Cell sheet manufacturing method

Abstract

The purpose of the present invention is to provide a method for producing a cell sheet, which is a simple method for producing a cell sheet and which is easy to handle. According to the present invention, it is possible to provide a method for producing a cell sheet and a cell sheet produced by the method, the cell sheet produced by the method being easy to handle, the method comprising: a step (1) in which one or more mammalian-derived cells are prepared; a step (2) in which the prepared cells are sown on the porous membrane of a first cell culture vessel having the porous membrane on the bottom surface; and a step (3) in which the first cell culture vessel is placed in a second cell culture vessel and cells are cultured.

Description

Technical Field

[0001] The present invention relates to a method for preparing a cell sheet from various types of cells, and more particularly to a method for preparing a cell sheet from various types of cells including retinal pigment epithelial cells, mesenchymal stem cells, fibroblasts, iPS cells, induced pluripotent stem cells and iPS-derived differentiated cells, ES cells and ES-cell-derived final cells. Background Art

[0002] In order to treat various diseases, cell culture sheet transplantation therapy has been reported. The so-called cell culture sheet transplantation therapy is a technology that cultured stem cells collected from the human body or artificially produced in a well plate (culture dish), and the obtained sheet-shaped cultured cells are placed in the defective part of the tissue caused by trauma or disease to repair the defective part.

[0003] The following method has been attempted: transplanting retinal pigment epithelial cells in a cell sheet state close to the morphology in vivo to treat retinal degeneration diseases. For example, for the treatment of age-related macular degeneration, a method of transplanting retinal pigment epithelial cells or retinal pigment epithelium to the defective part under the retina is an effective treatment method (Patent Document 1: Japanese Patent Publication No. 9-501303, Patent Document 2: Japanese Patent Publication No. 2008-173333). Although autologous tissue transplantation has been implemented in which a cell sheet made of retinal pigment epithelial cells (made into a layer accompanying the choroid) cut out from the retinal tissue of a patient with age-related macular degeneration is transplanted to the damaged macula, the cell sheet derived from the patient's tissue still has the following problems: in addition to the transplantation surgery, the invasive risk caused by the patient's retinal excision surgery is increased, the incidence of complications is high, and the proportion of confirmed improvement of macular function and stable maintenance of the effect after transplantation is low.

[0004] As a method of utilizing pigment epithelial cells such as retinal pigment epithelial cells and iris pigment epithelial cells cultured in vitro without relying on the collection of the patient's retina, it is known that a cell sheet made by culturing pigment epithelial cells on an artificial membrane or amniotic membrane is used for transplantation. However, the composition, properties, rigidity, etc. of the artificial membrane are different from the basement membrane formed by the pigment epithelial cells themselves in the organism, and are prone to inflammation or rejection reactions associated with inflammation, and are therefore not suitable for transplantation purposes. In view of this, a method for simply making a cell sheet has been reported, which is composed of a basement membrane formed by pigment epithelial cells cultured in vitro and the cells themselves (e.g., Patent Document 3: WO2011 / 142364). The cell sheet obtained by this method has a basement membrane formed on the side opposite to the flat substrate used for culture and the side in contact with the cell sheet. Therefore, there is no need to use special culture equipment such as temperature-responsive culture equipment or perform enzyme treatment. The cell sheet can be peeled off from the flat substrate, and the basement membrane can be brought into contact with the tissue of the transplant target without turning the sheet over, thereby simplifying the transplantation operation. In addition, it is accompanied by a basement membrane with the same composition as that of the organism, so it is easy to implant, has rigidity and excellent handleability, and is suitable for transplantation therapy.

[0005] However, a simpler and more efficient method for preparing a cell sheet is desired. In addition, the prepared cell sheet is peeled off from the culture dish for transplantation, but the peeled cell sheet separates from the support (flat substrate), so there is a problem that the shape is easily changed before transplantation at the medical site and it is difficult to handle.

[0006] Furthermore, for the purpose of supplying nutrients and oxygen to the transplanted pigment epithelium, a method for producing a cell sheet including a pigment epithelial cell layer and an angiogenic cell layer has been reported (Patent Document 4: WO2014 / 030749).

[0007] Mesenchymal stem cells (MSC) are stem cells with multi-differentiation and self-replication abilities. They have the ability to differentiate into cells derived from the mesoderm, such as osteoblasts, chondrocytes, adipocytes, and muscle cells. In addition, they are also reported to have the ability to differentiate into nerve cells, glial cells, and hepatocytes derived from the ectoderm. MSCs are also known to have homing effects, paracrine effects, and cell adhesion interactions. It is believed that MSCs, based on these effects, exert the repair / regeneration ability of target tissues and cells and immune control abilities such as anti-inflammatory, and the results show therapeutic effects on a variety of diseases.

[0008] Cell transplantation therapy using MSCs has been reported. For example, an attempt to treat kidney disease by applying a cell sheet composition containing MSCs to the kidney has been reported (Patent Document 5: Japanese Patent Application Laid-Open No. 2017-132744).

[0009] Although methods for producing cell sheets using various types of cells have been reported, there is no method for producing cell sheets that can be applied to various types of cells including MSCs or skin tissue cells, namely fibroblasts, and it is desired to establish a method for producing cell sheets that can be applied to a wide range of cell types.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Publication No. 9-501303

[0013] Patent Document 2: Japanese Patent Application Publication No. 2008-173333

[0014] Patent Document 3: WO2011 / 142364

[0015] Patent Document 4: WO2014 / 030749

[0016] Patent Document 5: Japanese Patent Application Publication No. 2017-132744 Summary of the invention

[0017] Problem that the invention aims to solve

[0018] The object of the present invention is to provide a method for preparing a cell sheet, which is a simple method for preparing a cell sheet and the cell sheet prepared by the method is easy to handle. The object of the present invention is also to provide a simple method for preparing a cell sheet, which can be applied to a variety of cells.

[0019] Solutions for solving problems

[0020] The inventors have devoted themselves to studying a method for preparing a cell sheet, which is a simple method for preparing a cell sheet and the cell sheet prepared by the method is easy to handle. As a result, they found that cells can be cultured using a cell culture container with a porous membrane on the bottom surface, thereby completing the present invention.

[0021] The present invention includes the following aspects.

[0022] [1] A method for preparing a cell sheet, comprising the following steps:

[0023] Step (1), preparing one or more mammal-derived cells;

[0024] Step (2), seeding the prepared cells on the porous membrane of a first cell culture container having a porous membrane on the bottom surface; and

[0025] Step (3) is to place the first cell culture container in a second cell culture container, culture cells, and prepare a cell sheet on the porous membrane.

[0026] [2] The method according to [1] above, wherein at least one of the culture medium in the first cell culture container or the culture medium in the second cell culture container contains serum and / or a Rho kinase (ROCK) inhibitor.

[0027] [3] The method according to [1] or [2] above, wherein in step (1), the mammal-derived cells are epithelial cells, endothelial cells, parenchymal cells or stem cells.

[0028] [4] The method as described in [3] above, wherein in step (1), the cells derived from mammals are cells selected from the group consisting of pigment epithelial cells (e.g., iris pigment epithelial cells, retinal pigment epithelial cells), fibroblasts and mesenchymal stem cells.

[0029] [5] The method according to any one of [1] to [4] above, wherein in step (2), the number of cells is 5000 cells / mm 2 (Preferably 10,000 cells / mm 2 ) The cells are seeded on the bottom surface.

[0030] [6] The method according to any one of [1] to [5] above, wherein in the cell sheet obtained by culture, a membrane containing an extracellular matrix is ​​formed on the side of the cells opposite to the side in contact with the porous membrane.

[0031] [7] The method according to [6] above, wherein the cell sheet obtained by culturing further has tight junctions formed between cells.

[0032] [8] The method according to [6] or [7] above, further comprising the following step (4):

[0033] In step (4), it is confirmed that a membrane containing an extracellular matrix is ​​formed on the side of the cell opposite to the side in contact with the porous membrane.

[0034] [9] The method according to any one of [1] to [8] above, further comprising the following step (5):

[0035] Step (5) is to confirm whether or not differentiation markers are expressed in the cultured cells obtained in step (3).

