Adherent sheet-like material for application to the surface of organs

Abstract

The objective of the present invention is to provide a bioadhesive sheet-like material to be used being adhered to an organ surface, a method for manufacturing the same, and a method for treating a disease using the same. The present invention provides a bioadhesive sheet-like material including an extracellular matrix layer, a sheet-like cell culture, and a biodegradable gel layer. The bioadhesive sheet-like material has the extracellular matrix layer on one surface thereof, and the biodegradable gel layer on the other surface thereof via the sheet-like cell culture. Providing, inter alia, the bioadhesive sheet-like material to be used with the extracellular matrix layer adhered to an organ surface solves the above problem.

Description

[Technical Field]

[0001] The present invention relates to an engraftment sheet-like material comprising an extracellular matrix layer, a sheet-shaped cell culture, and a biodegradable gel layer, the extracellular matrix layer being on one side and the biodegradable gel layer being on the other side, with the sheet-shaped cell culture sandwiched between them, for use by attaching the extracellular matrix layer to the surface of an organ; a method for producing the engraftment sheet-like material; and a method for treating a disease using the engraftment sheet-like material. [Background technology]

[0002] In recent years, attempts have been made to transplant various cells to repair damaged tissues, etc. For example, attempts have been made to use fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction (Non-Patent Document 1).

[0003] As an example of such an attempt, sheet-shaped cell cultures in which cells are molded into a sheet have been developed, some of which have reached the stage of clinical application. Examples of such applications include repair of dysfunctional myocardium by transplantation of a patient's own myocardial sheet (Non-Patent Document 2), application of a cell sheet containing hepatocytes induced from mesenchymal stem cells to the surface of the liver for the purpose of suppressing liver damage (Patent Document 1), reduction of liver fibrosis by transplantation of a mesenchymal stem cell sheet engineered with IC-2 (Non-Patent Document 3), and a cell sheet composition that is applied to a wound in a hollow organ to heal or prevent leakage from the wound in the hollow organ (Patent Document 1).

[0004] Furthermore, sheet-shaped cell cultures are fragile, making them difficult to manipulate for clinical applications. For this reason, for example, sheet-shaped cell cultures that have been improved to adhere well to tissues (Patent Document 3) and laminates of fibrin gel and sheet-shaped cell cultures (Patent Document 4) have been proposed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6008297 [Patent Document 2] International Publication No. 2017 / 130802 [Patent Document 3] International Publication No. 2006 / 093153 [Patent Document 4] Patent No. 6495603 [Non-patent literature]

[0006] [Non-Patent Document 1] Haraguchi et al., Stem Cells Trans1 Med. 2012 Feb;1(2):136-41 [Non-patent document 2] Imran A. Memon et al., The Journal of Thoracic and Cardiovascular Surgery, Volume 130, Number 5, 1333-1341 [Non-patent document 3] Noriko Itaba et al., SCIENTIFIC REPORT (2019) 9:6841 Summary of the Invention [Problem to be solved by the invention]

[0007] Recognizing that the development of new sheet-shaped cell cultures with greater operability is required to further popularize regenerative medicine, the present invention aims to provide an engraftment sheet-shaped material that has good adhesion to organ surfaces and good operability in clinical applications, a method for producing the same, and a method for treating diseases using the engraftment sheet-shaped material. [Means for solving the problem]

[0008] During intensive research into sheet-shaped cell cultures that can be successfully engrafted in various tissues, the inventors discovered for the first time that a myoblast cell sheet having an extracellular matrix layer on one side and a biodegradable gel layer on the other side can be successfully engrafted on the surface of the liver or large intestine by attaching the extracellular matrix layer side to the organ. Based on this finding, they continued their research and completed the present invention.

[0009] That is, the present invention relates to the following. [1] An engraftment sheet-like material comprising an extracellular matrix layer, a sheet-shaped cell culture, and a biodegradable gel layer, the sheet-shaped material having the extracellular matrix layer on one side and the biodegradable gel layer on the other side, with the sheet-shaped cell culture sandwiched between them, for use by attaching the extracellular matrix layer to the surface of an organ. [2] The engraftment sheet-like material according to [1], wherein the organ is a human organ. [3] The engraftment sheet-like material according to [1] or [2], wherein the attachment to the surface of the organ is performed under laparoscopy or thoracoscopy. [4] The engraftment sheet-shaped material according to any one of [1] to [3], wherein the sheet-shaped cell culture contains somatic cells. [5] The engraftment sheet-like material according to [4], wherein the somatic cells are skeletal myoblasts or mesenchymal stem cells. [6] The engraftment sheet-like material according to any one of [1] to [5], wherein the organ is an internal organ that can be accessed from outside the body using a laparoscope or a thoracoscope. [7] The engraftment sheet-like material according to any one of [1] to [6], further comprising a medicinal ingredient.

[0010] [8] A method for producing the engraftment sheet-like material according to any one of [1] to [7], seeding cells onto a substrate; forming the seeded cells into a sheet; and Step of peeling off the formed sheet-shaped cell culture from the substrate The method comprising: [9] A method for treating a disease that can be improved by application of the engraftment sheet-like material according to any one of [1] to [7], the method comprising applying an effective amount of the engraftment sheet-like material to an organ of a subject in need thereof, with the surface of the engraftment sheet-like material having an extracellular matrix layer.

[10] The method according to [9], wherein the application to the target organ is performed under laparoscopy or thoracoscope.

