Resin film containing x-ray contrast material

Abstract

The present invention is a resin film including a resin layer having a first surface including a region on which cultured cells are placed, wherein the resin film includes an X-ray contrast material, and has, in the first surface, a non-X-ray contrast material-containing region that does not contain any X-ray contrast material.

Description

Resin film containing X-ray contrast material

[0001] The present invention relates to a resin film comprising a resin layer having a first surface on which cultured cells are placed, and an X-ray contrast material.

[0002] In recent years, "cell sheet technology" has been developed as a method to improve tissue survival of transplanted cells, and research is being conducted into cell sheet transplantation therapy for various diseases such as ischemic cardiomyopathy, postoperative air leaks, and stenosis after endoscopic esophageal submucosal dissection, as well as for preventing postoperative complications.

[0003] Patent Document 1 describes a carrier for transporting a cell sheet for transplantation, which comprises a covering layer to which the cell sheet is adhered and a support layer made of a base material acceptable as a medical material.

[0004] Patent Document 2 describes a technology for imparting X-ray detectability to a tampon or medical suction tube made of a fleece material of cellulose fiber by embedding a contrast agent made of barium sulfate and a polymerized thermoplastic base material inside the fleece material by thermal bonding.

[0005] International Publication No. 2014 / 091796 Pamphlet Japanese Patent Application Laid-Open No. 63-143061

[0006] The above-mentioned Patent Document 2 describes that a tampon or medical suction body mistakenly left behind at a surgical site can be detected by X-ray using an X-ray contrast agent fixed to a fiber. The above-mentioned Patent Document 1 does not consider X-ray detection of a carrier for transporting a cell sheet for transplantation.

[0007] According to one aspect of the present invention, there is provided a resin film comprising the following X-ray contrast material:

[0008] 1. A resin film comprising: a resin layer having a first surface including an area for placing cultured cells; and a layer containing an X-ray contrast material present on the first surface of the resin layer, on a second surface opposite the first surface, and at least in part within the resin layer, wherein the area for placing cultured cells is present in at least a part of an X-ray contrast material-free area of ​​the first surface where the X-ray contrast material is not exposed. 2. The resin film described in 1., wherein the layer containing X-ray contrast material is an X-ray contrast layer including a pattern of the X-ray contrast material. 3. The resin film described in 1., wherein the layer containing X-ray contrast material is an X-ray contrast resin layer containing a plurality of filler-like X-ray contrast materials and a resin material. 4. The resin film described in any one of 1. to 3., wherein the thickness of the resin film is 5 μm or more and 250 μm or less. 5. 1. to 4. 5. The resin film according to any one of 1. to 5., wherein the resin layer comprises one or more selected from the group consisting of polyether ether ketone, polyethylene terephthalate, and polystyrene. 6. The resin film according to any one of 1. to 5., wherein the X-ray contrast material comprises an X-ray absorption contrast material. 7. The resin film according to any one of 1. to 6., wherein the X-ray contrast material comprises a metal or a metal compound. 8. The resin film according to any one of 1. to 7., wherein, when viewed from the normal direction of the first surface, the layer comprising the X-ray contrast material overlaps all or only a portion of the resin film. 9. The resin film according to 2., wherein the resin film comprises a protective layer that protects at least a portion of the surface of the X-ray contrast layer present on the first surface of the resin layer and / or on the second surface opposite the first surface. 10. The resin film according to 1. to 9. 10. The resin film according to claim 9, wherein the first surface in an area where the cultured cells are to be placed is subjected to a plasma treatment.11. The resin film according to any one of 1. to 10., wherein the water droplet contact angle θ measured by the θ / 2 method on the first surface in the region where the cultured cells are to be placed is 70° or less. 12. The resin film according to any one of 1. to 11., wherein the 60-degree specular gloss Gs(60°) of the outermost surface opposite the first surface, measured in accordance with the provisions of JIS Z8741:1997, is 700 or less. 13. The resin film according to any one of 1. to 12., wherein the resin film is used for culturing cells and / or transporting cells.

[0009] According to the present invention, a resin film having excellent X-ray detectability and cell adhesiveness can be provided.

[0010] FIG. 1 is a schematic diagram showing a first embodiment of a resin film. FIG. 2 is a schematic diagram showing a second embodiment of a resin film. FIG. 3 is a schematic diagram showing a third embodiment of a resin film. FIG. 4 is a schematic diagram showing a fourth embodiment of a resin film. FIG. 5 is a schematic diagram showing a fifth embodiment of a resin film. FIG. 6 is a schematic diagram showing a sixth embodiment of a resin film. FIG. 7 is a schematic diagram showing a patient when the appearance of the patient after cell transplantation in the body is examined with the naked eye. FIG. 8 is a schematic diagram showing a patient when the patient after cell transplantation in the body is examined by irradiating the patient with X-rays using a resin film of this embodiment. FIG. 9 is a schematic diagram showing a patient when the patient after cell transplantation in the body is examined by irradiating the patient with X-rays using a resin film not including an X-ray contrast material.

[0011] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all drawings, similar components are designated by similar reference numerals, and descriptions thereof will be omitted where appropriate. Furthermore, the drawings are schematic diagrams and do not correspond to actual dimensional proportions.

[0012] The resin film of this embodiment comprises: a resin layer having a first surface including an area on which cultured cells are placed; and a layer containing an X-ray contrast material present on the first surface of the resin layer, on a second surface opposite the first surface, and at least in part within the resin layer, wherein the area on which cultured cells are placed is present in at least a part of an X-ray contrast material-free area of ​​the first surface where the X-ray contrast material is not exposed on the surface.

[0013] The resin film of this embodiment has a layer containing an X-ray contrast material, making it detectable by X-ray even when it is located in a location difficult to visualize, such as inside a patient's body. Furthermore, in the X-ray contrast material-free region where the X-ray contrast material is not exposed on the surface, the cell sheet and the first surface of the resin layer can be tightly attached. This resin film has an area (cell-mounting region) in the X-ray contrast material-free region where cultured cells can be placed, allowing cells to be cultured on the first surface of the cell-mounting region. Furthermore, the adhesion between the cell sheet and the resin layer facilitates the transfer of the cell sheet. As a result, this facilitates the transfer of the cell sheet during cell transplantation and allows for easy detection of the used resin film after transplantation.

[0014] 7 is a schematic diagram showing the appearance of a patient 100 after cell transplantation when examined with the naked eye. The inside of the body of the patient 100 is difficult to see with the naked eye because visible light does not pass through or reflect therethrough.

[0015] 8 is a schematic diagram showing a patient 101 after cell transplantation using the resin film 102 of this embodiment, when the patient is examined by irradiating the patient with X-rays inside the body. The resin film 102 of this embodiment contains an X-ray contrast material, and thus can be visualized by irradiating it with X-rays. Even if the resin film is placed in a location that is difficult to see, such as inside the body of the patient 101, it can be detected by X-rays.

[0016] Specifically, in the method for detecting resin films, the X-ray contrast material absorbs / scatters X-rays, preventing the X-rays from passing through, and the X-ray non-transparent areas appear white in the X-ray image. X-ray inspection can be performed by multi-directional imaging, such as one-directional or two-directional imaging.

[0017] FIG. 9 shows a schematic diagram of a patient 111 after cell transplantation using a resin film 112 containing no X-ray contrast material in the body, as examined by X-ray irradiation. A transplantation surgery is performed using a resin film 112 containing no X-ray contrast material as a carrier for transplanting a cell sheet. If the resin film 112 is unintentionally placed inside the body, it is impossible to determine whether it is placed there or not from its appearance. Resin materials such as PEEK are highly transparent to X-rays. Because the resin film 112 made of such a resin material does not absorb or scatter X-rays, the resin film 112 appears black in an X-ray image. The surrounding body tissue also appears black, making it difficult to see the resin film 112.

[0018] The resin film of this embodiment contains an X-ray contrast material. This X-ray contrast material may be contained anywhere in the resin film, for example, on the surface of the resin layer, or may be contained inside the resin layer, as long as an X-ray contrast material-free region that does not contain any X-ray contrast material is provided in at least a region on the first surface where cells are placed, i.e., at least a portion of the first surface of the resin layer.

