Comparative Example 1, because there were no markings such as engravings on the film substrate, it was difficult to determine which side was the culture side when grasped with tweezers, and it was difficult to set the film in the culture vessel with the culture side facing upwards. Furthermore, when transplanting cell sheets after cell culture, it was sometimes difficult to determine which side of the film substrate the cell sheet was formed on.

[0132] The cell culture film substrates of Examples 1 and 2 showed superior handling compared to Comparative Example 1, including better operability and visibility (front / back distinguishability).

[0133] <Evaluation of Cell Observability> As shown in Table 3, Reference Examples 1 to 6 were prepared, consisting of a 25 μm thick PEEK film (high crystallinity type), a 9 μm thick PEEK film (high crystallinity type), a 25 μm thick PEEK film (low crystallinity type), a 9 μm thick PEEK film (low crystallinity type), a 25 μm thick PSS (polyphenylene sulfide) film, and a 25 μm thick PI (polyimide). The surfaces of the PEEK films in Reference Examples 1 to 4 were embossed to prevent sticking during winding. The film surfaces of Reference Examples 5 and 6 were smooth and did not have embossing. The total light transmittance (Tt) and haze of each film were measured using a Suga Test Instruments haze meter (HZ-V3). The total light transmittance (Tt) and haze were determined in accordance with JIS K 7361-1 (1997) and JIS K 7136 (2000). Table 3 shows the results for total light transmittance (%) and haze. Next, plasma treatment was performed on the surface of each film under the conditions shown in Table 1. After that, cell sheets were prepared on the film according to the above (culture conditions). Cell observability was evaluated using a phase-contrast microscope according to the following criteria: ・Observation (cells): Individual cells were observed 2 hours after the start of culture, before sheet formation. Good means that the shape and presence of cells can be confirmed without any problems, acceptable means that the shape of cells is somewhat unclear but the presence or absence can be confirmed, and unacceptable means that neither the shape nor the presence or absence of cells can be confirmed. ・Observation (cell sheet): Individual cells in the sheet were observed 48 hours after the start of culture. In the sheet as well, good means that the shape and presence or absence of cells can be confirmed without any problems, acceptable means that the shape of cells is somewhat unclear but the presence or absence can be confirmed, and unacceptable means that neither the shape nor the presence or absence of cells can be confirmed. As a result, as shown in Reference Example 5, it was demonstrated that even if the haze exceeds 55%, there is no impediment to cell observation in films without embossing on the surface. Reference Example 6 shows that in films without embossing on the surface, even if the haze is low, if the total light transmittance is low (the film has a yellowish tint), the cell observation performance is somewhat inferior.In contrast, Reference Examples 1 and 2 showed that in the case of crystalline polymers with embossed surfaces, even reducing the haze to about 35% did not provide sufficient cell observation capabilities. On the other hand, Reference Examples 3 and 4 showed that even with similar embossed surfaces, further reducing the haze by decreasing the crystallinity (amorphizing) of the film's constituent resin resulted in excellent cell observation capabilities.

[0134]

[0135] This application claims priority based on Japanese Patent Application No. 2024-212071, filed on 5 December 2024, and incorporates all of its disclosures herein.

[0136] 1 Cell culture film substrate 10 Culture surface 20 Outer circumference 30, 32 Imprint 40 Notch 50, 52 Protrusion 60, 62 Groove 70 Cutout 110 Culture vessel 150 Film substrate 170 Ring member 180 Cell sheet

Claims

1. A cell culture film substrate having a culture surface on at least one surface of the film substrate, comprising either one or both of the following structures: (a) a structure having an imprint including an asymmetrical shape on at least a portion of both surfaces; (b) a structure having one or more of notches or protrusions formed on at least a portion of the outer circumference of the film substrate, or cuts penetrating the front and back surfaces of the film substrate.

2. A cell culture film substrate according to claim 1, wherein the marking includes laser marking.

3. A cell culture film substrate according to claim 1 or 2, wherein the markings consist of one or more letters, symbols, or patterns.

4. A cell culture film substrate according to claim 1 or 2, wherein the markings are located near the outer periphery of the film substrate.

5. A cell culture film substrate according to claim 1 or 2, wherein a groove portion is formed between the imprint and the center of the culture surface, and a part of the surface is recessed.

6. A cell culture film substrate according to claim 1 or 2, wherein at least a portion of the outer peripheral surface of the film substrate, the notch, and the protrusion is a laser-processed surface.

7. A cell culture film substrate according to claim 1 or 2, wherein the cell culture film substrate is having two or more of the notches, protrusions, and cuts in combination.

8. A cell culture film substrate according to claim 1 or 2, wherein the protrusion has an asymmetrical shape on the front and back, the notch has an asymmetrical shape on the front and back, or two or more selected from the group consisting of the protrusion and the notch are provided in positions that are asymmetrical on the front and back.