[11] Applying it to the target organ The method according to [9] or

[10] , which is carried out using a device comprising a support for supporting the engrafted sheet-like material and a protective member with a low coefficient of friction for protecting one side of the support, and which can protect the support supporting the engrafted sheet-like material with the protective member, roll it up, and insert it into a cylindrical body. [Effects of the Invention]

[0011] By using the engraftment sheet-like material of the present invention, good engraftment can be achieved when the engraftment sheet-like material containing a sheet-shaped cell culture is applied to a target organ. Furthermore, the engraftment sheet-like material of the present invention can be applied to a target organ more easily. Furthermore, the engraftment sheet-like material of the present invention can be applied to a target organ using a delivery device, and can be applied to a target organ using a method that is less invasive to the subject, such as a laparoscope. Furthermore, the organ can be maintained in good condition after the engraftment sheet-like material is applied. [Brief explanation of the drawings]

[0012] [Figure 1] Figures 1A and 1B show hematoxylin-eosin (HE) stained images of tissue obtained from the stomach to which the engraftment sheet-like material was applied. The area enclosed by the dashed circle indicates the applied engraftment sheet-like material. The area enclosed by the straight circle indicates the area that is histologically continuous. Figure 1B is a further enlargement of a portion of Figure 1A. [Figure 2AB]Figures 2A and 2B show hematoxylin-eosin stained images of tissue obtained from the colon to which the engraftment sheet was attached. Figure 2B is a further enlargement of a portion of Figure 2A. [Figure 2C-D] Figures 2C and 2D show histological staining images of tissue obtained from the large intestine to which the engraftment sheet-like material was attached, immunostained with an anti-desmin antibody. Figure 2D is a further enlargement of a portion of Figure 2C. [Figure 3AB] Figures 3A and 3B show images of AZAN stained tissue obtained from a liver to which an engraftment sheet was attached. Figure 3B is a further enlargement of a portion of Figure 3A. The arrows in the image indicate areas that clearly show histological continuity. [Figure 3CD] Figures 3C and 3D show histological staining images of tissue obtained from a liver to which an engraftment sheet-like material had been applied, immunostained using an anti-desmin antibody. Figures 3C and 3D are from different fields of view within the same slide. The arrows in the figures indicate areas that clearly demonstrate histological continuity. The engraftment sheet-like material was applied to the side marked "Sheet" in the figures. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to an engraftment sheet-like material comprising an extracellular matrix layer, a sheet-shaped cell culture, and a biodegradable gel layer, the sheet-shaped cell culture having the extracellular matrix layer on one side and the biodegradable gel layer on the other side, with the sheet-shaped cell culture sandwiched between them, for use by attaching the extracellular matrix layer to the surface of an organ. In the present invention, the engraftment sheet-like material refers to a sheet-shaped material that is attached to a subject with good engraftment. The engraftment sheet-like material of the present invention comprises an extracellular matrix layer, a sheet-shaped cell culture, and a biodegradable gel layer, and has the extracellular matrix layer on one side of the sheet-shaped cell culture contained in the engraftment sheet-like material and the biodegradable gel layer on the other side, via the sheet-shaped cell culture. In the present invention, good engraftment refers, for example, to histological continuity between the engraftment sheet-like material and the organ to which it is attached. Histological continuity between the engraftment sheet-like material and the organ refers to continuous, gap-free contact between the engraftment sheet-like material and the organ. Here, "no gaps" refers to at least partial contact between the engraftment sheet-like material and the organ without any gaps between them. For example, the engraftment sheet-like material and the organ may be sutured to eliminate gaps between them. If gaps exist between the engraftment sheet-like material and the organ, other cells may infiltrate the gaps, reducing the effects of the sheet-shaped cell culture in the engraftment sheet-like material, such as preventing perforation, preventing fluid leakage from the organ, or treating fibrosis. The engraftment sheet-like material of the present invention has an extracellular matrix layer (also referred to as ECM) attached to the surface of the organ, so an extracellular matrix layer exists between the sheet-shaped cell culture in the engraftment sheet-like material and the organ. In areas where the engraftment sheet-like material is in contact with the organ, it is thought that cells constituting the engraftment sheet-like material will migrate into the organ, and conversely, cells constituting the organ will infiltrate the engraftment sheet-like material, with the extracellular matrix as a target for cell migration and infiltration.

[0014] In the present invention, the term "extracellular matrix layer" refers to a layer of extracellular matrix that contributes to the adhesion of the engraftment sheet-like material to the target organ and improves the engraftment rate of the engraftment sheet-like material at the application site when the engraftment sheet-like material is applied. Furthermore, because the extracellular matrix is ​​a cell-derived component, there is no problem in applying the engraftment sheet-like material of the present invention, including the extracellular matrix layer, to the target organ. In the present invention, the thickness of the extracellular matrix layer is 1 nm to 100 nm, and preferably 1 nm to 50 nm.

[0015] In the present invention, the term "sheet-shaped cell culture" refers to a sheet-like structure in which cells are interconnected. The cells may be interconnected directly (including via cellular elements such as adhesion molecules) and / or via an intervening substance. The intervening substance is not particularly limited as long as it can at least physically (mechanically) connect the cells, and examples thereof include extracellular matrix. The intervening substance is preferably derived from cells, particularly from the cells that constitute the cell culture. The cells are at least physically (mechanically) connected, but may also be functionally connected, for example, chemically or electrically. The sheet-shaped cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminated (multilayered) structure, e.g., two, three, four, five, six, etc.). The sheet-shaped cell culture may also have a three-dimensional structure with a thickness exceeding that of a single cell, without the cells exhibiting a distinct layer structure. For example, in a vertical cross section of a sheet-shaped cell culture, cells may not be uniformly aligned in the horizontal direction, but may be arranged unevenly (eg, in a mosaic pattern).