[0036] The aforementioned molecular markers are as follows: when the aforementioned cells derived from mammals are epithelial cells such as retinal pigment epithelial cells or iris pigment epithelial cells, it is preferably at least one selected from the group consisting of Bestrophin-1, RPE-65, pan-spectrum cytokeratin and any subtype of cytokeratin, occludin, ZO-1 (occluded occluded-1), elastin, actin, type 1 collagen, type 2 collagen and type 4 collagen; when the aforementioned cells derived from mammals are epithelial cells, it is preferably at least one selected from the group consisting of EpCAM and any subtype thereof, pan-spectrum cytokeratin and any subtype of cytokeratin; when the aforementioned cells derived from mammals are endothelial cells, it is preferably selected from the group consisting of CD31, CD34, CD45, ICAM-1 / CD54, LYVE-1, Tie2 / Tek, vWF (von Willebrand factor, von Willebrand factor When the cells derived from mammals are fibroblasts, it is preferably at least one selected from the group consisting of HSP47, Serpin H1, fibroblast-specific protein 1, CD248 (endosialin), DDR-2, CD280 (Endo180) and vimentin; when the cells derived from mammals are adipocytes, it is preferably PLN1 and / or fatty acid binding protein 4; when the cells derived from mammals are skeletal muscle cells, it is preferably troponin t-type 1 and / or myosin heavy chain; when the cells derived from mammals are smooth muscle cells, it is preferably at least one selected from the group consisting of transgelin, α-smooth muscle actin and calmodulin; when the cells derived from mammals are cardiomyocytes, it is preferably selected from the group consisting of fatty acid binding protein 3, cardiac troponin T and Nkx2.5; when the aforementioned cells derived from mammals are liver stem cells, it is preferably at least one selected from the group consisting of EpCAM, E-cadherin, CD133 and CD29; when the aforementioned cells derived from mammals are nerve cells, it is preferably collagen fibrillary acid protein, myelin basic protein and / or peripherin; when the aforementioned cells derived from mammals are mesenchymal stem cells, it is preferably at least one selected from the group consisting of CD73, CD90, CD105, CD11b, CD14, CD19, CD79α, CD34, CD45 and HLA-DR; when the aforementioned cells derived from mammals are T cells, it is preferably at least one selected from the group consisting of CD3, CD4 and CD8; when the aforementioned cells derived from mammals are B cells, it is preferably selected from At least one of the group consisting of CD19, CD20, CD10, CD34, CD38, CD40 and CD45R; when the aforementioned cells derived from mammals are natural killer cells, preferably at least one of the group consisting of CD16, CD56 and CD57; when the aforementioned cells derived from mammals are dendritic cells, preferably at least one of the group consisting of CD11c, HLA-DR, CD141, CD1c, CD11b, CD303, CD123 and CD1a; when the aforementioned cells derived from mammals are hematopoietic stem cells, preferably CD34 and / or CD45; when the aforementioned cells derived from mammals are macrophages, preferably at least one of the group consisting of CD16, CD32, CD64, CD68, CD80 and CD86. When the aforementioned mammal-derived cells are mesenchymal stem cells, they are more preferably positive for CD73 and / or CD90 and negative for CD11b or CD14, CD19 or CD79α, CD34, CD45 and HLA-DR.

[0037]

[10] The method according to any one of [1] to [9] above, further comprising the following step (5'):

[0038] Step (5') is to confirm whether the cultured cells obtained in step (3) secrete soluble Flt-1 (VEGF receptor 1) and / or TIMP-3.

[0039]

[11] The method as described in any one of [1] to

[10] above, wherein in step (1), the mammalian-derived cells are pigment epithelial cells such as retinal pigment epithelial cells or iris pigment epithelial cells, mesenchymal stem cells or fibroblasts, and the number of cells is 5000 / mm 2 (Preferably 10,000 cells / mm 2 )~40000 cells / mm 2The cells are seeded on the bottom surface.

[0040]

[12] A cell sheet produced by the method described in any one of [1] to

[11] above.

[0041]

[13] A transplant material for treating a disease, comprising a cell sheet produced by the method described in any one of [1] to

[11] above.

[0042]

[14] A method for producing a membrane containing an extracellular matrix (for example, a basement membrane when epithelial cells are used, and in particular, Bruch's membrane when retinal pigment epithelial cells are used), the method comprising the steps of: isolating the membrane containing the extracellular matrix formed on a cell sheet, the cell sheet being produced by the method described in any one of [1] to

[11] above.

[0043]

[15] A cell sheet comprising mammalian cells arranged on a porous membrane, wherein an extracellular matrix exists between the cells, and a membrane containing the extracellular matrix (for example, a basement membrane when epithelial cells are used, and particularly a Bruch's membrane when retinal pigment epithelial cells are used) is formed on the side opposite to the side in contact with the porous membrane.

[0044]

[16] The cell sheet according to

[15] above, wherein the cell sheet further has tight junctions formed between cells.

[0045]

[17] A cell sheet as described in

[15] or

[16] above, wherein the cell sheet is obtained by seeding one or more mammalian-derived cells on a porous membrane of a first cell culture container having a bottom surface of the porous membrane, and placing the first cell culture container in a second cell culture container for culturing.

[0046]

[18] The cell sheet according to

[17] above, wherein at least one of the culture medium in the first cell culture container or the culture medium in the second cell culture container is a culture medium containing serum and / or a Rho kinase (ROCK) inhibitor.

[0047]

[19] The cell sheet according to any one of

[15] to

[18] above, wherein the mammal-derived cells are epithelial cells, endothelial cells, parenchymal cells or stem cells.

[0048]

[20] The cell sheet as described in

[19] above, wherein the mammalian-derived cells are cells selected from the group consisting of pigment epithelial cells (e.g., retinal pigment epithelial cells, iris pigment epithelial cells), fibroblasts, and mesenchymal stem cells.

[0049]

[21] The cell sheet according to

[20] above, wherein the mammal-derived cells are retinal pigment epithelial cells or iris pigment epithelial cells.

[0050]

[22] A cell sheet as described in any one of

[15] to

[21] above, wherein the cells contained in the cell sheet express at least one molecular marker selected from the group consisting of the following substances: lutein-1, RPE-65, pan-cytokeratin and any subtype of cytokeratin, occludin, ZO-1, elastin, actin, type 1 collagen, type 2 collagen and type 4 collagen.

[0051]

[23] The cell sheet according to any one of

[15] to

[22] above, wherein

[0052] The cells contained in the aforementioned cell sheet secrete soluble Flt-1 (VEGF receptor 1) and / or TIMP-3.

[0053]

[24] A method for producing a multilayered cell sheet, comprising the following steps (1) to (4):

[0054] Step (1), preparing one or more mammal-derived cells;

[0055] Step (2), seeding the prepared cells on the porous membrane of a first cell culture container having a porous membrane on the bottom surface;

[0056] Step (3), placing the first cell culture container further into a second cell culture container, culturing cells, and preparing a cell sheet on the porous membrane; and

[0057] Step (4), repeating the following steps (a) and (b) once or more,

[0058] Step (a), seeding one or more mammal-derived cells on a cell sheet formed in a first cell culture container,

[0059] Step (b) is to place the first cell culture container in a second cell culture container, culture cells, and prepare a new cell sheet on the cell sheet.

[0060]

[25] A multilayer cell sheet produced by the method described in

[24] above.

[0061]

[26] A multilayer cell sheet arranged on a porous membrane, wherein an extracellular matrix exists between cells in each cell sheet included in the multilayer cell sheet, and a membrane containing the extracellular matrix is ​​formed on the side opposite to the side in contact with the porous membrane.

[0062] Effects of the Invention

[0063] According to the present invention, a method for preparing a cell sheet can be provided, which is a simple method for preparing a cell sheet, and the cell sheet prepared by the method is easy to handle. The cell sheet obtained by the method of the present invention can be easily separated from the cell container by applying a physical force, such as a water flow generated by pipetting, and also has rigidity, so that the cell sheet can be sucked or discharged using a fine hose, etc., so that the cell sheet can reach the target transplantation site during transplantation without performing a large-scale incision. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The results are shown in Figure 1 and Figure 2. The cells were placed in each container at 2×10 5 cells, 10×10 5 This is a photograph of the results of cell sheets produced by seeding and culturing cells.

[0065] Figure 2 This is a photograph of a cell sheet formed into a film being separated from a cell container by a water flow generated by pipetting.

[0066] Figure 3 This is a photo of a cell sheet placed in saline solution in a culture dish being sucked up and then discharged using a pipette. The numbers indicate the order of the operation.

[0067] Figure 4 This is a photograph showing a cross section of a cell sheet formed by mesenchymal stem cells derived from human adipose tissue. The bar showing the thickness is 39.0 μm.

[0068] Figure 5 These are the results of confirming the expression of each molecular marker in a cell sheet prepared using adipose tissue-derived mesenchymal stem cells by immunohistostaining.

[0069] Figure 6 The results of the test of the protective effect of the cell sheet formed by mesenchymal stem cells derived from human adipose tissue are shown. The photo on the left of the figure shows that the thickness of the outer granular layer (the layer of the nucleus of the visual cell) in the rats transplanted with the mesenchymal stem cell sheet prepared by the method of the present invention was maintained at the time point of three weeks after transplantation. The photo on the right of the figure shows that the outer granular layer in the subject group (sham operation group) without the transplantation of the mesenchymal stem cell sheet was thinned.

[0070] Figure 7 The results of using the method of the present invention to prepare a cell sheet using mesenchymal stem cells derived from adipose tissue. A, B, C, D, E, and F in the figure are cells in each container at 0.08×105 cells, 0.4×10 5 cells, 2×10 5 cells, 10×10 5 cells, 13×10 5 cells, 20×10 5 Photograph of the results after cells were seeded to make cell sheets.

[0071] Figure 8 The results of testing the phagocytic ability of cell sheets formed by iris pigment epithelial cells using the method of the present invention are shown. The upper half shows photographs of the results using a negative control, and the lower half shows photographs of the results using a cell sheet prepared using the method of the present invention.

[0072] Fig. 9 The results of immunohistostaining confirm that the cell sheets formed by iris pigment epithelial cells using the method of the present invention express elastin, type IV collagen, and type I collagen.

[0073] Fig.10 The results of the test of the retinal protection effect of the cell sheet formed by the iris pigment epithelial cells are shown. The photograph in the upper part of the figure shows that the thickness of the outer granular layer (the layer of the nucleus of the photoreceptor) in the rats transplanted with the iris pigment epithelial cell sheet prepared by the method of the present invention was maintained at the time point of four weeks after transplantation. The graph in the lower part of the figure shows that the photoreceptors in the rats transplanted with the iris pigment epithelial cell sheet are protected.

[0074] Fig.11 The graph shows the results of testing the phagocytic ability of cell sheets formed by fibroblasts.