[0019] As used herein, "on" in the context of the first or second surface refers to a position in contact with the surface or a position directly above the surface without contacting the surface. In the latter case, a laminate layer such as another resin layer may be present between the surface and the layer containing an X-ray contrast material formed on the surface. Furthermore, as used herein, "present in at least a portion of the resin layer" refers to the layer containing an X-ray contrast material being present in only a portion of the resin layer or in the entire resin layer in the thickness direction of the resin layer and / or the direction normal to the surface of the resin layer.

[0020] The resin film of this embodiment can be broadly divided into two types: (A) a type in which the layer containing an X-ray contrast material is an X-ray contrast layer containing a pattern of X-ray contrast material, and (B) a type in which the layer containing an X-ray contrast material is an X-ray contrast resin layer containing a plurality of filler-like X-ray contrast materials and a resin material. The type (A) corresponds to the first embodiment (FIG. 1), the second embodiment (FIG. 2), the fifth embodiment (FIG. 5), and the sixth embodiment (FIG. 6) described below, and the type (B) corresponds to the third embodiment (FIG. 3) and the fourth embodiment (FIG. 4) described below, respectively.

[0021] The X-ray contrast layer in form (A) can be formed by a known coating method such as printing using ink or paste containing an X-ray contrast material, while the X-ray contrast resin layer in form (B) can be formed by a known film molding method using a molding material containing a resin material and a plurality of filler-like X-ray contrast materials.

[0022] The X-ray contrast material may be any substance that appears significantly different from biological tissues when an X-ray transmission image is taken. It preferably contains an X-ray absorption contrast material, and more preferably contains a metal or a metal compound. Examples of suitable X-ray contrast materials include X-ray contrast fillers (filler-like X-ray contrast materials) and inks or pastes containing X-ray contrast fillers. Suitable X-ray contrast fillers include particles of zirconia, barium sulfate, barium oxide, and the like. The content of the X-ray contrast filler in inks or pastes containing the X-ray contrast filler is preferably approximately 1 to 70% by mass. Alternatively, metal materials such as titanium, gold, silver, and platinum, or metal oxides, are also suitable X-ray contrast materials. Metals and metal oxides may be formed by direct vapor deposition or other methods on resin layers, protective layers (described below), laminate layers, and the like, or may be added in the form of fine particles.

[0023] In the case of the (A) embodiment, an X-ray contrast layer (coating) can be formed by printing an ink or paste containing an X-ray contrast filler onto at least a portion of the second surface or onto at least a portion of the first surface other than the region where the cells are to be placed, using a printing technique and including an appropriate pigment as the ink. In addition to printing, the surface to be coated with the X-ray contrast material may be roughened, and the X-ray contrast material may be placed on the roughened surface, or may be adhered using an appropriate adhesive. Alternatively, the X-ray contrast layer may be formed by directly depositing a film by vapor deposition or other methods while masking areas other than the designated areas.

[0024] In the (B) embodiment, an X-ray contrast resin layer containing multiple filler-like X-ray contrast materials and a resin material can be obtained by mixing the X-ray contrast filler into a polymer material, for example by kneading it into the polymer material, and then forming a film. Conventional film forming methods, such as compression molding, extrusion molding, injection molding, calendar molding, and thermoforming, can be used to form a film from the polymer material kneaded with the X-ray contrast filler. This X-ray contrast resin layer can be a resin layer containing the X-ray contrast material therein or a laminate layer laminated on a resin layer. It is preferable that the X-ray contrast filler is prevented from being exposed on the surface of the X-ray contrast resin layer. For example, the X-ray contrast filler can be made spherical with little anisotropy, its particle size smaller than the thickness of the resin layer, and its wettability with the polymer material can be improved to prevent the X-ray contrast filler from being exposed on the surface. For example, it is preferable that the X-ray contrast filler is prevented from being exposed on at least a portion of the first surface of the resin layer formed by the X-ray contrast resin layer. The content of the X-ray contrast material in the resin layer containing the X-ray contrast material is preferably about 1 to 70% by mass.

[0025] When viewed from the normal direction of the first surface of the resin layer, the layer containing the X-ray contrast material may be present so as to overlap the entire or only a portion of the resin film. When the layer containing the X-ray contrast material is present so as to overlap the entire resin film, the X-ray detectability of the resin film can be further improved. On the other hand, when the resin layer contains a resin material having a predetermined level of resistance to tearing and pulling, the layer containing the X-ray contrast material may be present so as to overlap only a portion of the resin film. This reduces the manufacturing cost of the resin film compared to when the layer is formed over the entire resin layer. Furthermore, when viewed from the normal direction of the first surface (top view), the layer containing the X-ray contrast material may not be present near the outer periphery of the resin layer. This can suppress curling of the resin film. Curling is particularly effectively suppressed when the resin film has a circular shape when viewed from the top.

[0026] The resin film may also include a protective layer that protects at least a portion of the resin layer, for example, the surface of the portion containing the X-ray contrast material. Specifically, this protective layer may cover and protect at least a portion or the entire surface of the X-ray contrast layer present on the first surface of the resin layer and / or the second surface opposite to the first surface. The protective layer may be made of SiO Ｘ , SiN Ｘ , SnO Ｘ , TiO Ｘ , AlO Ｘ Suitable protective layers include inorganic protective layers containing inorganic oxides or inorganic nitrides such as those mentioned above, organic protective layers such as acrylate-based photocurable resins or thermosetting resins, and cationic polymerization ultraviolet-curable epoxy resins, and organic-inorganic hybrid / composite protective layers such as photocurable organic / inorganic hybrid hard coating agents. In particular, when the X-ray contrast layer is made of ink or paste, it is preferable to provide a protective layer thereon. The protective layer can improve the flatness of the surface of the X-ray contrast layer. Furthermore, the protective layer can suppress the elution of the X-ray contrast material into culture media, cryopreservation solutions, cell sheets, etc.

[0027] The resin film may also have a laminate layer containing an X-ray contrast material laminated on at least a portion of the surface of the resin layer. In this case, it is preferable that the laminate layer is provided on the second surface of the resin layer opposite to the first surface, and the entire first surface becomes an X-ray contrast material-free region. As the laminate layer, either a film of the same type as the resin film or a film of a different type can be preferably used. However, like the resin film, a laminate layer that is transparent and has a specific gravity of 1.0 g / cm ３ It is preferable that the material be larger and have excellent durability, mechanical strength, and processability, and therefore polyether ether ketone (PEEK) film and polyethylene terephthalate (PET) film are particularly suitable.

[0028] Furthermore, when using a resin film as a scaffold for cell culture, the materials used for the protective layer and laminate layer are preferably thin and have a high specific gravity, and are insoluble in the culture medium. The protective layer or laminate layer may be laminated directly to the resin layer, or may be laminated via an adhesive layer. Materials commonly used as adhesives for films can be used for such adhesive layers. For example, ester-based or ether-based adhesives can be used.

[0029] Furthermore, in order to ensure visibility, it is preferable that the surface of the resin film opposite the first surface has reduced gloss, assuming that the resin sheet carrying the cultured cells will be monitored with an endoscope during cell transplantation surgery. "Reduced gloss" refers to, for example, a 60-degree specular gloss Gs (60°) measured in accordance with JIS Z8741:1997 of 700 or less, preferably 400 or less. Measurement can be performed at any location on the surface of the resin film opposite the first surface. Examples of the opposite surface of the resin film include the surface of a resin layer composed of an X-ray contrast resin layer, a laminate layer laminated on a resin layer, the second surface of a resin layer having an X-ray contrast layer on the first surface, and the surface of a protective layer protecting the X-ray contrast layer formed on the second surface of the resin layer.

[0030] Furthermore, a portion or all of the region on the first surface of the resin layer where the cultured cells are placed may be subjected to a hydrophilization treatment such as plasma treatment. From the perspective of placing cultured cells, it is preferable that the first surface of the resin layer, at least the region where the cells are placed, be rendered hydrophilic by hydrophilization treatment. A hydrophilic surface refers to a surface having a water droplet contact angle θ measured by the θ / 2 method of 70° or less, preferably 65° or less, and more preferably 55° or less. The water droplet contact angle θ is measured by placing 2 μl of pure water on the first surface in a 25°C environment and measuring the angle between the water droplet and the first surface using a contact angle meter using the θ / 2 method in accordance with ISO 19403-2:2017. By creating such a surface, it becomes easier to ensure adhesion with the cultured cells.