[0016] The sheet-shaped cell culture preferably does not contain a scaffold (support). Scaffolds are sometimes used in the art to attach cells to their surface and / or inside and maintain the physical integrity of the sheet-shaped cell culture; for example, membranes made of polyvinylidene difluoride (PVDF) are known. However, the sheet-shaped cell culture contained in the engraftment sheet-shaped material of the present invention can maintain its physical integrity even without such a scaffold. Furthermore, the sheet-shaped cell culture contained in the engraftment sheet-shaped material of the present invention preferably consists only of substances derived from the cells that constitute the sheet-shaped cell culture, and does not contain any other substances.

[0017] In the present invention, the thickness of the sheet-shaped cell culture is not particularly limited. When a monolayer sheet is used as the sheet-shaped cell culture, the thickness is typically at least the thickness of one cell. The thickness varies depending on the type of cells forming the sheet-shaped cell culture. In one embodiment, the sheet-shaped cell culture of the present invention has a thickness of 30 μm or more, and in a preferred embodiment, a thickness of 50 μm or more. The thickness range of the sheet-shaped cell culture of the present invention is, for example, 30 μm to 200 μm, preferably 50 μm to 150 μm, and more preferably 60 μm to 100 μm. When a stacked sheet is used as the sheet-shaped cell culture, the thickness does not exceed the product of the thickness of the monolayer sheet multiplied by the number of stacked sheets. Therefore, in one embodiment, when a sheet consisting of five stacked monolayer sheets is used, the thickness is 150 μm or more, and in a preferred embodiment, 250 μm or more. In this case, the range of the thickness of the sheet-shaped cellular material is, for example, 150 μm to 1000 μm, preferably 250 μm to 750 μm, and more preferably 300 μm to 500 μm.

[0018] The cells contained in the engraftment sheet-like material can be derived from any organism to which the engraftment sheet-like material is to be attached, including, but not limited to, humans, primates, dogs, cats, pigs, horses, goats, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. The engraftment sheet-like material may contain only one type of cell, but two or more types of cells can also be used. In a preferred embodiment of the present invention, when the engraftment sheet-like material contains two or more types of cells, the content (purity) of the most abundant cell is 60% or more, preferably 70% or more, and more preferably 75% or more, at the end of the engraftment sheet-like material production.

[0019] Cells may be xenogeneic or allogeneic. Here, "xenogeneic cells" refers to cells derived from an organism of a different species from the recipient to which the engraftment sheet-like material is attached. For example, if the recipient is human, cells derived from monkeys or pigs are xenogeneic cells. Furthermore, "allogeneic cells" refers to cells derived from an organism of the same species as the recipient. For example, if the recipient is human, human cells are allogeneic cells. Allogeneic cells include autologous cells (also called autologous cells or autogenous cells), i.e., cells derived from the recipient, and allogeneic non-autologous cells (also called allogeneic cells). Autologous cells are preferred in the present invention because they do not induce rejection after transplantation. However, xenogeneic or allogeneic non-autologous cells can also be used. When xenogeneic or allogeneic non-autologous cells are used, immunosuppressive treatment may be required to suppress rejection. Note that, throughout this specification, cells other than autologous cells, i.e., xenogeneic and allogeneic non-autologous cells, are sometimes collectively referred to as non-autologous cells. In one embodiment of the invention, the cells are autologous or allogeneic. In one embodiment of the invention, the cells are autologous. In another embodiment of the invention, the cells are allogeneic.

[0020] The sheet-shaped cell culture contained in the engraftment sheet-shaped material of the present invention can be produced by any method known to those skilled in the art (see, for example, Patent Document 1, Japanese Patent Application Publication No. 2010-081829, Japanese Patent Application Publication No. 2011-110368, etc.). The method for producing a sheet-shaped cell culture typically includes, but is not limited to, the steps of seeding cells on a substrate, forming the seeded cells into a sheet, and detaching the formed sheet-shaped cell culture from the substrate. Cell freezing and thawing may be performed before the step of seeding the cells on the substrate. Furthermore, a cell washing step may be performed after the step of thawing the cells. Each of these steps can be performed by any known method suitable for producing a sheet-shaped cell culture. The step of producing a sheet-shaped cell culture may include, as substeps, one or more of the steps of the method for producing a sheet-shaped cell culture described above. In one embodiment, the step of growing cells after the step of thawing the cells and before the step of seeding the cells on the substrate is not included.

[0021] The substrate is not particularly limited as long as it allows cells to form cell cultures thereon, and includes, for example, containers made of various materials, solid or semi-solid surfaces within the container, and the like. The container is preferably made of a structure or material that is impermeable to liquids such as culture medium. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicone, polystyrene, glass, polyacrylamide, polydimethylacrylamide, and metals (e.g., iron, stainless steel, aluminum, copper, and brass). Furthermore, the container preferably has at least one flat surface. Examples of such containers include, but are not limited to, culture vessels with a bottom made of a substrate that allows cell cultures to be formed and liquid-impermeable sides. Specific examples of such culture vessels include, but are not limited to, cell culture dishes and cell culture bottles. The bottom of the container may be transparent or opaque. A transparent container bottom allows cells to be observed, counted, and the like from the back of the container. The container may also have a solid or semi-solid surface inside. Examples of solid surfaces include plates and containers made of the various materials described above, while examples of semi-solid surfaces include gels and soft polymer matrices. The substrate may be made using the materials described above, or commercially available substrates may be used. Preferred substrates include, but are not limited to, substrates with adhesive surfaces suitable for forming sheet-shaped cell cultures. Specific examples include substrates with hydrophilic surfaces, such as corona-discharge-treated polystyrene, substrates coated with hydrophilic compounds such as collagen gel or hydrophilic polymers, and substrates coated with extracellular matrices such as collagen, fibronectin, laminin, vitronectin, proteoglycans, and glycosaminoglycans, or cell adhesion factors such as members of the cadherin family, selectin family, and integrin family. Such substrates are commercially available (e.g., Corning® TC-Treated Culture Dish, Corning). The substrate may be entirely or partially transparent or opaque.