[0075] Fig.12 The diagram shows that a cell sheet formed by fibroblasts expresses type I collagen, type IV collagen, elastin, and ZO-1.

[0076] Fig.13 The results of the test of the retinal protective effect of the cell sheet formed by fibroblasts are shown. The left side is the result of the fibroblast sheet transplantation group, and the right side is the result of the sham operation group (control group). At the time point of four weeks after transplantation, it is shown that the thickness of the outer granular layer (the layer of the nucleus of the photoreceptor cell) in the rats transplanted with the fibroblast sheet prepared by the method of the present invention is maintained.

[0077] Fig.14 The results are as follows: the secretion of soluble Flt-1 (VEGF receptor 1) and TIMP-3 produced by a cell sheet formed from mesenchymal stem cells derived from human adipose tissue was measured.

[0078] Fig.15 The results of producing a laminated sheet composed of mesenchymal stem cells derived from human adipose tissue by the method of the present invention are shown.

[0079] Fig.16 The results of evaluating cell migration using a cell sheet prepared using mesenchymal stem cells by the method of the present invention are shown. In each figure, the left column shows the results using a cell suspension, and the right column shows the results using a cell sheet. DETAILED DESCRIPTION

[0080] Hereinafter, taking illustrative embodiments as an example, the preferred method and material that can be used when the present invention and the implementation of the present invention are described together, but the present invention is not limited to the following described methods. Furthermore, if there is no special description in the specification, all technical terms and scientific terms used in this specification have the same meaning as the meaning commonly understood by technical personnel in the technical field to which the present invention belongs. In addition, any material and method that is equal or the same as the method recorded in this specification can be used in the implementation of the present invention in the same manner. In addition, all publications and patents related to the present invention and cited in this specification, for example, can be used as a method that can be used in the present invention in addition to represent the method and material, and are cited in this specification and thus constitute a part of this specification.

[0081] In this specification, the description of "A to B" showing a numerical range means a numerical range including the endpoints A and B. The same applies to the description of "A to B". In addition, in this specification, the so-called "about" is used to indicate that ±10% is allowed.

[0082] The method for producing a cell sheet of the present invention comprises the following steps (1) to (3).

[0083] Step (1), preparing one or more mammal-derived cells;

[0084] Step (2), seeding the prepared cells on the porous membrane of a first cell culture container having a porous membrane on the bottom surface; and

[0085] Step (3) is to place the first cell culture container further into a second cell culture container, culture cells, and prepare a cell sheet on the porous membrane.

[0086] In the method of the present invention, one or more mammal-derived cells are typically prepared in the form of a cell suspension in step (1).

[0087] The mammalian cells prepared in step (1) are not particularly limited as long as they are mammalian cells, and examples thereof include cells derived from humans, monkeys, mice, rats, dogs, cows, horses, pigs, sheep, goats, cats, rabbits, hamsters, and guinea pigs, preferably cells derived from humans.

[0088] The cell types to be prepared are not particularly limited, and examples thereof include epithelial cells, endothelial cells, parenchymal cells, and stem cells. In addition, the cell types may be adhesive cells or non-adhesive cells, but are preferably adhesive cells. Specifically, although not limited to, examples include: parenchymal cells of the liver, i.e., liver cells; endothelial cells such as Kupffer cells, vascular endothelial cells, and corneal endothelial cells; epidermal cells such as fibroblasts, osteoblasts, osteoclasts, cells derived from the periodontal membrane, and epidermal basal cells; epithelial cells such as tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, conjunctival epithelial cells, corneal epithelial cells, iris pigment epithelial cells, and retinal pigment epithelial cells; mammary cells; pericytes; muscle cells such as smooth muscle cells and cardiomyocytes; kidney cells; pancreatic islet cells; neural cells such as peripheral nerve cells and optic nerve cells; chondrocytes; bone cells; undifferentiated stem cells such as pluripotent stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, hematopoietic stem cells, neural stem cells, myocardial stem cells, liver stem cells, skeletal muscle stem cells, epithelial stem cells, epidermal stem cells, retinal stem cells, and adipose stem cells, and cells derived from these stem cells. Preferred examples include corneal endothelial cells, tracheal epithelial cells, digestive tract epithelial cells, cervical epithelial cells, corneal epithelial cells, retinal pigment epithelial cells, fibroblasts, and mesenchymal stem cells; more preferably, corneal epithelial cells, corneal endothelial cells, retinal pigment epithelial cells, fibroblasts, and mesenchymal stem cells, iPS cells and differentiated cells derived from iPS cells, ES cells, and differentiated cells derived from ES cells; and most preferably, retinal pigment epithelial cells, fibroblasts, and mesenchymal stem cells can be used. These cells can be used to prepare cell sheets by the method of the present invention.

[0089] The cells to be prepared may be primary cells directly collected from tissues and organs, or cells obtained by subculturing these primary cells for several generations. Further, the cells may be stem cells, or cells obtained by inducing differentiation of these stem cells, including: undifferentiated cells, i.e., embryonic stem cells (ES cells), pluripotent stem cells such as mesenchymal stem cells with multi-differentiation ability, artificial pluripotent stem cells (iPS cells) made from somatic cells, or unipotent stem cells such as vascular endothelial progenitor cells with single differentiation ability. ES cells may be ES cells generated by nuclear reprogramming of autologous cells. iPS cells can be produced by introducing a certain recombinant substance (nucleic acid, protein, low molecular compound, etc.) into somatic cells, and are artificial stem cells derived from somatic cells with the same characteristics as ES cells. In addition, target cells can be prepared by inducing differentiation of stem cells such as artificial pluripotent stem cells (iPS cells) and ES cells. The conditions / medium for differentiating the aforementioned stem cells into the target differentiated cells can be carried out according to known conditions / medium, and those skilled in the art can set them appropriately. When the cell sheet prepared by the present invention is used for transplantation, iPS cells whose tissue compatibility antigens have been deleted or altered by using methods such as genome editing, or somatic cells of the object to be transplanted can be used as the source of iPS cells. Since the cell sheet obtained from these iPS cells will be a cell sheet without antigenicity to the object, it is preferable to use iPS cells.

[0090] When the cells derived from mammals used in the process (1) of the present invention are pigment epithelial cells such as retinal pigment epithelial cells or iris pigment epithelial cells, the mammals are the same as above, but preferably humans. In addition, the retinal pigment epithelial cells are differentiated cells derived from stem cells or cells derived from eyeballs. Retinal pigment epithelial cells derived from eyeballs can be obtained in the following manner: after removing the corpse eyeball, the eyeball is quickly divided at the equator, after removing the vitreous body and retina, scraped by a cell scraper or freed from Bruch's membrane with trypsin and EDTA solution and recovered, and then induced to adhere to the culture dish and proliferate by being placed in a culture solution, thereby making the required amount of cell proliferation, and appropriately subcultured with trypsin treatment, etc. to ensure the number of cells. When stem cells are subjected to differentiation induction, human ES cells or iPS cells are cultured using ES cell differentiation medium added with Dkk-1 (Wnt antagonist) and Lefty A (Nodal antagonist). By culturing for a predetermined period, retinal progenitor cell markers Rx, Pax6, and Mitf are expressed, and a polygonal morphology is confirmed by morphological observation under an optical microscope, thereby obtaining human retinal pigment epithelial cells.

[0091] When the mammal-derived cells used in step (1) of the present invention are fibroblasts, the mammal is the same as described above, preferably a human. The method for preparing the fibroblasts is not particularly limited, and a known method can be used. For example, various tissues derived from organisms can be separated and prepared by enzyme digestion, but it is not limited thereto. Non-limiting examples of the tissues derived from organisms include dermal tissues of mammals.

[0092] When the cells derived from mammals used in the process (1) of the present invention are mesenchymal stem cells, the mammals are the same as described above, preferably humans. In addition, the mesenchymal stem cells can be obtained from various tissues. It is not limited to this, but can be listed as follows: derived from bone marrow, derived from umbilical cord, derived from umbilical cord blood, derived from endometrium, derived from placenta, derived from amnion, derived from chorion, derived from exfoliated membrane, derived from dermis, derived from dental follicle, derived from periodontal ligament, derived from dental pulp, derived from tooth germ, derived from adipose tissue, derived from blood vessels (periphery), derived from skeletal muscle, or derived from synovium and other mesenchymal stem cells. The preparation method of mesenchymal stem cells is not particularly limited, and known methods can be used. For example, various tissues derived from organisms can be separated and prepared thereby using enzyme digestion, but it is not limited thereto. The non-limiting examples of the tissues derived from organisms include bone marrow, umbilical cord, and fat of mammals.

[0093] As the mammalian cells used in step (1) of the present invention, one or more cells can be used. When multiple cells are used, the combination is not particularly limited and can be appropriately selected within the scope of the purpose of the present invention. For example, cells derived from the same or different tissues, cells derived from the same or different animal species can be used in combination. The number of cell types combined is not particularly limited. For example, a combination of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more cell types can be used.

[0094] When the cells are prepared into a cell suspension, the suspension liquid is not particularly limited as long as it is a liquid having components commonly used when suspending living cells. The cell suspension liquid can be prepared using a known method used in the art. In one embodiment, the cell suspension liquid can be a cell culture fluid containing components for culturing cells. In one embodiment, the cell culture fluid containing components for culturing cells can be added to the first cell culture container after seeding the cells. In another embodiment, the cell culture fluid can be added to the first cell culture container in advance before seeding the cells.