[0031] Furthermore, it is preferable that a part or all of the region on the first surface where cells are placed is present in a region where X-ray contrast material is not present. Of the first surface, the region where X-ray contrast material is not present preferably accounts for 50% or more, more preferably 80% or more, and particularly preferably 100%. Note that when an X-ray contrast material is included on the first surface, if there is at least a portion of the region not subjected to hydrophilic treatment or a region where cells are not seeded, the X-ray contrast material may be placed on at least a part of this region.

[0032] Each embodiment will be described in detail below. FIG. 1 shows a first embodiment of the resin film of the present invention. The resin film 11 has a resin layer 12 and an X-ray contrast layer 13 containing an X-ray contrast material provided on a second surface 15 of the resin layer 12. Furthermore, an X-ray contrast material-free region 16 is provided on a first surface 14 of the resin layer 12. The X-ray contrast layer 13 is partially or entirely made of an X-ray opaque pattern. The spaces between the X-ray opaque patterns may be voids, or may be filled with a resin material. For example, in a cross-sectional view of the resin film 11, the X-ray contrast layer 13 may include a pattern made of a resin material and an X-ray opaque pattern, or may have a plurality of slit-shaped X-ray opaque patterns arranged at a distance from each other.

[0033] FIG. 1 shows an example in which the X-ray contrast layer 13 is provided over the entire second surface 15 of the resin layer 12. However, the X-ray contrast layer 13 may be provided only on a portion of the second surface 15, or a predetermined pattern may be formed. Examples of predetermined patterns include a design, figure, letter, symbol, etc. The pattern may have an asymmetric shape on the front and back sides, thereby improving the visibility of the front and back sides of the resin film. Examples of patterns include checkered, dotted, and striped patterns. From the viewpoint of preventing the resin film from curling, a pattern in which the individual patterns are spaced apart, specifically a dotted pattern, is preferred. Furthermore, the first surface 14 and / or the second surface 15 of the resin layer 12 may have a layer containing a substance whose color or state changes with temperature. Furthermore, the X-ray contrast material-free region 16 may be provided on a portion or the entire first surface 14, and this region can be made into a region on which cells are placed by subjecting this region to hydrophilic treatment.

[0034] 2 shows a second embodiment of the resin film of the present invention. Resin film 21 has resin layer 22 and X-ray contrast layer 23 containing an X-ray contrast material provided on a portion of first surface 24 of resin layer 22. Furthermore, first surface 24 of resin layer 22 is provided with X-ray contrast material-free region 26. In this embodiment, no X-ray contrast material is provided on second surface 25 of resin layer 22.

[0035] 2, the radiopaque material may form a predetermined pattern on a first surface 24 of the resin layer 22. Regions 26 free of the radiopaque material can be made into regions on which cells can be placed by subjecting the region 26 to hydrophilic treatment.

[0036] 3 shows a third embodiment of the resin film of the present invention. Resin film 31 has resin layer 32 containing radiopaque filler 37 embedded therein as an radiopaque material. Furthermore, a first surface 34 of resin layer 32 is provided with an area 36 free of radiopaque material.

[0037] 3, because the radiopaque filler 37 is embedded inside the resin layer 32, the radiopaque filler 37 is not exposed at least on the first surface 34 side, and it is possible to form an radiopaque material-free region 36. This radiopaque material-free region 36 may be provided on part or the entire first surface 34, and this region can be made into a region on which cells are placed by subjecting it to a hydrophilic treatment.

[0038] 4 shows a fourth embodiment of the resin film of the present invention. Resin film 41 has a resin layer 42 and a laminate layer 47 containing an X-ray contrast material laminated on a second surface 45 of resin layer 42. In addition, a first surface 44 of resin layer 42 is provided with an X-ray contrast material-free region 46.

[0039] 4 shows an example in which the laminate layer 47 is laminated over the entire second surface 45 of the resin layer 42, but it may be laminated so as to cover only a portion of the second surface 45. Furthermore, the X-ray contrast material-free region 46 may be provided over a portion or the entire first surface 44, and this portion can be made into a region on which cells are placed by subjecting it to a hydrophilic treatment.

[0040] 5 shows a fifth embodiment of the resin film of the present invention. Resin film 51 includes a resin layer 52, an X-ray contrast layer 53 containing an X-ray contrast material provided on a second surface 55 of resin layer 52, and a laminate layer 57 laminated so as to cover X-ray contrast layer 53. In addition, an X-ray contrast material-free region 56 is provided on a first surface 54 of resin layer 52.

[0041] 5 shows an example in which the X-ray contrast layer 53 is provided over the entire second surface 55 of the resin layer 52, but it may be provided only over a portion of this second surface 55, or may form a predetermined pattern. Similarly, the laminate layer 57 may be laminated so as to cover a portion of the second surface 55. In this case, the laminate layer 57 may cover all or part of the X-ray contrast layer 53. Furthermore, the X-ray contrast material-free region 56 may be provided over a portion or the entire first surface 54, and this region can be made into a region on which cells are placed by subjecting it to a hydrophilic treatment.

[0042] 6 shows a sixth embodiment of the resin film of the present invention. Resin film 61 has a resin layer 62, an X-ray contrast layer 63 containing an X-ray contrast material provided on a second surface 65 of resin layer 62, and a protective layer 67 that protects X-ray contrast layer 63. In addition, a first surface 64 of resin layer 62 is provided with an X-ray contrast material-free region 66.

[0043] 6 shows an example in which the X-ray contrast layer 63 is provided over the entire second surface 65 of the resin layer 62, but as in the embodiment of FIG. 1, it may be provided over only a portion of the second surface 65, or a predetermined pattern of X-ray contrast material may be formed. It is sufficient for the protective layer 67 to cover at least the portion where the X-ray contrast layer 63 is provided. Furthermore, the X-ray contrast material-free region 66 may be provided over a portion or the entire first surface 64, and this region can be made into a region for placing cells by subjecting it to hydrophilic treatment.

[0044] An example of a method for manufacturing a resin film of the present invention will be described. (Method for forming a resin film containing an X-ray contrast material in the resin layer) The manufacturing methods for the first embodiment shown in FIG. 1, the second embodiment shown in FIG. 2, the third embodiment shown in FIG. 3, and the sixth embodiment shown in FIG. 6 will be described. One surface of the material used as the resin layer, for example, a PEEK film, is hydrophilized. This hydrophilization treatment can be performed, for example, by UV ozone treatment or plasma treatment. This provides an area on which cultured cells can be placed, ensuring cell adhesion. The hydrophilization treatment may be performed after the X-ray contrast material is impregnated on the surface of the resin layer, after a protective layer is formed, or after the X-ray contrast material is impregnated inside the resin layer.

[0045] Next, an X-ray contrast material is impregnated into at least a portion of the first surface of the resin layer other than the region where cells are to be placed, and / or at least a portion of the second surface, where the region of the first surface that does not contain the X-ray contrast material is designated as an X-ray contrast material-free region.

[0046] In particular, when an X-ray contrast material is contained on the surface of the resin layer, it is preferable to provide a protective layer to protect the portion containing the X-ray contrast material. The protective layer may be provided by forming a vapor-deposited film by vapor deposition so as to selectively protect the portion containing the X-ray contrast material, or, in particular, when the X-ray contrast material is placed on the backside of the first surface, by applying a polymer material to form the protective layer as a coating liquid to this surface, followed by removing the solvent to form a coating film.

[0047] (Method of forming a resin film using a laminate layer) (When a laminate layer containing an X-ray contrast material is used) A manufacturing method of the fourth embodiment shown in Figure 4 will be described. A film containing an X-ray contrast material as a laminate layer is produced. The X-ray contrast material may be contained on the surface or inside of this film, and a film containing an X-ray contrast material can be obtained by a method similar to the method of containing an X-ray contrast material on the surface of a resin layer or the method of containing an X-ray contrast material inside a resin layer as described above.

[0048] Next, a film containing an X-ray contrast material is laminated as a laminate layer on the resin layer including the area where cells are to be placed, to obtain a resin film. The surface of the resin layer opposite the surface on which the laminate layer is provided is then hydrophilized. This ensures adhesion to the cells and provides an area where cultured cells are to be placed. Since no laminate layer is laminated on the first surface, the first surface of the resin layer is an area free of X-ray contrast material. An adhesive may also be used when laminating the resin layer and the laminate layer. (When a laminate layer without an X-ray contrast layer is used) A manufacturing method for the fifth embodiment shown in Figure 5 will be described. The provision of an X-ray contrast layer on the second surface of the laminate layer resin layer is as described above.