[0022] The surface of the substrate may be coated with a material whose physical properties change in response to a stimulus, for example, temperature or light. Examples of such materials include, but are not limited to, (meth)acrylamide compounds, N-alkyl-substituted (meth)acrylamide derivatives (e.g., N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide, etc.), N,N-dialkyl-substituted (meth)acrylamide derivatives (e.g., N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide, etc.), (meth)acrylamide derivatives having a cyclic group (e.g., 1-(1- Examples of materials that can be used include known temperature-responsive materials made of homopolymers or copolymers of 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine, or vinyl ether derivatives (e.g., methyl vinyl ether); light-absorbing polymers having azobenzene groups; copolymers of vinyl derivatives of triphenylmethane leucohydroxide and acrylamide monomers; and photoresponsive materials such as N-isopropylacrylamide gels containing spirobenzopyran (see, for example, Japanese Patent Application Laid-Open Nos. 2-211865 and 2003-33177). By applying a specific stimulus to these materials, their physical properties, such as hydrophilicity or hydrophobicity, can be changed, thereby facilitating the detachment of cell cultures attached to the materials. Culture dishes coated with temperature-responsive materials are commercially available (e.g., UpCell (registered trademark) from CellSeed Inc. and Cepallet (registered trademark) from DIC Corporation), and these can be used in the manufacturing method of the present disclosure.

[0023] The substrate may have various shapes, but is preferably flat. The area of ​​the substrate is not particularly limited, but may be, for example, about 1 cm. 2 ~about 200cm 2 , about 2 cm 2 ~Approx. 100cm 2 , about 3cm 2 ~about 50cm 2 For example, the substrate may be a circular culture dish with a diameter of 10 cm. In this case, the area is 56.7 cm. 2 This becomes: The substrate may be coated with serum. By using a serum-coated substrate, a sheet-shaped cell culture can be formed at a higher density. "Coated with serum" refers to a state in which serum components are attached to the surface of the substrate. This state can be obtained, for example, by treating the substrate with serum, without being limited thereto. Treatment with serum involves contacting the substrate with serum and, if necessary, incubating for a predetermined period of time.

[0024] As the serum, xenoserum and / or allogeneic serum can be used. When an engraftment sheet-like material is used, xenoserum refers to serum derived from an organism of a different species from the recipient. For example, when the recipient is human, serum derived from cows or horses, such as fetal bovine serum (FBS, FCS), calf serum (CS), and horse serum (HS), corresponds to xenoserum. Furthermore, "allogeneic serum" refers to serum derived from an organism of the same species as the recipient. For example, when the recipient is human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum (also called autologous serum), i.e., serum derived from the recipient, and allogeneic serum derived from an individual of the same species other than the recipient. Note that, in this specification, serum other than autologous serum, i.e., xenoserum and allogeneic serum, are sometimes collectively referred to as non-autologous serum. Serum for coating the substrate is commercially available or can be prepared by a standard method from blood collected from a desired organism. Specifically, for example, collected blood is left at room temperature for about 20 to about 60 minutes to clot, and then centrifuged at about 1000 × g to about 1200 × g to collect the supernatant.

[0025] When incubating on a substrate, serum may be used undiluted or diluted. Dilution can be performed with any medium, including, but not limited to, water, physiological saline, various buffer solutions (e.g., PBS, HBSS, etc.), various liquid media (e.g., DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.). The dilution concentration is not particularly limited as long as it allows serum components to adhere to the substrate, and is, for example, about 0.5% to about 100% (v / v), preferably about 1% to about 60% (v / v), and more preferably about 5% to about 40% (v / v).

[0026] The incubation time is not particularly limited as long as the serum components can be attached to the substrate, and is, for example, about 1 hour to about 72 hours, preferably about 2 hours to about 48 hours, more preferably about 2 hours to about 24 hours, and even more preferably about 2 hours to about 12 hours. The incubation temperature is also not particularly limited as long as the serum components can be attached to the substrate, and is, for example, about 0°C to about 60°C, preferably about 4°C to about 45°C, and more preferably room temperature to about 40°C.

[0027] After incubation, the serum may be discarded. Conventional liquid disposal techniques, such as aspiration using a pipette or decantation, can be used to discard the serum. In a preferred embodiment of the present disclosure, after discarding the serum, the substrate may be washed with a serum-free washing solution. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components attached to the substrate. Examples of the serum-free washing solution include, but are not limited to, water, physiological saline, various buffer solutions (e.g., PBS, HBSS, etc.), and various liquid media (e.g., DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, and RITC80-7). Conventional substrate washing techniques can be used, such as, but not limited to, adding a serum-free washing solution to the substrate, stirring for a predetermined time (e.g., about 5 to about 60 seconds), and then discarding the solution.

[0028] In the present invention, the substrate may be coated with a growth factor. Here, "growth factor" refers to any substance that promotes cell proliferation compared to the absence of the growth factor, and includes, for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), etc. The methods for coating, discarding, and washing the substrate with the growth factor are essentially the same as those for serum, except that the dilution concentration during incubation is, for example, about 0.0001 μg / mL to about 1 μg / mL, preferably about 0.0005 μg / mL to about 0.05 μg / mL, and more preferably about 0.001 μg / mL to about 0.01 μg / mL.