[0095] In step (2), the prepared cells are sown on a porous membrane of a first cell culture container having a porous membrane on the bottom surface, and a culture solution is added. When the prepared cells are a cell suspension in which cells are suspended in a culture medium at a concentration suitable for culture, the culture solution may not be added. The shape of the first cell culture container is not particularly limited as long as it can hold the prepared cells and the porous membrane can be set on the bottom surface. The material of the porous membrane is not particularly limited, and it can be any of resin, metal, and glass.

[0096] Examples of the resin forming the porous membrane include various polyethylenes such as low-density polyethylene, high-density polyethylene, and ultra-high molecular weight polyethylene, or polyethylene naphthalate; polypropylene, polymethyl methacrylate, methyl methacrylate-styrene copolymer, polystyrene; fluororesins such as polytetrafluoroethylene and polyvinylidene fluoride; ethylene-vinyl acetate copolymers, polyamides, styrene-acrylonitrile copolymers, styrene-butadiene-acrylonitrile terpolymers, polycarbonates, polyvinyl chloride, etc. Among these, porous resin membranes formed of fluororesins such as polytetrafluoroethylene and polyvinylidene fluoride are preferred because they have excellent absorption of the storage solution and excellent visibility of cells under microscopic observation.

[0097] Examples of metals forming the porous film include copper, copper alloys, aluminum, aluminum alloys, gold, gold alloys, silver, silver alloys, tin, zinc, lead, titanium, nickel, stainless steel, etc. Furthermore, porous films formed of metal oxides such as silicon dioxide, aluminum oxide, and zirconium can also be used. In addition, the porous metal and porous metal oxide films can be porous films containing two or more of the above-mentioned metals and metal oxides, respectively.

[0098] The pore size of the porous body in the porous membrane of porous resin, porous metal, porous metal oxide and porous glass is not particularly limited as long as it is a size that the cells used will not pass through. For example, a porous membrane with a pore size of less than 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm can be used. In one embodiment, it is preferably a pore size of less than 3 μm.

[0099] The porous membrane may be subjected to surface treatment for the purpose of improving the adhesion to the adhesive cells, but it may not be subjected to treatment in order to facilitate the process of peeling the cell sheet of the present invention from the flat substrate. When treatment is required, the surface treatment may be performed, for example, with collagen, gelatin, Matrigel, poly-L-lysine, poly-D-lysine, laminin, fibronectin, etc.

[0100] When cells are to be seeded on a porous membrane, the shrinkage of the sheet can be suppressed by seeding the cells at a high density. The so-called "high density" in this description refers to a density above the cell density observed in normal tissues derived from the prepared cells. In addition, its upper limit can be appropriately determined by a person skilled in the art, for example, a density at which cells are closely attached to each other and a cell density at which sheet formation defects and cell death are not caused due to excessive seeding. More specifically, the so-called "high density" is preferably set to more than 2.5 times the recommended seeding density during normal culture, for example, it refers to a cell density of 1 to 100 times, preferably 1.2 to 50 times, and more preferably about 2.5 to 30 times, relative to the cell density observed in normal tissues. The cell density observed in normal tissues will vary depending on the type of tissue, for example, it can be set to 1000 cells / mm 2 Above, 2000 cells / mm 2 Above, 3000 cells / mm 2 Above, 4000 cells / mm 2 Above, 5000 cells / mm 2 More specifically, it is preferably set as follows: about 3000 cells / mm for the corneal endothelium 2 The above is about 4000 cells / mm for retinal pigment epithelial cells. 2 For mesenchymal stem cells, the number of cells is about 200 / mm 2 For fibroblasts, the number of cells is about 5000 / mm 2 above.

[0101] When the mammalian-derived cells are retinal pigment epithelial cells, "high density" means "a density higher than the cell density observed in a normal eyeball". Specifically, such a density is at least 4000 cells / mm 2 Above. However, even if the cell density is higher than the cell density observed in a normal eyeball, when the cell sheets are sown on a flat substrate at a density below the prescribed density, sometimes the cell sheets formed themselves will be affected by the contraction force and cannot maintain the area at the time of sowing. It is speculated that this is due to the following reasons that the area at the time of sowing cannot be maintained: in order to cover the serum contact surface of the retinal pigment epithelium, a certain number of cells will be mobilized, there is uneven cell density, and the survival rate of the cells themselves. However, even such a shrunk sheet can still be used for the purposes described below without any special problems. Therefore, the so-called "density higher than the cell density observed in a normal eyeball" is preferably about 5000 cells / mm when the cell sheet formed is allowed to shrink from the area at the time of sowing. 2 More preferably, about 10,000 cells / mm 2When the cell sheet is maintained at the area of ​​the seeding, the number of cells is preferably about 20,000 cells / mm 2 In addition, as the upper limit, it is a density that does not induce sheet formation defects or partial cell death due to excessive cell seeding. When such an upper limit is also considered, the so-called "density above the cell density observed in a normal eyeball" can preferably be about 5000 cells / mm 2 ~About 200,000 cells / mm 2 , more preferably about 10,000 cells / mm 2 ~About 120,000 cells / mm 2 , particularly preferably about 20,000 cells / mm 2 ~About 40,000 cells / mm 2 .

[0102] When the cells derived from mammals are mesenchymal stem cells, the term "high density" means "a density higher than the density at which cells are seeded in general cell culture". For example, in the case of mesenchymal stem cells, the density is specifically limited to about 200 cells / mm. 2 , preferably about 1000 cells / mm 2 , more preferably about 1200 cells / mm 2 , more preferably about 5000 cells / mm 2 As an upper limit, for example, about 200,000 cells / mm 2 , preferably about 120,000 cells / mm 2 , more preferably about 60,000 cells / mm 2 , more preferably about 40,000 cells / mm 2 The preferred range may be about 1000 cells / mm 2 ~About 200,000 cells / mm 2 , more preferably about 3000 cells / mm 2 ~About 120,000 cells / mm 2 , more preferably about 5000 cells / mm 2 ~About 60,000 cells / mm 2 , particularly preferably about 10,000 cells / mm 2 ~About 40,000 cells / mm 2 When the mammal-derived cells are other stem cells such as iPS cells and ES cells, the so-called "high density" is the same as the "high density" in mesenchymal stem cells.

[0103] When the mammalian-derived cells are fibroblasts, the term "high density" means "a density higher than the density at which cells are seeded in general cell culture". Specifically, such a density may be about 5,000 cells / mm 2 ~About 200,000 cells / mm 2 , more preferably about 10,000 cells / mm 2 ~About 12,000 cells / mm 2 , more preferably about 10,000 cells / mm 2 ~About 60,000 cells / mm 2 , particularly preferably about 20,000 cells / mm 2 ~About 40,000 cells / mm 2 .

[0104] In step (3), the first cell culture container is placed in the second cell culture container. In this step, the addition of the cell culture solution to the first cell culture container and the addition to the second cell culture container can be performed at various time points. For example, after the culture solution is added to the first cell culture container in which the cells have been seeded, the first cell culture container can be placed in the second culture container in which the culture solution has been pre-added. In another embodiment, after the first cell culture container in which the cells have been seeded is placed in the second culture container in which the culture solution has been pre-added, the culture solution can be added to the first cell culture container. Thereafter, by culturing the cells, a single-layer or multi-layer cell colony can be formed, and a cell sheet can be produced.

[0105] As a culture medium, any cell culture medium commonly used in the technical field can be used without particular limitation. For example, depending on the type of cells used, the basic culture medium described on page 581 of "The Third Edition of the Technology of Tissue Culture Edited by the Japanese Society of Tissue Culture" published by Asakura Bookstore can be used: F-10 culture medium, F12 culture medium, MEM, BME culture medium, DMEM, αMEM, IMD culture medium, ES culture medium, DM-160 culture medium, Fisher culture medium, WE culture medium and RPMI1640 culture medium, etc. Further, serum (fetal bovine serum, etc.), various growth factors, antibiotics, amino acids, etc. can be added to the above-mentioned basic culture medium, and a culture medium with serum and / or Rho kinase (ROCK) inhibitor is preferably used. By adding Rho kinase (ROCK) inhibitor, cell apoptosis can be prevented even at high density culture, and cell sheets of good quality can be obtained. The pH of the culture medium is preferably about 6 to about 8. The culture is usually carried out at about 30 to about 40°C and about 6 to about 168 hours. The culture medium can be replaced and / or supplemented, ventilated or stirred as needed.

[0106] Rho kinase (ROCK) inhibitor is a phosphorylated serine-threonine protein kinase, which is identified as a target protein of the low molecular weight GTP-binding protein Rho. Its inhibitor, i.e., ROCK inhibitor, is commercially available, and these commercial products can be used in the present invention without limitation. As ROCK inhibitors used in the present invention, it is not limited thereto, and examples include Y27632 and HA1077, preferably HA1077. The concentration of the Rho kinase inhibitor used in the present invention can be, for example, 1 to 50 μM, preferably 2 to 30 μM, and more preferably 5 to 20 μM.

[0107] The second cell culture container is not particularly limited as long as it can be poured with culture fluid and is a common cell culture container, and examples thereof include: culture dish, petri dish, tissue culture dish, multidish, microplate, microwell plate, multiplate, multiwall plate, chamber slide, glass culture dish, tray, etc. The material of the culture container includes, for example, inorganic materials such as metal, glass, ceramic, silicon, and organic materials represented by elastomers, plastics (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, vinyl chloride resin, etc.), but is not limited thereto.