[0049] Next, a film is laminated on the side of the resin layer where the X-ray contrast layer is provided, covering the X-ray contrast layer, to obtain a resin film. The surface of the resin layer opposite the surface where the laminate layer is provided is then hydrophilized. This provides an area where cultured cells can be placed, ensuring adhesion to the cells. Since the X-ray contrast layer is provided on the second surface opposite the first surface, the first surface of the resin layer becomes an area where the X-ray contrast material is not present. An adhesive may also be used when laminating the resin layer and the laminate layer.

[0050] When preparing a resin film using a laminate layer, in any case, if the X-ray contrast material is exposed on the back surface opposite to the lamination surface of the laminate layer when the laminate layer is laminated on the resin layer, a protective layer may be further provided by the method described above.

[0051] Each component of the resin film will be described in detail below.

[0052] The resin film of this embodiment is a resin film including a resin layer having a first surface on which cultured cells are placed. The first surface has an area for placing cells, and the area for placing cells preferably occupies 50% or more of the first surface, more preferably 80% or more, and particularly preferably 100%.

[0053] Here, the cells placed on the first surface are preferably a cell sheet to facilitate transplantation. Furthermore, such cells may be cells cultured elsewhere in the form of a sheet, or may be cells cultured on the first surface. When culturing cells on the first surface, the first surface functions as a scaffold for culturing the cells, and the resin film is preferably transparent to facilitate observation of the culture status using transmitted light observation under a microscope, for example.

[0054] The resin film of this embodiment is used by placing cultured cells on a first surface provided in an X-ray contrast material-free region that does not contain an X-ray contrast material. Specifically, in cell transplantation procedures for various diseases, such as skin or ischemic cardiomyopathy, postoperative air leaks in the lungs, and stenosis after endoscopic esophageal submucosal dissection, or for preventing postoperative complications, cultured cells, such as a cell sheet, are placed on the resin film and transferred to the transplantation site. In this case, sufficient adhesion between the resin film and the cultured cells is ensured, and the resin film functions as a self-supporting membrane, facilitating cell transfer. Then, after the cells are detached from the resin film at the transplantation site, the used resin film is removed.

[0055] The resin film may contain an organic polymer material, or may be configured not to contain a biological membrane such as a collagen film or amniotic membrane. When a biological membrane is used as the substrate, the cell sheet formed on the biological membrane tends to be difficult to peel off immediately. In contrast, a resin film containing an organic polymer material allows the cell sheet formed on the cell-mounting region (culture surface) to be peeled off immediately after being attached to the transplantation destination. The time from attachment to peeling may be 10 minutes or less.

[0056] Specific examples of materials constituting the resin film (hereinafter also referred to as resin materials) include organic polymer materials such as polyether ether ketone (PEEK), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polystyrene (PS), polycarbonate (PC), modified polyphenylene ether (mPPE), polyphenylene sulfide (PPS), polysulfone (PSU), polyarylate (PAR), liquid crystal polymer (LCP), polyethylene (PE), polypropylene (PP), nylon 66 (N66), ethylene-tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), acrylonitrile-butadiene-styrene copolymer (ABS), polyethersulfone (PES), silicone, polyvinylidene fluoride (PVDF), polyacetal (POM), polyimide (PI), polyamide (PA), polyglycolic acid (PGA), polylactic acid (PLA), fibroin, cellulose, regenerated cellulose, cyclic olefin polymer, gelatin, and collagen. In particular, examples of resin materials include synthetic polymer materials such as 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 polymer. Examples of materials that can be used for the resin layer include transparent polymers having a specific gravity of 1.0 g / cm. ３Preferably, the resin film is a material that is larger and has excellent durability, mechanical strength, and processability. An example of a resin film may be a film containing at least one of these resin materials as a main component, preferably a polyether ether ketone film, a polyethylene terephthalate film, or a polystyrene film, more preferably a polyether ether ketone film. The resin film preferably includes one or more resin layers made of the above-mentioned resin materials. The two or more resin layers may contain different materials. The resin film may be configured as a resin substrate including a resin layer made of the above-mentioned material, or as a laminate substrate including a resin layer made of the above-mentioned material and a resin layer and / or inorganic layer made of another material. However, the resin layer does not need to include a layer formed by chemical vapor deposition such as parylene or a wet coating layer. Furthermore, the inorganic layer in the laminate substrate preferably does not include glass and may include metal foil. Furthermore, the resin film preferably does not include a nonwoven fabric or a fiber substrate on the cell-mounting region side. A resin film containing PEEK is preferable to one containing PET from the viewpoints of a low linear expansion coefficient, solvent resistance, and heat resistance. Furthermore, the resin film is preferably made of a material with a higher specific gravity than the culture medium.

[0057] The resin film may be transparent. Transparent means that the haze value is 30% or less. Even a transparent resin film has a contrast ratio, and therefore the visibility of the markings 30 is excellent.

[0058] The surface of the resin film, including the cell-mounting region, is preferably subjected to a hydrophilic treatment. This improves cell adhesion. Examples of hydrophilic treatment include UV ozone treatment and plasma treatment. Among these, it is preferable to use a resin film in which the cell-mounting region is plasma-treated. In this specification, a hydrophilized surface refers to 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 on the hydrophilized 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 in a 25°C environment and measuring the angle between the water droplet and the substrate using a contact angle meter using the θ / 2 method in accordance with ISO 19403-2:2017. Furthermore, the lower limit of the surface roughness Ra of the hydrophilic-treated 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, and more preferably 2 nm or less. The surface roughness Ra here refers to the arithmetic mean roughness in a square area with one side of 100 nm, obtained by using surface shape data measured with an atomic force microscope (AFM).

[0059] The shape of the resin film in the normal direction to the surface is preferably circular, but may be other shapes, such as a regular polygon such as a square, regular pentagon, regular hexagon, or regular octagon, an oval, or a rectangle.

[0060] The thickness of the resin film may be, for example, 5 to 250 μm from the viewpoint of ease of handling in accordance with the intended use. If the resin film is too thin, it may be difficult to handle while maintaining its sheet shape by simply holding one edge of the resin film with tweezers. If the resin film is too thick, it may be difficult to properly fit the film to an affected area with a high degree of curvature. The thickness of the resin film refers to the thickness of the resin layer alone, or, if a protective layer or laminate layer is present, the total thickness including the resin layer and the protective layer or laminate. The lower limit of the substrate thickness is preferably 5 μm or more, more preferably 6 μm or more, even more preferably 8 μm or more, 10 μm or more, or 12 μm or more. Meanwhile, the upper limit of the substrate thickness is preferably 100 μm or less, 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 resin film is 6 μm or more to 50 μm or less, 8 μm or more to 30 μm or less, or 10 μm or more to 20 μm or less. Furthermore, by setting the thickness to be equal to or greater than the lower limit, the resin film can be prevented from tearing or curling during transport, improving handleability. By setting the thickness to be equal to or less than the upper limit, the resin film can be improved in its ability to conform to the affected area during transplantation. Note that the resin film may have a sheet-like shape with a flat surface as a whole when viewed in cross section in the thickness direction.

[0061] Furthermore, from the viewpoint of functioning as a free-standing membrane during culture, the lower limit of the resin film substrate thickness is preferably greater than 10 μm, more preferably 11 μm or greater, and even more preferably 12 μm or greater. Thus, by using a resin film substrate thickness greater than 10 μm, it is possible to produce a cell sheet during the cell culture process without fixing or adhering the resin film to the inside of the culture vessel. A free-standing membrane is preferably a membrane that maintains the planar state of the cell-mounting region of the resin film even in culture medium, and more preferably maintains this planar state even after a cell sheet is formed on the cell-mounting region. Because the planar state of the cell-mounting region is maintained, a resin film that does not curl can improve cell culture characteristics in culture medium without adhering. Furthermore, this free-standing membrane may be capable of retaining its sheet shape to a certain extent when lifted by pinching one end with tweezers. When viewed from the surface normal direction, the resin film preferably does not have an opening penetrating from the front to the back in the region where the cell-mounting region is formed, and preferably does not have an opening that exposes the bottom of the culture vessel when placed in the culture vessel. That is, it is preferable that the resin film does not have a ring structure, and the region where the cell-mounting region is formed is solid.