[0029] In the present invention, the substrate may be coated with a steroid. Here, "steroid" refers to a compound having a steroid nucleus that can adversely affect the living body, such as causing adrenocortical insufficiency or Cushing's syndrome. Examples of such compounds include, but are not limited to, cortisol, prednisolone, triamcinolone, dexamethasone, and betamethasone. The methods for coating, disposing of, and washing the substrate with the steroid are essentially the same as those for serum, except that the dilution concentration during incubation is, for example, about 0.1 μg / mL to about 100 μg / mL, preferably about 0.4 μg / mL to about 40 μg / mL, and more preferably about 1 μg / mL to about 10 μg / mL, as dexamethasone.

[0030] The substrate may be coated with serum, growth factors, and steroids, or any combination thereof, i.e., serum and growth factors, serum and steroids, serum, growth factors, and steroids, or growth factors and steroids. When coated with multiple components, these components may be mixed and coated simultaneously or in separate steps.

[0031] The substrate may be coated with serum or the like and then immediately seeded with cells, or may be stored after coating and then seeded with cells. The coated substrate can be stored for a long period of time by, for example, keeping it at about 4°C or below, preferably about -20°C or below, and more preferably about -80°C or below.

[0032] Cells can be seeded onto the substrate using any known method and conditions. For example, cells can be seeded onto the substrate by injecting a cell suspension prepared by suspending cells in a culture medium into the substrate (culture vessel). The cell suspension can be injected using a tool suitable for injecting a cell suspension, such as a dropper or pipette.

[0033] In one embodiment, the seeding is about 7.1 x 10 5 pieces / cm 2 ~Approx. 3.0×10 6 pieces / cm 2, about 7.3×10 5 pieces / cm 2 ~Approx. 2.8×10 6 pieces / cm 2 , about 7.5×10 5 pieces / cm 2 ~Approx. 2.5×10 6 pieces / cm 2 , about 7.8×10 5 pieces / cm 2 ~Approx. 2.3×10 6 pieces / cm 2 , about 8.0×10 5 pieces / cm 2 ~Approx. 2.0×10 6 pieces / cm 2 , about 8.5×10 5 pieces / cm 2 ~Approx. 1.8×10 6 pieces / cm 2 , about 9.0×10 5 pieces / cm 2 ~Approx. 1.6×10 6 pieces / cm 2 , about 1.0×10 6 pieces / cm 2 ~Approx. 1.6×10 6 pieces / cm 2 It can be done at a density of

[0034] In the present invention, the term "biodegradable gel layer" refers to a layer of biodegradable gel, which is a gel that decomposes in the body, is absorbed into the body, metabolized, and excreted. Biodegradable gels include, but are not limited to, fibrin gel and gels formed by Adspray (registered trademark) (manufactured by Terumo Corporation). The biodegradable gel preferably refers to a gel that becomes viscous by mixing two types of liquid. For example, fibrin gel is a highly strong gel that is formed by mixing a liquid containing fibrinogen (hereinafter referred to as fibrinogen solution) with a liquid containing thrombin (hereinafter referred to as thrombin solution), and the thrombin acts on the fibrinogen. Furthermore, since the biodegradable gel can be degraded in vivo, the engraftment sheet-like product of the present invention can be applied to a target organ together with the biodegradable gel layer contained therein. In the present invention, the thickness of the biodegradable gel layer is 5 μm to 1300 μm, preferably 50 μm to 300 μm.

[0035] In one embodiment, the biodegradable gel layer has a uniform thickness. Here, "uniform" means that the difference in thickness between the thickest and thinnest parts is 20% or less. "Uniform" preferably means that the difference in thickness is 10% or less, and more preferably 5% or less.

[0036] The engraftment sheet-like material of the present invention is used by attaching the extracellular matrix layer contained in the engraftment sheet-like material to the surface of an organ. The organ to which the engraftment sheet-like material of the present invention is attached may be an organ of any individual organism, but is preferably an animal organ, more preferably a mammalian organ, and even more preferably a human organ. In the present invention, an organ refers to an organ located within a body cavity and having a specific form and function, such as a digestive system (e.g., liver, gallbladder, large intestine, stomach, duodenum), a circulatory system (e.g., heart, spleen), a respiratory system (e.g., lungs), a urinary system (e.g., kidneys), a reproductive system (e.g., prostate, uterus, ovaries), and an endocrine system (e.g., adrenal glands). In order for a sheet-shaped cell culture to successfully engraft on an organ and maintain the organ in good condition, it is believed that the sheet-shaped cell culture must be supplied with oxygen and nutrients. Since the engraftment sheet-like material of the present invention contains an extracellular matrix layer, it is believed that it releases greater amounts of various cytokines, such as VEGF and HGF, which have angiogenic effects.