[0108] As a combination of a first cell culture container and a second cell culture container having a porous membrane on the bottom surface that can be used for the method of the present invention, for example, commercial products can also be used. As the first and second cell culture containers, cell culture plug-ins (the bottom surface of the cell culture plug-in is composed of a porous membrane) and culture containers that can be placed in the plug-in can be listed, and these containers are provided by Corining International Inc., Thermo Scientific Inc., Greiner Bio-One International, etc. As the first cell culture container, for example, there are Transwell (registered trademark) permeable supports, Snapwell (trademark) plug-ins, Netwell (trademark) plug-ins, Falcon cell culture plug-ins, etc. made by Corining, and can be applied to the present invention. In addition, as the second cell culture container, for example, there are Falcon cell culture plates, Falcon multi-cell culture plates (Corining) made by Corining, etc., and can be applied to the present invention. In the method of the present invention, the surface that can adhere to cells adopts a structure of a porous membrane, although not limited to this, it is believed that it is helpful to culture from the point of being able to supply nutrition and oxygen to the cell sheet from top to bottom. In addition, the cell sheet produced by the culture is held on the porous membrane on the bottom surface of the first cell culture container, so it can be moved and transported together with the cell culture insert, and can be moved and transported in a state of stably holding the cells. Therefore, when used for transplantation, the cell sheet can be peeled off from the bottom surface of the cell culture insert for use in the medical field where the transplantation is performed, which is easy to operate.

[0109] The cell sheet obtained by the method of the present invention has an extracellular matrix, so the sheet becomes tough and easy to maintain its shape. Therefore, although not limited to this, it becomes possible to use a pipette or other fine tube for suction / discharge, and the sheet can be transplanted to a place that is difficult to reach from the surface of the individual.

[0110] The cell sheet obtained by the method of the present invention may also have a membrane containing an extracellular matrix formed on the side opposite to the side of the cell in contact with the porous membrane. The so-called side opposite to the side in contact with the porous membrane is the contact surface side of the cell and the culture medium. Therefore, the method of the present invention may further include the following step (4).

[0111] In step (4), it is confirmed that a membrane containing an extracellular matrix is ​​formed on the side of the cells opposite to the side in contact with the porous membrane.

[0112] When pigment epithelial cells such as retinal pigment epithelial cells or iris pigment epithelial cells are used, step (4) is a step for confirming the formation of Bruch's membrane. The formation of the membrane containing the extracellular matrix can be confirmed by observing the expression of elastin, type 1 collagen or type 4 collagen on the cell surface by immunostaining and observing the expression of protein by Western blotting.

[0113] In the cell sheet obtained by the method of the present invention, tight junctions can also be formed. Tight junction formation can be confirmed by observing the hexagonal and mutually close cell morphology and observing the expression of proteins and the like between cells by immunostaining. The unique proteins and the like that are expressed vary depending on the cell type used, and can be appropriately selected with reference to known information. For example, when epithelial cells are used, the formation of tight junctions can be confirmed by observing the expression of occludin, ZO-1, etc.

[0114] The method of the present invention may further include the following step (5).

[0115] Step (5) is to confirm whether or not differentiation markers are expressed in the cultured cells obtained in step (3).

[0116] By confirming the expression of differentiation markers in the cultured cells obtained in step (3) in step (5), the completion of the cell sheet can be determined. In this specification, differentiation markers may be expressed at any location of the cells (e.g., cytoplasm, cell membrane, nuclear membrane, etc.).

[0117] The term "differentiation marker" as used herein includes transcription products, translation products, or degradation products of genes that are specifically expressed in differentiated cells, or whose expression is increased or decreased compared to other differentiated cells, undifferentiated cells, or precursor cells. Examples of differentiation markers are listed below.

[0118] When the cells derived from mammals are pigment epithelial cells such as retinal pigment epithelial cells or iris pigment epithelial cells, it is preferred that at least one of the proteins be selected from the group consisting of lutein-1, RPE-65, pan-spectrum cytokeratin and any subtype of cytokeratin, occludin, ZO-1, elastin, actin, type 1 collagen, type 2 collagen and type 4 collagen.

[0119] When the mammal-derived cell is an epithelial cell, it is preferably at least one selected from the group consisting of EpCAM and any subtype thereof, pan-cytokeratin and any subtype of cytokeratin.

[0120] When the cells derived from mammals are endothelial cells, they are preferably at least one selected from the group consisting of CD31, CD34, CD45, ICAM-1 / CD54, LYVE-1, Tie2 / Tek, vWF (von Willebrand factor), CD144, VCAM-1 / CD106, VE-cadherin and VEGF-R2, more preferably CD31 and / or CD34.

[0121] When the mammal-derived cell is a fibroblast, at least one selected from the group consisting of HSP47, SerpinH1, fibroblast-specific protein 1, CD248 (endosialin), DDR-2, CD280 (Endo180) and vimentin is preferred.

[0122] When the aforementioned mammal-derived cells are adipocytes, PLN1 and / or fatty acid binding protein 4 are preferred.

[0123] When the mammal-derived cells are skeletal muscle cells, troponin t type 1 and / or myosin heavy chain are preferred.

[0124] When the mammal-derived cell is a smooth muscle cell, at least one selected from the group consisting of transcollin, α-smooth muscle actin and calmodulin is preferred.

[0125] When the mammal-derived cell is a cardiomyocyte, at least one selected from the group consisting of fatty acid binding protein 3, cardiac troponin T, and Nkx2.5 is preferred.

[0126] When the mammal-derived cells are liver stem cells, at least one selected from the group consisting of EpCAM, E-cadherin, CD133 and CD29 is preferred.

[0127] When the mammal-derived cells are nerve cells, fibrillary acidic protein, myelin basic protein and / or peripherin are preferred.

[0128] When the mammal-derived cell is a T cell, it is preferably at least one selected from the group consisting of CD3, CD4, and CD8.

[0129] When the mammal-derived cell is a B cell, it is preferably at least one selected from the group consisting of CD19, CD20, CD10, CD34, CD38, CD40, and CD45R.

[0130] When the mammal-derived cell is a natural killer cell, it is preferably at least one selected from the group consisting of CD16, CD56 and CD57.

[0131] When the mammal-derived cell is a dendritic cell, it is preferably at least one selected from the group consisting of CD11c, HLA-DR, CD141, CD1c, CD11b, CD303, CD123, and CD1a.

[0132] When the mammal-derived cells are hematopoietic stem cells, CD34 and / or CD45 are preferred.

[0133] When the mammal-derived cell is a macrophage, at least one selected from the group consisting of CD16, CD32, CD64, CD68, CD80, and CD86 is preferred.

[0134] When the mammal-derived cells are mesenchymal stem cells, they are preferably at least one selected from the group consisting of CD73, CD90, CD105, CD11b, CD14, CD19, CD79α, CD34, CD45, and HLA-DR. In particular, they are more preferably positive for CD73 and / or CD90 and negative for CD11b, CD14, CD19, CD79α, CD34, CD45, and HLA-DR.

[0135] When the mammal-derived cells are differentiated cells derived from iPS cells or differentiated cells derived from ES cells, the differentiation marker can be appropriately selected depending on the type of differentiated cells to be produced or the purpose of use of the cell sheet.

[0136] In addition, as neural cell differentiation markers, there are: tubulin (especially β-tubulin), MAP2, neurofilaments, neuron-specific enolase; as adipocyte differentiation markers, there are: aP2, glycerol phosphate dehydrogenase, human adipsin, leptin; as osteoblast differentiation markers, such as procollagen 1α1, RUNX2, alkaline phosphatase, osteopontin, osteocalcin, etc.

[0137] The sample used for "confirming the presence or absence of expression of differentiation markers" is a sample derived from the cells cultured in step (3) and is not particularly limited as long as it contains differentiation markers (e.g., RNA, protein, degradation products thereof, etc.).

[0138] When the sample is cells, the cells cultured in step (3) can be immunostained, and the expression level of the differentiation marker can be measured using, for example, flow cytometry.

[0139] When the sample is RNA, an RNA (e.g., total RNA, mRNA) fraction is prepared from the cells cultured in step (3), and the transcription product of the marker gene contained in the fraction is detected, or the marker gene product in the cell is directly detected without extracting RNA from the cell, thereby investigating the expression of the differentiation marker gene. The preparation of the RNA (e.g., total RNA, mRNA) fraction from the cell can be carried out according to a general method, for example, it can be carried out using a commercially available RNA extraction kit. As a means of detecting the transcription product of the differentiation marker gene in the RNA fraction, a method using hybridization (immunoblotting (northern blotting), dot blot, DNA chip analysis, etc.) or a method using PCR (RT-PCR, competitive PCR, real-time PCR, etc.) can be cited, but quantitative PCR or DNA chip analysis is preferred. When the marker gene is detected without extracting RNA from the cell, in situ hybridization can be used as a detection means, which is suitable for the present invention because the expression of the differentiation marker can be directly confirmed on the serum contact surface of the cell.

[0140] Alternatively, the expression of differentiation marker genes can be confirmed by investigating as follows: preparing a protein fraction from the cell and detecting the translation product of the marker gene contained in the fraction (i.e., marker protein); or directly detecting the translation product of the marker gene in the cell without extracting protein from the cell. The detection of marker proteins can be performed by immunological determination methods (e.g., ELISA, FIA, RIA, Western blotting, etc.) using antibodies against each protein, and for proteins with measurable physiological activity such as enzymes, the physiological activity can also be measured using known methods for each marker protein. Alternatively, the detection of marker proteins can also be performed using mass spectrometry such as MALDI-TOFMS.