[0062] The area of ​​the resin film is not particularly limited, but may be, for example, 0.3 cm ２ More than 1000cm ２ The lower limit of the area of ​​the resin film is 0.6 cm ２ More than 1.6 cm ２ 3cm or more ２ More than 4cm ２ More than 5cm ２ More than 6cm ２ More than 7cm ２ More than 8cm ２ or more or 10 cm ２ On the other hand, the upper limit of the area of ​​the resin film is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably, 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm２ For example, the area of ​​the resin film may be 0.6 cm or less. ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ The following is the result.

[0063] When the cell-mounting area of ​​the resin film is measured with a laser microscope, there are three or fewer holes with a diameter of 1 μm to 100 μm and a depth of 0.5 μm to 100 μm within a 100 μm square area. The diameter of the holes is, for example, 1 μm to 100 μm, preferably 2 μm to 50 μm, and more preferably 3 μm to 30 μm. The depth of the holes is, for example, 0.5 μm to 100 μm, preferably 1 μm to 50 μm, and more preferably 2 μm to 20 μm. The combinations of the ranges of the hole diameter and the hole depth are, 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, a hole diameter of 1 μm or more and 10 ... The hole diameter is 2 μm to 50 μm and the hole depth is 1 μm to 50 μm, the hole diameter is 2 μm to 50 μm and the hole depth is 2 μm to 20 μm, the hole diameter is 3 μm to 30 μm and the hole depth is 0.5 μm to 100 μm, the hole diameter is 3 μm to 30 μm and the hole depth is 1 μm to 50 μm, and the hole diameter is 3 μm to 30 μm and the hole depth is 2 μm to 20 μm. The upper limit of the porosity of the resin film 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 resin film is not particularly limited, but may be 0% or more. By having three or less holes in the cell mounting region and / or by setting the porosity of the resin film below the upper limit, the adhesion to the cell sheet can be moderate. The presence or absence of holes may be measured on the surface of the resin layer where the resin film is formed on the cell-mounting region side. 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)} x 100.

[0064] The resin film does not need to have a layer containing a temperature-responsive polymer formed on the surface where the cell-mounting region is formed. This prevents a decrease in adhesion between the cell sheet and the resin film in low-temperature environments, such as during cryopreservation. The temperature-responsive polymer is a material that exhibits cell adhesiveness at the temperature used for cell culture and exhibits cell non-adhesive properties by changing the temperature from that temperature, allowing for easy detachment of the cell sheet. A temperature range in which the temperature-responsive polymer exhibits cell adhesiveness of 10°C to 45°C, particularly 33°C to 40°C, is preferred because it allows stable cell culture. Furthermore, a temperature range in which the temperature-responsive polymer exhibits cell non-adhesive properties of 1°C to 36°C, particularly 4°C to 32°C, is preferred because it reduces damage to the detachment of the cell sheet. Specific examples of materials constituting the temperature-responsive polymer include temperature-responsive polymers such as poly-N-isopropylacrylamide (PNIPAAm), poly-N-n-propylacrylamide, poly-N-n-propylmethacrylamide, poly-N-ethoxyethylacrylamide, poly-N-tetrahydrofurfurylacrylamide, poly-N-tetrahydrofurfurylmethacrylamide, and poly-N,N-diethylacrylamide, with PNIPAAm, poly-N-n-propylmethacrylamide, and poly-N,N-diethylacrylamide being particularly preferred. The bottom surface of the culture vessel may also be configured so as not to contain a temperature-responsive polymer.

[0065] Next, a cell sheet-attached resin film and a method for producing the same will be described.

[0066] The cell sheet-attached resin film of this embodiment includes the above-mentioned resin film and a cell sheet provided on the cell-mounting region of the first surface of the resin film.

[0067] An example of a method for manufacturing a resin film with a cell sheet according to this embodiment includes an installation step of placing the resin film described above in a culture vessel, a culture step of placing a culture medium P in the culture vessel, seeding cells, and culturing a cell sheet on the cell-mounting region on the first surface of the resin film, and a removal step of removing the resin film with a cell sheet from the culture vessel.

[0068] The placing step may include placing the ring member on the resin film in the culture vessel, and the removing step may include removing the ring member from the resin film.

[0069] The cells are not particularly limited as long as they are clinically useful cells for treating or preventing symptoms associated with cell, tissue, or organ deficiency, dysfunction, or dysfunction, or culturable cells for use in non-clinical trials, and are cells isolated from a living organism. Examples of cells include biological tissue cells, mesenchymal stem cells capable of differentiating into cells belonging to mesenchymal tissue, pluripotent stem cells capable of differentiating into various biological tissues, and stem cells and progenitor cells that can be induced to differentiate. The cells may be adherent cells or suspension cells.

[0070] Specific examples of biological tissue cells include fibroblasts, myofibroblasts, corneal epithelial cells, retinal cells, nerve cells, muscle cells, cardiac muscle cells, myoblasts, bone cells, osteoblasts, chondrocytes, adipocytes, hepatocytes, pancreatic cells, kidney cells, gingival cells, periosteal cells, skin cells, and endothelial cells. Specific examples of mesenchymal stem cells include adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells. Specific examples of pluripotent stem cells include induced pluripotent stem cells, embryonic stem cells, nuclear transfer embryonic stem cells, embryonic tumor cells, and embryonic germ cells. These cells may be cultured alone or in combination of two or more types. These cells may be appropriately selected from known types depending on the intended use of the cells.

[0071] The origin of the cells is not particularly limited, and examples thereof include mammals, birds, amphibians, fish, insects, plants, microorganisms, etc. Specific examples of mammals and birds include humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, chickens, etc.

[0072] A cell sheet has a sheet structure in which cells are physically and functionally connected to each other via adhesion molecules, extracellular matrix, etc. The cell sheet may have a single-layer structure consisting of one cell layer, or a laminated structure consisting of two or more cell layers. The laminated structure is not particularly limited, but examples include multi-layer structures such as two-layer, three-layer, four-layer, and five-layer structures.

[0073] When a cell sheet has a multilayer structure, the multilayer structure may be obtained by culturing the cell sheet on a resin film, or it may be obtained by stacking cell sheets with a single layer structure. In particular, a cell sheet with a multilayer structure can be obtained by preparing multiple cell sheet-attached resin films of the present invention, overlaying one cell sheet on another, and peeling off the culture carrier substrate from the other cell sheets.

[0074] The thickness of the cell sheet is not particularly limited, but is, for example, 0.001 mm or more and 2.0 mm or less. The lower limit of the thickness of the cell sheet is more preferably 0.01 mm or more, even more preferably 0.03 mm or more, and particularly preferably 0.05 mm or more. On the other hand, the upper limit of the thickness of the cell sheet is more preferably 1.5 mm or less, even more preferably 1.2 mm or less, and particularly preferably 1.0 mm or less. The thickness of the cell sheet may be, for example, 0.001 mm to 2.0 mm, 0.001 mm to 1.5 mm, 0.001 mm to 1.2 mm, 0.001 mm to 1.0 mm, 0.01 mm to 2.0 mm, 0.01 mm to 1.5 mm, 0.01 mm to 1.2 mm, 0.01 mm to 1.0 mm, 0.03 mm to 2.0 mm, 0.03 mm to 1.5 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, or 0.05 mm to 1.0 mm. By keeping the thickness of the cell sheet within the above ranges, high cell activity within the cell sheet and excellent shape retention ability, which is advantageous for cell transplantation, can be achieved.

[0075] The area of ​​the cell sheet is not particularly limited, but may be, for example, 0.3 cm ２More than 1000cm ２ The lower limit of the cell sheet area is 0.6 cm ２ More than 1.6 cm ２ More than 3cm ２ More than 4cm ２ More than 5cm ２ More than 6cm ２ More than 7cm ２ More than 8cm ２ or more or 10 cm ２ On the other hand, the upper limit of the area of ​​the cell sheet is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably, 500 cm ２ is less than or equal to 100 cm ２ Below, 50cm ２ Less than or equal to 20cm ２ For example, the area of ​​the cell sheet may be 0.6 cm or less. ２ More than 900cm ２ Below, 1.6cm ２ More than 800cm ２ Below, 3cm ２ More than 500cm ２ Below, 6cm ２ More than 100cm ２ Below, 8cm ２ More than 50cm ２ Less than or equal to 10 cm ２ 20cm or more ２ Conventionally, when a cell sheet is transplanted alone, the strength of the cell sheet is low, and therefore there is a high possibility that the cell sheet will break during transportation if it is large in area. However, in the present disclosure, the cell sheet is supported by a culture carrier substrate, which prevents the cell sheet from breaking during transplantation. ２ It is also possible to produce a larger cell sheet and adjust it to the size of the affected area as needed. In the cell sheet-attached resin film, it is preferable that the entire underside of the cell sheet overlaps the surface of the resin film (cell-mounting area).