[0037] In one embodiment, the engrafted sheet-like material of the present invention is applied to the surface of an organ via laparoscopy or thoracoscopy. Here, laparoscopic or thoracoscopic application refers to the delivery of the engrafted sheet-like material to the vicinity of the target organ through an instrument passageway (e.g., a laparoscopic port, a thoracoscopic port, etc.) prepared for laparoscopic or thoracoscopic surgery in the subject, and then application to the surface of the organ. When applying the sheet-like material via laparoscopy or thoracoscopy, the engrafted sheet-like material may be curved using forceps or the like to pass through the instrument passageway, unfolded near the organ, and then applied to the organ. Furthermore, a delivery device or the like may be used to pass the sheet-like material through the instrument passageway in this manner. The delivery device is not limited to, but examples of usable delivery devices include therapeutic substance delivery and administration devices (see JP 2009-000511 A). Therefore, in one embodiment, the organ to which the engraftment sheet-like material of the present invention is applied is an organ that can be the subject of laparoscopic or thoracoscopic surgery, i.e., an internal organ that can be accessed from outside the body using a laparoscope or thoracoscope, and examples thereof include, but are not limited to, the liver, gallbladder, spleen, large intestine, stomach, duodenum, prostate, kidneys, adrenal glands, uterus, ovaries, and heart. Because the engraftment sheet-like material of the present invention may adhere to the abdominal wall in the small intestine, the organ to which the engraftment sheet-like material of the present invention is applied is preferably an organ selected from the group consisting of the stomach, large intestine, duodenum, and liver.

[0038] The cells contained in the engraftment sheet-shaped material of the present invention are not particularly limited as long as they are capable of forming a sheet-shaped cell culture and producing an extracellular matrix, and include, for example, adhesive cells (adherent cells). Adherent cells include, for example, adhesive somatic cells (e.g., cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc.) and stem cells (e.g., myoblasts, tissue stem cells such as cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.). Somatic cells may also be differentiated from stem cells, particularly iPS cells (iPS cell-derived adhesive cells). Non-limiting examples of cells that may be contained in the cell culture of the present invention include myoblasts (e.g., skeletal myoblasts, etc.), mesenchymal stem cells (e.g., those derived from bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue, endometrium, placenta, umbilical cord blood, etc.), cardiomyocytes, mesothelial cells, stromal cells (e.g., fibroblasts, adipose-derived cells), alveolar tissue cells, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (e.g., small intestinal epithelial cells, oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (e.g., vascular endothelial cells, etc.), hepatocytes (e.g., hepatic parenchymal cells, hepatic progenitor cells, etc.), pancreatic cells (e.g., pancreatic islet cells, pancreatic progenitor cells, etc.), kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, immune cells (T cells, NK cells), skin cells (e.g., epidermal keratinocytes), and the like. Here, iPS cells are cells induced by gene introduction that have pluripotency and self-renewal capabilities. Non-limiting examples of iPS cell-derived adhesive cells include iPS cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, and chondrocytes. The cells contained in the engraftment sheet-like material of the present invention are preferably mesenchymal stem cells or skeletal myoblasts, and more preferably skeletal myoblasts.

[0039] Skeletal myoblasts are well known in the art and can be prepared from skeletal muscle by any known method (e.g., the method described in Japanese Patent Application Laid-Open No. 2007-89442). Alternatively, they can be commercially obtained, for example, as Lonza Japan Co., Ltd. (Cat. No. CC-2580) or Cosmo Bio Co., Ltd. (Product Code 3520). Skeletal myoblasts can be identified by markers such as, but not limited to, CD56, α7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx), MyoD, Myf5, Myf6, myogenin, desmin, and PAX3. In one embodiment, the skeletal myoblasts are CD56-positive. In one embodiment, the skeletal myoblasts are CD56-positive and desmin-positive.

[0040] When skeletal myoblasts are prepared from striated muscle tissue, the prepared cell population contains fibroblasts. When a cell population containing skeletal myoblasts prepared from striated muscle tissue is used to produce the cell culture of the present invention, the cell population will contain a certain amount of fibroblasts. Fibroblasts are well known in the art and can be identified by markers such as TE-7 (see, for example, Rosendaal et al., J. Cell Sci. 1994; 107(Pt1): 29-37; Goodpaster et al. J. Histochem Cytochem. 2008; 56(4): 347-358). In one embodiment, the cells constituting the cell culture of the present invention comprise skeletal myoblasts prepared from striated muscle tissue. Thus, the cell population used in producing the cell culture of the present invention may comprise skeletal myoblasts and fibroblasts. In one embodiment, the cell population used in producing the cell culture of the present invention may have a CD56 positivity rate of 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, preferably 60% or more.

[0041] When the engraftment sheet-like material of the present invention contains skeletal myoblasts, the cell population used in the production of the engraftment sheet-like material may also contain fibroblasts, but an excessively high fibroblast content is undesirable because it reduces the skeletal myoblast content. Thus, in one embodiment, the cell population used in the production of the cell culture of the present invention may have a TE-7 positivity rate of 50% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, preferably 40% or less. The cell population used in the production of the engraftment sheet-shaped material of the present invention may contain cells other than skeletal myoblasts and fibroblasts, but the fewer such cells, the better. Therefore, the higher the total CD56 positivity rate and TE-7 positivity rate, the better, and may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc., and preferably 90% or more.

[0042] In one embodiment, the engraftment sheet-like material of the present invention further contains a medicinal component. Here, the medicinal component refers to any component that treats abnormalities in the organ to which the engraftment sheet-like material has been engrafted. Abnormalities in the organ include, but are not limited to, inflammation occurring in the organ, tumors formed in the organ, ulcers formed in the organ, etc., and medicinal components that treat such abnormalities in the organ include, but are not limited to, anti-inflammatory agents, antibacterial agents, antifungal agents, antihistamines, adrenocortical hormones, anticancer agents, and any combinations thereof. In addition to the extracellular matrix layer, sheet-shaped cell culture, biodegradable gel layer, and / or medicinal ingredients, the engraftment sheet-shaped material of the present invention may contain various additional components, such as a pharmaceutically acceptable carrier, components that enhance the viability, engraftment, and / or function of the engraftment sheet-shaped material, and components that supplement medicinal ingredients useful for treating abnormalities in the target organ. Any known additional components can be used as such additional components, and those skilled in the art are familiar with these additional components. Furthermore, the engraftment sheet-shaped material of the present invention can be used in combination with components that enhance the viability, engraftment, and / or function of the engraftment sheet-shaped material, and components that supplement medicinal ingredients useful for treating abnormalities in the target organ.