9. A cell culture film substrate according to claim 1 or 2, wherein a groove portion is formed between the protruding portion and the center of the culture surface, and a part of the surface is recessed.

10. A cell culture film substrate according to claim 1 or 2, wherein the outer edge of the film substrate, as viewed from the surface, has a circular shape, and at least one protrusion is located between two notches formed by a portion of the circular outer edge being missing.

11. A cell culture film substrate according to claim 1 or 2, wherein the cell culture film substrate has an embossed surface, a haze of 35% or less, and a total light transmittance of 40% or more.

12. A cell culture film substrate according to claim 1 or 2, wherein the thickness of the cell culture film substrate is 5 μm or more and 100 μm or less.

13. A cell culture film substrate according to claim 1 or 2, wherein the cell culture film substrate comprises an organic polymer material.

14. A cell culture film substrate according to claim 1 or 2, wherein no layer containing a temperature-responsive polymer is formed on the surface on which the culture surface is formed.

15. A method for producing a cell sheet-attached film substrate, comprising: an installation step of placing the cell culture film substrate according to claim 1 or 2 into a culture vessel; a culture step of placing a culture medium into the culture vessel, seeding cells, and culturing a cell sheet on the cell culture film substrate; and an removal step of removing the cell sheet-attached film substrate from the culture vessel.

16. A method for manufacturing a cell sheet-attached film substrate according to claim 15, wherein the installation step includes the step of installing a ring member on the film substrate of the cell culture film substrate in the culture vessel, and the removal step includes the step of removing the ring member from the film substrate.

17. A method for manufacturing a cell sheet-attached film substrate according to claim 15, wherein in the installation step, the cell culture film substrate according to claim 1 or 2 having at least one of a notch, a protrusion, and a cut portion is used, and the removal step includes a step of holding the portion of the film substrate near the notch, the protrusion, or the cut portion.

18. A cell culture kit comprising a culture vessel and a cell culture film substrate, wherein the cell culture film substrate is the cell culture film substrate described in claim 1 or 2.

19. A cell culture kit according to claim 18, comprising a ring member.

20. A cell culture kit according to claim 19, wherein the cell culture film substrate is the cell culture film substrate according to claim 1 or 2, having a protrusion formed on at least a part of the outer circumference of the film substrate, the outer edge of the film substrate as viewed from the surface is circular, and has at least one of the protrusions formed between two notches formed by a part of the circular outer edge being missing, and satisfies the relationship L1 ≥ L2 when the maximum width of the bottom surface of the culture container is L1 and the maximum width of the film substrate including the protrusions is L2.

21. A cell culture kit according to claim 19, wherein the cell culture film substrate is the cell culture film substrate according to claim 1 or 2, having a protrusion formed on at least a part of the outer circumference of the film substrate, the outer edge of the film substrate being circular when viewed from the surface, and having at least one protrusion projecting from the circular outer edge, and satisfying the relationship L3 ≤ L1 < L2' when L1 is the maximum width of the bottom surface of the culture container, L2' is the maximum width of the film substrate including the protrusion, and L3 is the maximum width of the film substrate excluding the protrusion.

22. A cell culture kit according to claim 19, wherein the cell culture film substrate has a notch formed on at least a part of the outer circumference of the film substrate, and the width of the notch in the radial direction of the circular film substrate is less than or equal to the ring width of the ring member.

23. A cell culture kit according to claim 22, wherein the width of the notch in the radial direction of the circular film substrate is 5 mm or less.

24. A film substrate with a cell sheet, comprising a cell culture film substrate according to claim 1 or 2, and a cell sheet provided on the culture surface of the cell culture film substrate.

25. A method for manufacturing a cell culture film substrate, comprising: a surface treatment step of applying plasma treatment to at least one surface of a resin sheet to form a culture surface; a cutting step of cutting out one or more film substrates having the culture surface from the resin sheet; and a step of forming either one or both of the following structures (a) or (b): (a) a structure having an imprint including an asymmetrical shape on at least a portion of both of the surfaces; (b) a structure having one or more notches or protrusions formed on at least a portion of the outer circumference of the film substrate, or cuts that penetrate the front and back surfaces of the film substrate.

26. A method for producing a cell culture film substrate according to claim 25, wherein the step of forming the structure is carried out by laser treatment.

27. A method for producing a cell culture film substrate according to claim 26, wherein the step of forming the structure, including the formation of the imprint, is performed before the cutting step.

28. A method for producing a cell culture film substrate according to claim 26, wherein the step of forming the structure, which includes forming at least one of the notches or the protrusions, is carried out in the same step as the cutting step.

29. A method for producing a cell culture film substrate according to claim 26, wherein the step of forming the structure, which includes forming the markings, includes a step of removing a portion of the surface of the film substrate in the depth direction by laser treatment to form a groove between the markings and the culture surface.

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