[0076] The cell culture is not particularly limited, and conventional means used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as basic technical fields such as regenerative medicine and bioengineering, can be used.

[0077] The culture conditions are not particularly limited as long as they can bring the cultured cells into the desired state. Typical culture conditions include, for example, culture at 37°C, 5% CO using a prepared basal medium. ２ The cell culture period is not particularly limited as long as the cultured cells reach the desired state. The cell culture period is, for example, 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 of time, the medium may be replaced. The frequency and method of medium replacement are not particularly limited. In general, it is preferable to replace the medium every 1 to 7 days. It is particularly preferable to replace the medium every 1 to 5 days. At this time, the entire medium may be replaced, or a portion of the medium may be left and new medium may be added.

[0078] 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 intended use of the cultured cells. For example, ２ cells / cm ２ 1x10 or more ９ cells / cm ２ The lower limit of the cell density is more preferably 1 × 10 ３ cells / cm ２ More preferably, 5×10 ３ cells / cm ２ More preferably, 5 × 10 ４ cells / cm ２ On the other hand, the upper limit of the cell density is more preferably 1 × 10 ８ cells / cm ２ More preferably, 5 × 10 ７ cells / cm ２ Particularly preferably, 1 × 10 ７ cells / cm ２ The density of the cells to be cultured is, for example, 5 × 10 ２ cells / cm ２ 1x10 or more ９ cells / cm２ Below, 5×10 ２ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 5×10 ２ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 5×10 ２ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 1×10 ３ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 5×10 ７ Cells / cm ２ Below, 5×10 ３ Cells / cm ２ Above 1×10 ７ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ９ Cells / cm ２ Below, 5×10 ４ Cells / cm ２ Above 1×10 ８ Cells / cm ２ Below, 5×10４ cells / cm ２ 5x10 or more ７ cells / cm ２ Below, 5 x 10 ４ cells / cm ２ 1x10 or more ７ cells / cm ２ The following is the result.

[0079] The medium is not particularly limited as long as it is suitable for the cells to be cultured. Examples of medium components include sugars, amino acids, vitamins, inorganic salts, trace metals, additives, etc. These medium components may be blended alone or in combination of two or more. These medium components may be appropriately selected from known ones depending on the cells to be cultured, etc.

[0080] 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.

[0081] 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.

[0082] Specific examples of vitamins include sodium L-ascorbate, L-ascorbic acid-diphosphate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0083] 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, magnesium carbonate, etc. Specific examples of trace metals include iron sulfate, iron nitrate, copper sulfate, copper nitrate, zinc sulfate, etc.

[0084] Specific examples of additives include serum 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.

[0085] Examples of media containing the above-mentioned medium components include AIM V medium, HFDM-1 medium, equilibrated buffers such as Dulbecco's phosphate buffered saline (D-PBS) and Hank's balanced 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 suitable basal media include basal media such as PBS medium, Williams' medium E, Brinster's BMOC-3 medium, and E8 medium. These basal media may be used alone or in combination of two or more. Furthermore, medium components may be added, removed, increased, or decreased in amount depending on the type and condition of the cells. These basal media may be appropriately selected from known media depending on the cells to be cultured, etc.

[0086] <Cell Culture Kit> Next, the cell culture kit will be described.

[0087] An example of the cell culture kit of this embodiment includes: a culture vessel; and the above-described resin film.

[0088] The culture vessel is a vessel for culturing cells in a medium, and is not particularly limited as long as it is suitable for the type of cells to be cultured and the intended use. Examples of the culture vessel 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, roller bottles, etc.

[0089] The material of the culture vessel is not particularly limited as long as it is impermeable to the culture medium, and examples thereof include polystyrene, polyethylene, polypropylene, polyvinyl alcohol, polyethylene terephthalate, polyacetal, polyvinyl chloride, acrylic resin, polycarbonate, polyether ether ketone, polyether sulfone, polytetrafluoroethylene, polyimide, polyamide, cycloolefin polymer, cellulose, silicone, nylon 6,6, glass, and metals such as stainless steel and aluminum.

[0090] The area of ​​the culture vessel is not particularly limited, but any commercially available culture vessel can be used without any problems. For example, a culture vessel having an area of ​​0.3 cm ２ More than 1000cm ２ The lower limit is more preferably 0.35 cm. ２ More preferably, 1.0 cm or more ２ More than 1.9 cm, particularly preferably ２ On the other hand, the upper limit is more preferably 900 cm ２ More preferably, 800 cm or less ２ Particularly preferably, 500 cm ２ The following is the result.

[0091] The cell culture kit may also include a ring member that is removably housed in the interior space of the culture vessel and that can press and fix a resin film placed inside the culture vessel.

[0092] The ring member is, for example, cylindrical or annular. Note that the shape of the ring member is not limited to cylindrical or annular, and may be a ring having an appropriate predetermined shape, such as an ellipse, rectangle, or polygon when viewed from above. Furthermore, the ring member does not need to be completely cylindrical or annular, and may be partially missing, or may be U-shaped.

[0093] The specific gravity of the ring member is greater than 1.0. The specific gravity of the ring member is preferably 1.05 or greater so that the ring member sinks stably in the culture medium.

[0094] The material of the ring member may include one or more of, for example, resin, glass, ceramic, metal, etc. Resin is suitable for the ring member from the viewpoints of moldability and productivity. The resin may include one or more of, for example, polystyrene, ABS resin, AS resin, polyphenyl ether, nylon 66, MS resin, nylon 6, methacrylic resin, acrylic resin, polycarbonate resin, polysulfone resin, polyetherimide resin, polyurethane resin, PBT resin, PET resin, vinyl chloride resin, PBT resin, polyacetal resin, PET resin, fluororesin, etc. The glass may be soda glass, borosilicate glass, quartz glass, etc. The metal may include one or more of stainless steel, aluminum, etc. Furthermore, the ring member may contain various additives based on the materials exemplified here.

[0095] <Preservation> An example of the cryopreservation method of this embodiment includes a cooling step of cooling the cell sheet-attached resin film in a cryopreservation solution. The cryopreservation of the cell sheet-attached resin film may be performed inside a culture vessel.

[0096] A cryopreservation solution is a solution for reducing cell damage due to cryopreservation. The cryopreservation solution is not particularly limited as long as it is suitable for cryopreserving cells, but it may contain medium components contained in the medium, a cryoprotectant, etc. The medium components, such as sugars, amino acids, vitamins, inorganic salts, trace metals, and additives, may be any that satisfy the description in the above section (Culture Medium). Furthermore, it is preferable that the cryopreservation solution has a solidification onset temperature in the range of -15°C or higher and -5°C or lower.

[0097] Cryoprotectants are substances used to reduce damage to cells caused by freezing and thawing during cryopreservation. Examples of cryoprotectants include cell-impermeant cryoprotectants and cell-permeant cryoprotectants. Specific examples of cell-impermeant cryoprotectants include albumin, sucrose, trehalose, dextran, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and polylysine. Specific examples of cell-permeant cryoprotectants include dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, and propanediol. These cryoprotectants may be formulated alone or in combination of two or more. These cryoprotectants may be appropriately selected from known cryoprotectants depending on the type of cell, the composition of the cryopreservation solution, and the like.

[0098] Examples of commercially available cryopreservation solutions that do not contain DMSO include StemCellBanker (registered trademark) DMSO-free GMP grade (Nihon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) DMSO-free (GC Lymphotec Co., Ltd.), Cryoscarless (registered trademark) DMSO-free (BioVerde Co., Ltd.), StemCellKeep (BioVerde Co., Ltd.), CryoNovo (registered trademark) X12 (Akron BioProducts LCC), CryoNovo (registered trademark) P24 (Akron BioProducts LCC), DMSO-free cell cryopreservation solution for cryopreservation of human ES / iPS cells (ReproCell Co., Ltd.), Cell Reservoir One (Nacalai Tesque Co., Ltd.), ThelioKeep (registered trademark: BioVerde Co., Ltd.), and Cellvation (registered trademark: Protide Co., Ltd.). Examples include ReproCryo RM (ReproCell Pharmaceuticals), and SOFORO Cryo (Saraya). In addition, examples of commercially available cryopreservation solutions containing DMSO include Stem Cell Banker (registered trademark) GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Stem Cell Banker (registered trademark) EX GMP Grade (Nippon Zenyaku Kogyo Co., Ltd.), Bambanker (registered trademark) hRM (GC Lymphotec), Bambanker (registered trademark) (GC Lymphotec), iStock (GC Lymphotec), CryoStor CS5 (Charles River Laboratories Cell Solutions, inc.), and CryoStor CS10 (Charles River Laboratories Cell Solutions, inc.).