[0141] In a preferred embodiment, the cells contained in the cell sheet prepared by the method of the present invention can secrete factors that inhibit angiogenesis. For example, although not limited thereto, the cell sheet prepared by the method of the present invention from iris pigment epithelial cells can secrete factors that inhibit choroidal angiogenesis, namely soluble Flt-1 (VEGF receptor 1) and TIMP-3.

[0142] In another preferred embodiment, the cells included in the cell sheet produced by the method of the present invention are unlikely to induce cell migration, and detachment of cells from the cell sheet due to cell migration is hardly observed.

[0143] The cell sheet produced by the method of the present invention has rigidity and is not likely to cause migration of cells from the cell sheet. The cell sheet of the present invention can be applied to a local part of a living body that has been administered using a cell suspension so far, and is not likely to cause migration of cells that cause problems with the suspension. Therefore, the cell sheet of the present invention can be used for target diseases that are preferably administered locally.

[0144] Hereinafter, a description will be given of a method of producing a cell sheet using pigment epithelial cells such as retinal pigment epithelial cells or iris pigment epithelial cells by the method of the present invention, but the present invention is not limited thereto.

[0145] When using retinal pigment epithelial cells and making a cell sheet by the method of the present invention, the membrane containing the extracellular matrix in the above-mentioned step (4) is a Bruch's membrane in one mode. The so-called "Bruch's membrane" is a thin film located between the retinal pigment epithelium and the choroid, and is a layer that lines the retinal pigment epithelium. The Bruch's membrane is an acellular structure with collagen fibers as the main body, the choroid adheres to the pigment epithelial cells, and can play the role of transporting substances from the choroid to the flow channel of the outer layer of the retina without blood vessels. In the method of the present invention, by forming a membrane containing the extracellular matrix produced by the cell itself, such as a basement membrane, a Bruch's membrane containing elastic fibers, a strength that can withstand a transplant operation can be obtained. In addition, in the method of the present invention, the membrane containing the extracellular matrix can be formed on the opposite side of the cell in contact with the porous membrane, and therefore, it is easy to separate the cell sheet from the first cell culture container in the form of a membrane containing the extracellular matrix as a whole.

[0146] The present invention also provides an in vitro method for preparing a membrane containing an extracellular matrix, wherein the method is characterized in that, in the method for preparing the cell sheet, the membrane containing the extracellular matrix formed is separated from the cell sheet. The membrane containing the extracellular matrix formed on the retinal pigment epithelium can be peeled off and recovered from the sheet by treating with EDTA or by performing pipetting and suction operations on the sheet surface using a culture medium without adding magnesium or potassium. In addition, the membrane can also be peeled off and recovered by the following method: suctioning the culture solution to contact it with air, and applying a dehydrating effect to the sheet surface by diluting alcohol treatment.

[0147] The present invention also relates to a cell sheet obtained according to the above-mentioned cell sheet preparation method, preferably a pigment epithelial cell sheet such as a retinal pigment epithelial cell sheet or an iris pigment epithelial cell sheet. The cell sheet of the present invention can be used as a biological use, for example, for screening purposes, toxicity test purposes and various uses. In addition, the cell sheet of the present invention can be appropriately transplanted to an object requiring tissue transplantation as a transplant material for disease treatment. In particular, the retinal pigment epithelial cell sheet of the present invention is suitable as a transplant material for retinal treatment of patients with eye diseases. As eye diseases, for example: age-related macular degeneration, retinitis pigmentosa, retinal angioid streaks, etc. can be listed. In addition, the retinal pigment epithelial cell sheet of the present invention can also be used for various screening purposes such as drug efficacy screening and toxicity evaluation for the above-mentioned eye diseases. For example, according to the method described in Japanese Patent Publication No. 2007-517210, the cell sheet of the present invention can be applied to the screening of toxic substances. Furthermore, the retinal pigment epithelial cell sheet of the present invention can also be used as an invitro model to evaluate the various functions of retinal pigment epithelial cells in vivo, such as the phagocytic ability of photoreceptor outer segments, neuroprotective effects and other functions related to the maintenance of photoreceptors, pumping effects, and retinal vascular barrier functions generated by tight junctions.

[0148] The present invention is also a cell sheet, which is obtained according to the aforementioned cell sheet preparation method and is in a state of being maintained on a porous membrane, preferably a retinal pigment epithelial cell sheet. The prepared cell sheet is in a state of being maintained (fixed) on the membrane, and can prevent shape changes such as shrinkage, flipping or rolling up. In addition, since it is maintained on the membrane, even if physical stimulation such as movement and transportation is given, the aforementioned shape change can be prevented, and a good state can be maintained for transplantation.

[0149] The disease treatment transplant material of the present invention can be used to treat diseases in humans and mammals other than humans (eg, monkeys, mice, rats, dogs, cows, horses, pigs, sheep, goats, cats, rabbits, hamsters, guinea pigs, etc.).

[0150] The range of the disease site to which the disease treatment transplant material of the present invention can be applied can vary depending on the target disease, the type of animal to be administered, age, sex, weight, symptoms, etc. For example, when applied to age-related macular degeneration, the range of the disease site to be transplanted is generally 0.07 cm 2 ~0.28cm 2 range.

[0151] The disease treatment transplant material of the present invention can be transplanted once or several times. The number of transplant applications can be determined by medical personnel according to the treatment guidelines based on the disease. For example, when applied to age-related macular degeneration, the retinal pigment epithelial cell sheet of the present invention can be transplanted more than twice depending on the severity. In addition, when multiple transplants are performed, the interval is not particularly limited and can also be set to a period of several days to several weeks.

[0152] The disease treatment transplant material of the present invention can be transplanted by medical personnel according to treatment instructions and according to appropriate transplantation methods. For example, for the treatment site in the chest or abdominal cavity, thoracotomy or laparotomy can be applied, and in addition, endoscopic methods can also be used. In addition, when the retinal pigment epithelial cell sheet is transplanted as the disease treatment transplant material of the present invention under the retina, a transplantation method can be performed along the water flow from the injection needle, and the injection needle is inserted into the transplant site under the retina of the eyeball, and in addition, it can also be performed by a dedicated delivery treatment device.

[0153] The present invention further provides a membrane comprising an extracellular matrix, which is obtained by the aforementioned method for making a membrane comprising an extracellular matrix. The making of such a membrane, in addition to the method of peeling off the membrane from the sheet after making a cell sheet using the method of the present invention, can obtain the membrane by a method of removing cells. The removal of cells can be carried out using methods such as freeze-drying and freeze-thaw. Therefore, the method for making a membrane comprising an extracellular matrix of the present invention can further include a process of removing cells and obtaining the membrane by a freeze-drying process in the manufacturing method of the cell sheet of the present invention mentioned above. The membrane comprising an extracellular matrix of the present invention is a basement membrane without cells, and since the situation of transplanting a membrane is less restricted in terms of specifications than the situation of transplanting cells, the difficulty of practical application is low. In addition, it is also helpful for research purposes such as functional analysis.

[0154] The above describes the method of using retinal pigment epithelial cells and making a cell sheet by the method of the present invention. When another cell type is used as a cell derived from a mammal, a person skilled in the art can clearly understand that the object of administration, the target disease, the transplantation method, etc. can be appropriately changed according to the cells used, referring to the treatment instructions. When fibroblasts are used as cells derived from mammals, the target disease is not limited to this, and skin-related diseases can be listed. In addition, when mesenchymal stem cells are used as cells derived from mammals, the target disease is not limited to this, and retinal epithelial deformation diseases and nerve damage diseases can be listed.

[0155] In one embodiment, the present invention provides a multilayer cell sheet and a method for manufacturing the same, wherein the multilayer cell sheet comprises a plurality of cell sheets. The multilayer cell sheet can be manufactured by repeating the following steps: on cells that have been sheeted by the above-mentioned method for manufacturing a cell sheet, cells are sown again to form a cell sheet. Each cell sheet included in the multilayer cell sheet may be a cell sheet containing a single cell type or a cell sheet containing multiple cell types. Each cell sheet included in the multilayer cell sheet may contain different cell types or the same cell type.

[0156] Combining cell sheets composed of cells with different functions, or multi-layering cell sheets containing cells with different functions, can form artificial tissues (Allergy, 2013, Vol. 62, pp. 25-32). For example, artificial lymphoid tissue or artificial bone marrow can be produced by forming a cell sheet or a multi-layer cell sheet using a cell suspension, wherein the cell suspension is a mixture of stromal cells or mesenchymal stem cells and hematopoietic cells such as antigen-presenting cells and lymphocytes or hematopoietic stem cells. Such a multi-layer cell sheet can be used, for example, for the treatment of hematopoietic failure such as immunodeficiency, severe infection, and malignant tumors.

[0157] By forming mesenchymal stem cells into sheets, the expression of their marker CD105 is reduced or eliminated, and a CD105-negative mesenchymal subtype with a strong immunosuppressive property can be induced. The multilayered cell sheet formed by multilayering such a mesenchymal stem cell sheet can be used, for example, to prevent or suppress rejection reactions after organ transplantation, to prevent or suppress rejection reactions during allogeneic transplantation of mesenchymal stem cells, and to treat autoimmune diseases.

[0158] Therefore, in one embodiment, the present invention provides a method for producing a multilayered cell sheet, comprising the following steps (1) to (4).