[0043] Another aspect of the present invention relates to a method for producing an engrafted sheet-like material, comprising the steps of seeding cells on a substrate, forming the seeded cells into a sheet, and peeling the formed sheet-like cell culture from the substrate.

[0044] The sheet-shaped cell culture contained in the engraftment sheet-shaped material of the present invention can be produced by any method known to those skilled in the art, as described above (see, for example, Patent Document 1, JP 2010-081829 A, JP 2011-110368 A, etc.). The method for producing a sheet-shaped cell culture typically includes, but is not limited to, the steps of seeding cells on a substrate, forming the seeded cells into a sheet, and peeling the formed sheet-shaped cell culture from the substrate.

[0045] The method for producing an engraftment sheet-like material of the present invention may further include a step of forming an extracellular matrix layer. In the present invention, the extracellular matrix forming the extracellular matrix layer is produced by cells contained in the sheet-shaped cell culture by culturing the cells for 24 hours or more, e.g., 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours, depending on various conditions, after seeding. The produced extracellular matrix is ​​formed, for example, in a layer between the sheet-shaped cell culture and the substrate. In this case, in the subsequent step of detaching the formed sheet-shaped cell culture from the substrate, the sheet-shaped cell culture is preferably detached from the substrate without destroying the formed extracellular matrix layer. Methods for detaching the sheet-shaped cell culture from the substrate without destroying the formed extracellular matrix layer include, but are not limited to, using a culture dish coated with a temperature-responsive material for culture. The method for producing an engraftment sheet-like material of the present invention may further include the step of forming a biodegradable gel layer. Formation of the biodegradable gel layer may be, but is not limited to, a method in which a fibrinogen solution is dropped onto a sheet-like cell culture, followed by spraying a thrombin solution to form a fibrin gel layer (see Japanese Patent No. 6495603). The method for producing an engraftment sheet-like material of the present invention may further include the step of adding a medicinal ingredient. The step of adding the medicinal ingredient may be performed before or after the step of forming the seeded cells into a sheet, or after the step of forming the biodegradable gel layer.

[0046] Yet another aspect of the present invention relates to a method for treating a disease that can be improved by application of an engrafted sheet-like material, comprising applying an effective amount of the engrafted sheet-like material, with the side of the sheet-like material having an extracellular matrix layer attached to an organ of a subject in need thereof. In the present invention, the term "subject" means any individual organism, preferably an animal, more preferably a mammal, and even more preferably a human individual.

[0047] The term "treatment" is also intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions aimed at curing, temporarily ameliorating, or preventing a disease. For example, the term "treatment" encompasses medically acceptable interventions for various purposes, including delaying or halting the progression of a disease, causing regression or elimination of lesions, preventing the onset of the disease, or preventing recurrence of the disease.

[0048] In the present invention, an "effective amount" refers to, for example, an amount that can suppress the onset or recurrence of a disease, alleviate symptoms, or delay or halt progression (e.g., the number of cells contained in the engraftment sheet-like material, the size and weight of the sheet-shaped cell culture contained in the engraftment sheet-like material, etc.), and is preferably an amount that prevents the onset or recurrence of the disease or cures the disease. Furthermore, an amount that does not cause adverse effects that outweigh the benefits of administration is preferred. Such amounts can be appropriately determined, for example, by tests using laboratory animals or disease model animals such as mice, rats, dogs, or pigs, and such test methods are well known to those skilled in the art. Furthermore, the size of the tissue lesion to be treated can be an important indicator for determining the effective amount.

[0049] The treatment method of the present invention may further comprise a step of producing the engraftment sheet-like material of the present invention according to the production method of the present invention. The treatment method of the present invention may further comprise a step of collecting cells (e.g., skin cells, blood cells, etc. when iPS cells are used) or tissue that will serve as a source of cells (e.g., skin tissue, blood, etc. when iPS cells are used) from the subject to produce the sheet-shaped cell culture contained in the engraftment sheet-like material, prior to the step of producing the engraftment sheet-like material. In one embodiment, the subject from which the cells or tissue that will serve as a source of cells is collected is the same individual as the subject to which the engraftment sheet-like material will be applied. In another embodiment, the subject from which the cells or tissue that will serve as a source of cells is collected is a different individual of the same species as the subject to which the engraftment sheet-like material will be applied. In yet another embodiment, the subject from which the cells or tissue that will serve as a source of cells is collected is an individual of a different species than the subject to which the engraftment sheet-like material will be applied.

[0050] A typical application method is direct application to tissue, for example, direct application to the target organ. The engrafted sheet-like material of the present invention can also be applied via laparoscopy or thoracoscopy. Here, application via laparoscopy or thoracoscopy refers to delivery of the engrafted sheet-like material to the vicinity of the target organ through the passageway of an instrument prepared for laparoscopic or thoracoscopic surgery (e.g., a laparoscopic port, a thoracoscopic port, etc.) and application to the surface of the target organ. Such delivery to the vicinity of the target organ may be performed using a delivery device. The delivery device may include, for example, a support for supporting the engrafted sheet-like material and a protective member with a low coefficient of friction for protecting one side of the support, and may be a device in which the support supporting the engrafted sheet-like material can be protected by the protective member, rolled up, and inserted into a cylindrical body. [Example]

[0051] The present invention will now be described in more detail with reference to the following examples, which illustrate particular embodiments of the present invention and are not intended to limit the invention thereto.