[0099] In the cooling step, the cell sheet-attached resin film is preferably cooled in a cryopreservation solution using a non-throughflow cooling device, which allows the cell sheet, the object to be cooled, to be cooled at a uniform temperature, thereby minimizing damage to the cells and suppressing a decrease in cell activity.

[0100] In the cooling step, the cooling rate at 0 to -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. By setting the cooling rate at or above the lower limit, unnecessary contact time between the liquid cryoprotectant and the cells can be reduced. By setting the cooling rate at or below the upper limit, intracellular ice crystal formation can be suppressed.

[0101] In the cooling step, the temperature of the freezing treatment is not particularly limited as long as it can freeze the cultured cells and the cryopreservation solution. The freezing temperature is, for example, −196°C or higher and −25°C or lower. The lower limit is more preferably −180°C or higher, even more preferably −160°C or higher, and particularly preferably −150°C or higher. On the other hand, the upper limit is more preferably −25°C or lower, even more preferably −30°C or lower, and particularly preferably −35°C or lower. The temperature for the freezing treatment is, for example, −196°C or higher and −25°C or lower, −196°C or higher and −30°C or lower, −196°C or higher and −35°C or lower, −180°C or higher and −25°C or lower, −180°C or higher and −30°C or lower, −180°C or higher and −35°C or lower, −160°C or higher and −25°C or lower, −160°C or higher and −30°C or lower, −160°C or higher and −35°C or lower, −150°C or higher and −25°C or lower, −150°C or higher and −30°C or lower, or −150°C or higher and −35°C or lower.

[0102] The cooling device used for the freezing process is not particularly limited, and examples include quick-freezing devices and cryogenic refrigeration devices. As a cooling device, a freezing device that does not come into contact with a heat transfer device and sprays cold air onto the culture vessel from multiple directions, preferably all directions, rather than one direction, to freeze the culture vessel and cells, is preferable from the viewpoint of freezing cells at a uniform temperature and increasing the survival rate of the cells after thawing. Specific examples of freezing devices that spray cold air onto the culture vessel include cooling devices that cool the object to be cooled by circulating cold air using a cooling fan, such as the non-through-flow cooling device equipped with a cooling fan disclosed in Japanese Patent Laid-Open No. 2005-127666. The term "non-through-flow system" refers to a system in which the majority of the through-flow air from the object to be cooled does not pass through (through-flow) the cooler.

[0103] The cryopreservation method preferably includes a cryopreservation step in which the cell sheet-attached resin film is stored after the cooling step, for example, at −196°C to −60°C, preferably −180°C to −65°C, and more preferably −150°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] The cryopreservation method is not particularly limited as long as the cells can be stably cryopreserved. Examples of cryopreservation methods include contact with the liquid or gas phase of a coolant, and use of an ultra-low temperature freezer. A preferred cryopreservation method is contact with the liquid or gas phase of a coolant from the viewpoint of temperature. 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 the cells can be stably cryopreserved. The cryopreservation temperature may be, for example, within any range of −196°C or higher to −60°C or lower, −196°C or higher to −134°C or lower, or −134°C or higher to −60°C or lower. The cryopreservation temperature may be the surface temperature of the cell sheet to be frozen. The surface temperature can be measured, for example, using a K thermocouple.

[0106] The frozen cell sheet-attached resin film contains a resin film and a cell sheet formed on the surface (cell-mounting area) of the resin film. In the cell sheet-attached resin film, the resin film and the cell sheet are in a frozen state.

[0107] In the above-mentioned frozen product, the resin film and cell sheet are frozen at the above-mentioned cryopreservation temperature, specifically, preferably at or below -60°C, more preferably at or below -135°C. The frozen product may also be frozen at a temperature between -134°C and -60°C. The frozen product is frozen at or above -196°C.

[0108] The frozen cell sheet-attached culture carrier substrate frozen inside a culture vessel or cryopreservation container can be packaged in a packaging material either while frozen in the culture vessel or cryopreservation container or after being removed from the container. The sealed packaging prevents microbial contamination of the cell sheet and allows the cell sheet to be stored and distributed without contamination. For example, a frozen product package can be obtained by packaging a cell sheet-attached culture carrier substrate and a container containing a cryopreservation solution in a film-like packaging material and freezing the container in a sealed state. 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, fluororesins such as perfluoroalkoxy fluororesins and tetrafluoroethylene-hexafluoropropylene copolymer (FEP), nylon, cellophane, and paper. These materials may be used alone or in combination of two or more. When sterilization requiring gas permeability, such as EOG sterilization or high-pressure steam sterilization, is performed, packaging materials combining paper or nonwoven fabric materials with the above-mentioned materials are preferably used. The packaging material may be multi-packaged, or may be further packaged after sterilization, and the outermost part of the package is preferably a resin film-like packaging material. This package may also be sterilized. Sterilization methods include electron beam sterilization, gamma ray sterilization, EOG sterilization, high-pressure steam sterilization, etc.

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

[0110] The thawing method is not particularly limited, and conventional means used in technical fields such as medicine, pharmaceuticals, quasi-drugs, cosmetics, food, and veterinary medicine, as well as basic technical fields such as regenerative medicine and bioengineering, can be used. Thawing methods include, for example, using a water bath, bead bath, incubator, hot plate, defroster, etc., immersing in a melting liquid at a temperature higher than the freezing temperature, or leaving in an environment at a temperature higher than the freezing temperature. The environmental temperature in contact with the frozen material during thawing is not particularly limited, as long as it is higher than the freezing temperature and lower than 50°C. The upper limit of the environmental temperature is, for example, 49°C or lower, preferably 45°C or lower, and more preferably 40°C or lower. Meanwhile, the lower limit of the environmental 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 environmental 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. Thawing temperatures of 50°C or higher are not preferred due to the possibility of thermal damage to cells. Furthermore, thawing may be temporarily performed in an environment at a temperature below the freezing point. For example, frozen material stored at -80°C can be exposed to an environmental temperature of -30°C and then thawed at an environmental temperature above the freezing point. The time required to thaw a frozen material is not particularly limited as long as freezing-induced damage to cells does not occur. Typically, thawing can be performed without problems within a time period of more than 10 seconds and less than 60 minutes. The lower limit of the time required to thaw a frozen product is sufficient if it is more than 10 seconds, preferably 20 seconds or more, more preferably 30 seconds or more, and even more preferably 1 minute or more, and the upper limit is sufficient if it is 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 time required for thawing is more than 10 seconds and less than 60 minutes, more than 10 seconds and less than 50 minutes, more than 10 seconds and less than 40 minutes, more than 10 seconds and less than 30 minutes, 20 seconds and less than 60 minutes, 20 seconds and less than 50 minutes, 20 seconds and less than 40 minutes, 20 seconds and less than 30 minutes, 30 seconds and less than 60 minutes, 30 seconds and less than 50 minutes, 30 seconds and less than 40 minutes, 30 seconds and less than 30 minutes, 1 minute and less than 60 minutes, 1 minute and less than 50 minutes, 1 minute and less than 40 minutes, or 1 minute and less than 30 minutes. If thawing occurs too quickly, the frozen material may crack due to thermal shock caused by the temperature difference, potentially damaging the cell sheet. If thawing occurs too slowly, water molecules may recrystallize under sub-zero conditions, causing ice crystals to grow larger and causing serious damage to the cells, which is undesirable. During the thawing time, the environmental temperature may be set to a constant temperature or may be set to fluctuate, such as by gradually increasing.

[0111] The thawing solution is not particularly limited as long as it does not damage the cultured cells. Examples of components contained in the thawing solution include sucrose, glucose, maltose, trehalose, and fructose. The thawing solution may also contain the components described above in the section (Culture Medium).