[0159] Step (1), preparing one or more mammal-derived cells;

[0160] Step (2), seeding the prepared cells on the porous membrane of a first cell culture container having a porous membrane on the bottom surface;

[0161] Step (3), placing the first cell culture container further into a second cell culture container, culturing cells, and preparing a cell sheet on the porous membrane; and

[0162] Step (4), repeating the following steps (a) and (b) once or more,

[0163] Step (a), seeding one or more mammal-derived cells on a cell sheet formed in a first cell culture container,

[0164] Step (b) is to place the first cell culture container in a second cell culture container, culture cells, and prepare a new cell sheet on the cell sheet.

[0165] In one embodiment, the present invention provides a multilayered cell sheet produced by the above method and a transplant material for treating a disease comprising the multilayered cell sheet.

[0166] In one embodiment, the present invention provides a multilayer cell sheet, which is arranged on a porous membrane, wherein an extracellular matrix exists between cells in each cell sheet included in the multilayer cell sheet, and a membrane containing the extracellular matrix is ​​formed on the side opposite to the side in contact with the aforementioned porous membrane. Furthermore, in the multilayer cell sheet, the membrane containing the extracellular matrix may be formed between each layer in addition to being formed on the side opposite to the side in contact with the aforementioned porous membrane of the cell sheet.

[0167] [Example]

[0168] Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to the following examples.

[0169] Example 1

[0170] Formation of mesenchymal stem cell sheets derived from human adipose tissue

[0171] Mesenchymal stem cells derived from human adipose tissue (PromoCell GmbH Sickingenstr. 63 / 6569126 Heidelberg Germany) obtained from human tissue were suspended in a culture medium for mesenchymal stem cells. The prepared cell suspension was added to the porous membrane (6.5 mm diameter Transwell with 0.4 μm porous polyester membrane) on the bottom of the cell culture insert, with 2.0×10 5 cells or 10×10 5 Cell seeding. Cultivate the cells while immersing them in the wells of the cell culture insert that have been pre-filled with culture medium, and confirm the formation of a cell sheet ( Figure 1 ). It was confirmed that any number of cells can be used to prepare a cell sheet. 5 Cells / container (bottom diameter 6.5mm) is equivalent to about 6000 cells / mm 2 By seeding cells at such a high density, it was observed that a cell sheet that did not shrink could be obtained. Therefore, in the following experiments, 2.0×10 5 The cells are seeded at a cell number greater than that of the cells to prepare a cell sheet.

[0172] When the obtained cell sheet was observed under a microscope, it was observed that a membrane containing an extracellular matrix was formed on the opposite side of the membrane, and the cell sheet could be easily separated from the cell container by applying a water flow generated by physical pipetting. Figure 2 The figure shows a state where a cell sheet formed into a film is separated from a cell container by a water flow generated by pipetting.

[0173] The cell sheet separated from the cell container was transferred to a culture dish filled with physiological saline by pipetting. The cell sheet was sucked up in the culture dish using a pipette and then discharged. It was confirmed that the cell sheet could maintain its shape and had rigidity, and that it was easy to move the cell sheet during transplantation. Figure 3 The pipetting situation is shown in the figure.

[0174] The cell sheets separated from the cell container were fixed with 10% triformaldehyde, and frozen sections with a thickness of 14 μm were prepared, and then the cell nuclei were stained with DAPI. As a result, it was confirmed that the obtained cell sheets had a thickness of 20-40 μm ( Figure 4 ).

[0175] The cell sheets separated from the cell container were stored at 2-8°C for 96 hours, and then cultured for 48 hours to measure the secretion of PEDF involved in protecting photoreceptors and VEGF essential for maintaining choroidal microvessels by the cell sheets by ELISA. The results are shown in Table 1 below.

[0176] [Table 1]

[0177]

[0178] In addition, the expression of each molecular marker was confirmed by immunohistochemical staining according to the conventional method. It was confirmed that the mesenchymal stem cell sheet prepared by the method of the present invention can express elastin, type IV collagen, type I collagen, Zo-1 ( Figure 5 ).

[0179] Furthermore, when the prepared mesenchymal stem cell sheet was transplanted into retinal degeneration rats (Royal College Surgeon [RCS] rats), it was confirmed that the retina protection effect could be obtained. In observations 3 weeks after transplantation, the rats transplanted with the mesenchymal stem cell sheet prepared by the method of the present invention could still maintain the thickness of the outer granular layer (the layer where the nuclei of the visual cells are arranged) ( Figure 6 On the other hand, the outer granular layer of the diseased rats that did not receive the cell sheet was thinned ( Figure 6Right photo). When the thickness of the outer granular layer (i.e., the degree of protection of visual cells) was scored by comparing the treated eye (right) with the untreated eye (left), a statistically significant difference was found between the mesenchymal stem cell sheet transplantation group (n=10) and the Sham group (n=5) (p<0.01, Wilcoxon rank sum test).

[0180] In addition, as shown in the following Table 2, the photoreceptors of rats transplanted with the mesenchymal stem cell sheet prepared by the method of the present invention were protected. It was confirmed that transplantation of the mesenchymal stem cell sheet prepared by the method of the present invention can inhibit the progression of lesions and protect photoreceptors.

[0181] [Table 2]

[0182]

[0183] As a safety test of the mesenchymal stem cell sheet produced by the method of the present invention, a general toxicity test (GLP test) was performed using immunodeficient rats. No implantation defects / early shedding and no other harmful phenomena were found.

[0184] Example 2: Study on the number of cells to be seeded

[0185] A cell sheet was prepared using human adipose tissue-derived mesenchymal stem cells in the same manner as in Example 1. A cell suspension was added to a container having a porous membrane with a diameter of 6.5 mm on the bottom surface so that the following cell numbers were obtained. The number of cells per container was 0.08×10 5 cells, 0.4×10 5 cells, 2.0×10 5 cells, 10×10 5 cells, 13×10 5 cells, 20×10 5 The cell concentration of each container's bottom area was equivalent to about 240 cells / mm 2 Bottom area, about 1200 cells / mm 2 Bottom area, about 6000 cells / mm 2 Bottom area, 30,000 cells / mm 2 Bottom area, about 40,000 cells / mm 2 Bottom area, about 60,000 cells / mm 2 The formation of cell sheets was confirmed at all cell concentrations. The results are shown in Figure 7 .

[0186] Example 3

[0187] Formation of iris pigment epithelial cell sheets

[0188] A cell sheet was prepared in the same manner as in Example 1 using iris pigment epithelial cells obtained from human iris tissue.

[0189] In vitro experiments showed that the obtained iris pigment epithelial cell sheets exhibited (1) phagocytic ability and (2) secretion of PEDF and VEGF possessed by normal retinal pigment epithelial cells, and (3) had a membrane structure containing an extracellular matrix that could function as a scaffold for cell regeneration, similar to Bruch's membrane.

[0190] After the cell sheets were stored at 2-8°C for 96 hours, PEDF involved in protecting photoreceptors and VEGF essential for maintaining choroidal microvessels secreted by the cell sheets were measured by ELISA at 48 hours of culture. The measurement results are shown in Table 3 below.

[0191] [Table 3]

[0192]

[0193] exist Figure 8 The data showing the phagocytic ability of the cell sheets cultured in the same manner are shown in Figure 2. It should be noted that the phagocytic ability test was performed using a Phagocytosis assay kit manufactured by Cayman Chemical (US). The test was performed by mixing the cell sheets with fluorescently labeled latex beads and culturing them, then washing with trypan blue, and then observing the phagocytic latex beads using a fluorescent microscope. The sheet in which the fluorescent label that was not bound to the beads was reacted with a secondary antibody was used as a negative control group. Figure 8 In the figure, the upper two photographs show the data of the negative control, and the lower two photographs show the data of the cell sheet prepared by the method of the present invention.

[0194] The expression of each molecular marker was confirmed by conventional methods. It was confirmed that the retinal pigment epithelial cell sheet prepared by the method of the present invention expressed elastin, type IV collagen, type I collagen ( Fig. 9 ).

[0195] When the iris pigment epithelial cell sheet prepared by the method of the present invention was transplanted into retinal deformity rats (RCS rats), it was confirmed that a retinal protective effect was obtained ( Fig.10 At 4 weeks after transplantation, rats transplanted with the retinal pigment epithelial cell sheet prepared by the method of the present invention can still maintain the thickness of the outer granular layer (the layer of the nucleus of the visual cell) ( Fig.10 In addition, the photoreceptors of rats transplanted with the retinal pigment epithelial cell sheets prepared by the method of the present invention were protected ( Fig.10chart below).

[0196] As a safety test of the iris pigment epithelial cell sheet produced by the method of the present invention, a general toxicity test (GLP test) was performed using immunodeficient rats, and no implantation defects / early shedding and other harmful phenomena were found. It should be noted that the transplantation method, which is considered to be the biggest problem in sheet-shaped products, has been continuously tested on primates and established.

[0197] Example 4

[0198] Fibroblast sheet formation

[0199] A cell sheet was prepared in the same manner as in Example 1 using fibroblasts obtained from human tissue.

[0200] In vitro experiments showed that the obtained fibroblast sheets exhibited (1) phagocytic ability and (2) secretion of PEDF and VEGF possessed by normal retinal pigment epithelial cells, and (3) had a membrane structure containing an extracellular matrix that could function as a scaffold for cell regeneration similar to Bruch's membrane.

[0201] After the cell sheets were stored at 2-8°C for 96 hours, PEDF and VEGF secreted by the cell sheets were measured by ELISA at 48 hours of culture. The results are shown in Table 4 below. Similarly, data showing that the cultured cell sheets have phagocytic ability are shown in Fig.11 .