[0052] Example 1. Preparation of sheet-shaped cell culture Striated muscle from the lower limb of a pig was collected under general anesthesia, and the collected tissue was treated with an enzymatic digestion solution containing collagenase and trypsin to disperse into single cells. The cells were cultured in MCDB131 medium containing 20% ​​FBS at 37°C and 5% CO2 until confluent, and then the cells were harvested. The harvested cells were cultured in a 60 mm temperature-responsive culture dish (UpCell®, 6 cm dish, CS3006, CellSeed Co., Ltd.) at a density of 2.2 × 10 7The cells were seeded and cultured in 20% FBS-containing DMEM / F12 medium for 12 hours to form a sheet and an extracellular matrix layer. The temperature was then lowered to 20°C, allowing the sheet-shaped cell culture to be detached from the culture dish and collected while still retaining the extracellular matrix layer. 500 μL of fibrinogen solution (Bolheal® Tissue Adhesive (Teijin Pharma)) was dropped onto the surface of the sheet-shaped cell culture opposite the surface bearing the extracellular matrix layer, resulting in a fibrinogen concentration of 80 mg / mL. Next, 800 μL of thrombin solution (Bolheal® Tissue Adhesive (Teijin Pharma)) was sprayed onto the surface opposite the surface bearing the extracellular matrix layer, resulting in a dissolution of the contents of vial 1 (lyophilized fibrinogen powder) with the contents of vial 2 (fibrinogen solution). The thrombin concentration was 250 units / mL. By dripping fibrinogen solution and then spraying thrombin solution, a biodegradable gel layer of fibrin gel was formed on the side of the sheet-shaped cell culture opposite the side bearing the extracellular matrix layer, resulting in an engrafted sheet-like material.

[0053] Example 2: Attaching the engraftment sheet to an organ and collecting tissue The engraftment sheet-like material prepared in Example 1 was applied under general anesthesia to the pig from which the striated muscle was harvested in Example 1. The application was carried out so that the extracellular matrix layer of the engraftment sheet-like material was applied to the surfaces of the stomach, large intestine, and liver. The pig to which the engraftment sheet-like material had been applied underwent abdominal surgery again under general anesthesia on the third day after surgery, and the application site was observed, after which tissue was harvested.

[0054] Example 3. Observation of stained tissue images from collected tissue Histological staining images were obtained from each collected tissue. Images of tissues obtained from the stomach stained with hematoxylin and eosin are shown in Figures 1A and 1B. Images of tissues obtained from the colon stained with hematoxylin and eosin are shown in Figures 2A and 2B, and images of tissues immunostained with anti-desmin antibody are shown in Figures 2C and 2D. Images of tissues obtained from the liver stained with Azan stain are shown in Figures 3A and 3B, and images of tissues immunostained with anti-desmin antibody are shown in Figures 3C and 3D. The stained tissue images in Figures 1 to 3 confirmed that the engrafted sheet-like material had survived in the stomach, large intestine, and liver, and that the organs and the engrafted sheet-like material were histologically continuous. In particular, the histological continuity between the organs and the engrafted sheet-like material was clearly confirmed in the areas circled in Figure 1B and the areas indicated by arrows in Figures 3B, 3C, and 3D.

Claims

1. An engraftment sheet-like material comprising an extracellular matrix layer, a sheet-shaped cell culture, and a biodegradable gel layer, the sheet-shaped material having the extracellular matrix layer on one side and the biodegradable gel layer on the other side, with the sheet-shaped cell culture sandwiched between them, the sheet-shaped material being used by attaching the extracellular matrix layer to the surface of an organ; The organ is the large intestine, The sheet-shaped cell culture comprises skeletal myoblasts, and The engraftment sheet-like material, wherein the biodegradable gel layer is obtained by mixing a liquid containing thrombin with a liquid containing fibrinogen.

2. 2. The engraftment sheet-like material according to claim 1, which is used to maintain histological continuity between the engraftment sheet-like material and the organ to which the engraftment sheet-like material is attached, and which is used without causing the engraftment sheet-like material to adhere to the abdominal wall.

3. The engraftment sheet-like material according to claim 1 or 2, wherein the organ is a human organ.

4. The engraftment sheet-like material according to any one of claims 1 to 3, wherein the attachment to the surface of an organ is performed under a laparoscope or a thoracoscope.

5. The engraftment sheet-like material according to any one of claims 1 to 4, wherein the organ is an internal organ that can be accessed from outside the body using a laparoscope or a thoracoscope.

6. The engraftment sheet-like material according to any one of claims 1 to 5, further comprising a medicinal ingredient.

7. The engraftment sheet-like material according to any one of claims 1 to 6, for use in a method for treating a disease that can be improved by application of an engraftment sheet-like material, the method comprising attaching an effective amount of the engraftment sheet-like material to an organ of a subject in need thereof, the surface of the engraftment sheet-like material having an extracellular matrix layer thereon, The organ is the large intestine, The sheet-shaped cell culture comprises skeletal myoblasts, and The biodegradable gel layer is obtained by mixing a liquid containing thrombin with a liquid containing fibrinogen. The engraftment sheet-like material.

8. The engraftment sheet-like material according to claim 7, wherein the attachment to the target organ is carried out under laparoscopy or thoracoscopy.

9. It can be attached to the target organ, The engraftment sheet-like material according to claim 7 or 8, which is implemented using a device that includes a support for supporting the engraftment sheet-like material and a protective member with a low coefficient of friction for protecting one side of the support, and that can protect the support supporting the engraftment sheet-like material with the protective member, roll it up, and insert it into a cylindrical body.

Images