[0112] The temperature of the melting liquid is not particularly limited as long as it is higher than the freezing temperature. The temperature of the melting liquid is, for example, 0°C or higher and 45°C or lower. The lower limit is more preferably 4°C or higher, even more preferably 25°C or higher, and particularly preferably 28°C or higher. On the other hand, the upper limit is more preferably 40°C or lower, even more preferably 39°C or lower, and particularly preferably 38°C or lower. The temperature of the melting liquid is, for example, 0°C or higher and 45°C or lower, 0°C or higher and 40°C or lower, 0°C or higher and 39°C or lower, 0°C or higher and 38°C or lower, 4°C or higher and 45°C or lower, 4°C or higher and 40°C or lower, 4°C or higher and 39°C or lower, 4°C or higher and 38°C or lower, 25°C or higher and 45°C or lower, 25°C or higher and 40°C or lower, 25°C or higher and 39°C or lower, 25°C or higher and 38°C or lower, 28°C or higher and 45°C or lower, 28°C or higher and 40°C or lower, 28°C or higher and 39°C or lower, and 28°C or higher and 38°C or lower.

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

[0114] In the present disclosure, since the cell-mounting area does not contain a temperature-responsive polymer, it is possible to prevent the cell sheet from peeling off from the resin film in a low-temperature environment such as during the cryopreservation process, and it is possible to obtain a cell-sheet-attached resin film that maintains the adhesiveness between the cell sheet and the film even after undergoing the cryopreservation process and the thawing process.

[0115] <Transplantation> The transplantation method of this embodiment includes a transplantation step of attaching a cell sheet formed in a cell-mounting region of a resin film to a transplantation site, and then peeling off the resin film from the cell sheet.

[0116] The transplantation site may be at least a portion of the body of a recipient, such as a mammal, bird, amphibian, fish, insect, plant, or microorganism. 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. However, the transplantation site may exclude the interior and exterior of the human body. Examples of transplantation sites other than the recipient include other cell sheets, medical devices, and tissues or organs isolated from the recipient. Examples of tissues or organs isolated from the recipient include skin, oral tissue, esophagus, trachea, bronchi, lungs, lung lobes, stomach, duodenum, pancreas, spleen, small intestine, large intestine, muscle tissue, and bone (excluding tissues that are to be returned to the same recipient for treatment).

[0117] Cell transplantation therapy suppresses or prevents the onset and recurrence of symptoms associated with cell, tissue, or organ loss, dysfunction, or dysfunction. Examples of diseases that can be treated with cell transplantation therapy include spinal cord injury, knee articular cartilage injury, ischemic heart disease, age-related macular degeneration, corneal epithelial stem cell deficiency, aplastic anemia, severe limb ischemia, refractory skin ulcers, and the prevention of postoperative complications (e.g., various organ suture failures, bronchial stump fistulas, pancreatic fistulas, and bile leaks), burns, and the like. Furthermore, cells used in cell transplantation therapy may be autologous, allogeneic non-autologous, or xenogeneic. Autologous cells are preferred from the standpoints of clinical application and safety, while non-autologous cells are preferred from the standpoints of clinical application and productivity.

[0118] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations can be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications, improvements, etc., within the scope of achieving the object of the present invention, are included in the present invention. Examples of reference embodiments are listed below. 1. A resin film including a resin layer having a first surface including an area for placing cultured cells, the resin film including an X-ray contrast material, and an X-ray contrast material-free area in the first surface that does not include the X-ray contrast material, the area for placing cultured cells being provided in at least a portion of the X-ray contrast material-free area. 2. The resin film described in 1., wherein the surface of the area for placing cultured cells has a water droplet contact angle θ measured by the θ / 2 method of 70° or less. 3. The resin film described in 1. or 2., wherein an X-ray contrast layer including an X-ray contrast material is formed on the surface of the resin layer. 4. 3. 1. The resin film according to claim 1, wherein the X-ray contrast layer is formed on a second surface of the resin layer opposite to the first surface. 5. The resin film according to any one of claims 1 to 4, wherein the X-ray contrast material is contained inside the resin layer. 6. The resin film according to any one of claims 1 to 5, further comprising a laminate layer laminated on the surface of the resin layer. 7. The resin film according to claim 6, wherein the laminate layer is laminated on the second surface of the resin layer opposite to the first surface. 8. The resin film according to claim 6 or 7, wherein the laminate layer comprises the X-ray contrast material. 9. The resin film according to any one of claims 1 to 8, further comprising a protective layer that protects at least a portion of the surface. 10. The resin film according to any one of claims 1 to 9, wherein the thickness of the resin film is 5 to 250 μm. 11. The resin film according to claim 1 to 10. 10. The resin film according to any one of claims 1 to 9, wherein the X-ray contrast material comprises a metal or a metal compound.12. The resin film according to any one of 1. to 11., wherein the 60-degree specular gloss Gs(60°) of the surface opposite to the first surface, measured in accordance with JIS Z8741:1997, is 700 or less. 13. The resin film according to any one of 1. to 12., wherein the X-ray contrast material comprises an X-ray contrast filler. 14. The resin film according to any one of 1. to 13., wherein the resin layer comprises polyether ether ketone (PEEK). 15. The resin film according to any one of 1. to 14., wherein the first surface is a scaffold for culturing cells.

[0119] This application claims priority based on Japanese Patent Application No. 2024-009966, filed January 26, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0120] REFERENCE SIGNS LIST 11 Resin film 12 Resin layer 13 X-ray contrast layer 14 Surface 15 Surface 16 Region without X-ray contrast material 21 Resin film 22 Resin layer 23 X-ray contrast layer 24 Surface 25 Surface 26 Region without X-ray contrast material 31 Resin film 32 Resin layer 34 Surface 36 Region without X-ray contrast material 37 X-ray contrast filler 41 Resin film 42 Resin layer 44 Surface 45 Surface 46 Region without X-ray contrast material 47 Laminate layer containing X-ray contrast material 51 Resin film 52 Resin layer 53 X-ray contrast layer 54 Surface 55 Surface 56 Region without X-ray contrast material 57 Laminate layer 57 Laminate layer 61 Resin film 62 Resin layer 63 X-ray contrast layer 64 Surface 65 Surface 66 Nylon 66 N 66 X-ray contrast material-free region 67 Protective layer 100 Patient 101 Patient 102 Resin film 111 Patient 112 Resin film

Claims

1. A resin film comprising a resin layer having a first surface including a region for placing cultured cells, and a layer containing an X-ray contrast material present on the first surface of the resin layer, on a second surface opposite to the first surface, and at least partially within the resin layer, wherein the region for placing the cultured cells is present in at least a part of the X-ray contrast material non-existence region on the first surface where the X-ray contrast material is not surface-exposed.

2. The resin film according to claim 1, wherein the layer containing the X-ray contrast material is an X-ray contrast layer including a pattern of the X-ray contrast material.

3. The resin film according to claim 1, wherein the layer containing the X-ray contrast material is an X-ray contrast resin layer including a plurality of filler-like X-ray contrast materials and a resin material.

4. The resin film according to any one of claims 1 to 3, wherein the thickness of the resin film is 5 μm or more and 250 μm or less.

5. The resin film according to any one of claims 1 to 3, wherein the resin layer includes one or more selected from the group consisting of polyetheretherketone, polyethylene terephthalate, and polystyrene.

6. The resin film according to any one of claims 1 to 3, wherein the X-ray contrast material includes an X-ray absorption contrast material.

7. The resin film according to any one of claims 1 to 3, wherein the X-ray contrast material includes a metal or a metal compound.

8. The resin film according to any one of claims 1 to 3, wherein when viewed from the normal direction of the first surface, the layer containing the X-ray contrast material is present so as to overlap only the whole or a part of the resin film.

9. The resin film according to claim 2, further including a protective layer that protects at least a part of the surface of the X-ray contrast layer present on the first surface of the resin layer and / or on the second surface opposite to the first surface.

10. The resin film according to any one of claims 1 to 3, wherein the first surface of the region for placing the cultured cells is subjected to plasma treatment.

11. The resin film according to any one of claims 1 to 3, wherein the water droplet contact angle θ measured by the θ / 2 method on the first surface of the region on which the cultured cells are placed is 70° or less.

12. The resin film according to any one of claims 1 to 3, wherein the 60-degree specular glossiness Gs(60°) measured in accordance with the provisions of JIS Z8741:1997 on the outermost surface on the opposite side of the first surface is 700 or less.

13. The resin film according to any one of claims 1 to 3, which is used for culturing cells and / or transporting cells.

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