[0202] [Table 4]

[0203]

[0204] The expression of each molecular marker was confirmed by immunohistochemical staining according to the conventional method. It was confirmed that the fibroblast sheet prepared by the method of the present invention can express type I collagen, type IV collagen, elastin, ZO-1 ( Fig.12 ).

[0205] When the fibroblast retinal pigment epithelial cell sheet prepared by the method of the present invention was transplanted into retinal deformity rats (RCS rats), it was confirmed that the retinal protective effect could be obtained (Table 5). At the time point of 4 weeks after transplantation, it was confirmed that the preservation (maintenance of thickness) of the outer granular layer (layer of the nucleus of the visual cell) was significantly thinned ( Fig.13 ).

[0206] [Table 5]

[0207]

[0208] The target cell purity of the cell sheet was determined by confirming the expression status of CD90, vimentin, and CK18. As shown in Table 6 below, the results confirmed that the target cell purity was maintained at a high level in the cell sheets used for toxicity tests and efficacy tests. In addition, it was also confirmed that the sample after transportation still maintained a high purity.

[0209] [Table 6]

[0210] Isotype Benchmark CD90 Vimentin CK18 Toxicity test 1 sample 98.04％ 98.16％ 0.00％ Toxicity test 2 sample 97.72％ 92.95％ 0.14％ Efficacy test samples 99.87％ 98.60％ 0.10％ Sample after transportation 88.29％ 85.11％ 11.71％

[0211] As a safety test of the fibroblast sheet produced by the method of the present invention, a general toxicity test (GLP test) was performed using immunodeficient mice. No implantation defects / early shedding and no other harmful phenomena were found.

[0212] Example 5

[0213] Confirmation of secretion of angiogenesis inhibitory factors from mesenchymal stem cell sheets

[0214] The cell sheet was prepared using human adipose tissue-derived mesenchymal stem cells obtained from human tissue in the same manner as in Example 1. As a comparative example, human adipose tissue-derived mesenchymal stem cells obtained from human tissue were used for planar culture. Planar culture is to culture cells by seeding them at the most appropriate density according to the culture area in a resin cell culture container and allowing adherent cells to adhere to the culture surface.

[0215] After the culture, the soluble Flt-1 (soluble VEGF receptor 1) and TIMP-3 secreted into the culture medium were measured by ELISA. The results are shown in Fig.14 The amount of soluble Flt-1 secreted from the cell sheet was significantly increased when cultured by the method of the present invention compared to the planar culture. Furthermore, TIMP-3 secretion was not detected in the planar culture, but TIMP-3 secretion was detected from the cell sheet cultured / made by the method of the present invention. This shows that the cell sheet made by the method of the present invention has the potential to inhibit the generation of choroidal neovascularization.

[0216] Example 6

[0217] Fabrication of mesenchymal stem cell laminates

[0218] The same operation as in Example 1 was performed to prepare a cell sheet (monolayer) of mesenchymal stem cells on the membrane. Furthermore, new mesenchymal stem cells were seeded on the prepared cell sheet at the same concentration and cultured to prepare a laminated sheet. The results are shown in Fig.15 The color tone shows that the stacked cell sheet is thicker than the single cell sheet on the left.

[0219] Example 7

[0220] Evaluation of migration of cells from mesenchymal stem cell sheets

[0221] Mesenchymal stem cells derived from human adipose tissue were used to evaluate the migration of cells in a cell suspension and a cell sheet made of cells according to the present invention. The migration of cells was determined by using a cell counting kit-8 (Cell Counting Kit-8, Dojindo, Cat. CK04) and a 1% crystal violet solution (SIGMA, Cat. V5265-250ML) to determine whether cells on the inner side of the cell culture plug migrate to the outer side of the plug through the membrane. The pore size of the membrane was 8 μm. 20% or 40% fetal bovine serum albumin was added as a cell attractant.

[0222] Cell suspension: Add cell attractant (or control group) and basal medium to the cell culture plate (wells and cell culture inserts), then seed the cell suspension on the inserts. After 24 hours, remove the cells in the inserts and evaluate the number of cells in the wells. No attractant is added to the control group.

[0223] Cell sheet: The cell sheet prepared by the method of the present invention is used for evaluation in a state of being maintained in the first culture container (insert). After the medium in the insert containing the cell sheet and the medium in the cell culture plate (well) are replaced with the basal medium for adjustment, the medium in the insert is replaced with the basal medium, and the medium in the cell culture plate (well) is replaced with the basal medium containing the cell attractant (or control group). After 24 hours, the cells in the insert are removed and the number of cells in the well is evaluated.

[0224] The evaluation was performed with n=3 in each group. The cell count was evaluated using Cell Counting Kit-8 (Dojindo) and 1% crystal violet solution (SIGMA). The results are shown in Fig.16 While cell migration was observed in the cell suspension, cell migration was not observed in the cell sheet formed by the method of the present invention.

[0225] The above detailed description is only used to illustrate the purpose and object of the present invention, and does not limit the scope of the claims. Without departing from the scope of the claims, those skilled in the art can make various changes and substitutions to the described embodiments based on the teachings described in this specification.

[0226] This application is based on Japanese Patent Application No. 2022-73877 (filing date: April 27, 2022) and Japanese Patent Application No. 2023-34995 (filing date: March 7, 2023) filed in Japan, and all the contents of these applications are included in this specification.

[0227] Industrial Applicability

[0228] The present invention provides a method for preparing a cell sheet, which is a simple method for preparing a cell sheet, and the cell sheet prepared by the method is easy to handle. The present invention is helpful for preparing a cell sheet, and the cell sheet can be applied to various cells including MSCs and skin tissue cells, i.e., fibroblasts, and the cell sheet prepared by the method of the present invention can be used as a therapeutic transplant material and also for various purposes such as screening purposes and toxicity test purposes.

Claims

1. A method for preparing a cell sheet, comprising the following steps: Step (1), preparing one or more mammal-derived cells; Step (2), seeding the prepared cells on the porous membrane of a first cell culture container having a porous membrane on the bottom surface; and Step (3) is to place the first cell culture container in a second cell culture container, culture cells, and prepare a cell sheet on the porous membrane.

2. The method of claim 1, wherein: At least one of the culture medium in the first cell culture container or the culture medium in the second cell culture container is a culture medium containing serum and / or a Rho kinase (ROCK) inhibitor.

3. The method of claim 1, wherein: In step (1), the mammal-derived cells are epithelial cells, endothelial cells, parenchymal cells or stem cells.

4. The method of claim 3, wherein: The mammal-derived cells are cells selected from the group consisting of pigment epithelial cells, fibroblasts and mesenchymal stem cells.

5. The method of claim 4, wherein: In step (2), 5000 cells / mm 2 The cells are seeded on the bottom surface.

6. The method according to any one of claims 1 to 5, wherein: In the cell sheet obtained by culture, a membrane containing an extracellular matrix is ​​formed on the side of the cells opposite to the side in contact with the porous membrane.

7. The method of claim 6, wherein: The cell sheet obtained by culture further has tight junctions formed between cells.

8. The method according to claim 6, further comprising the following step (4): In step (4), it is confirmed that a membrane containing an extracellular matrix is ​​formed on the side of the cell opposite to the side in contact with the porous membrane.

9. The method according to any one of claims 1 to 5, further comprising the following step (5): Step (5) is to confirm whether or not differentiation markers are expressed in the cultured cells obtained in step (3).

10. The method according to any one of claims 1 to 5, further comprising the following step (5'): Step (5') is to confirm whether the cultured cells obtained in step (3) secrete soluble Flt-1 (VEGF receptor 1) and / or TIMP-3.

11. The method according to any one of claims 1 to 4, wherein: In step (1), the mammalian-derived cells are retinal pigment epithelial cells, iris pigment epithelial cells, mesenchymal stem cells or fibroblasts, and in step (2), the number of cells is 5000 / mm 2 ~40,000 cells / mm 2 The cells are seeded on the bottom surface.

12. A cell sheet produced by the method according to any one of claims 1 to 5.

13. A transplant material for treating a disease, comprising a cell sheet produced by the method according to any one of claims 1 to 5. 14 . A method for producing a membrane containing an extracellular matrix, comprising the step of isolating the membrane containing an extracellular matrix formed on a cell sheet produced by the method according to claim 1 .

15. A cell sheet comprising mammalian-derived cells arranged on a porous membrane, wherein an extracellular matrix exists between cells and a membrane containing the extracellular matrix is ​​formed on the side opposite to the side in contact with the porous membrane.

16. The cell sheet according to claim 15, wherein The cell sheet further has tight junctions formed between cells.

17. The cell sheet according to claim 15, wherein The cell sheet is obtained by seeding one or more mammal-derived cells on a porous membrane on the bottom of a first cell culture container, and placing the first cell culture container in a second cell culture container for culturing.

18. The cell sheet according to claim 17, wherein At least one of the culture medium in the first cell culture container or the culture medium in the second cell culture container is a culture medium containing serum and / or a Rho kinase (ROCK) inhibitor.

19. The cell sheet according to claim 17 or 18, wherein The mammal-derived cells are epithelial cells, endothelial cells, parenchymal cells or stem cells.

20. The cell sheet according to claim 19, wherein The mammal-derived cells are cells selected from the group consisting of pigment epithelial cells, fibroblasts and mesenchymal stem cells.

21. The cell sheet according to claim 19, wherein The mammal-derived cells are retinal pigment epithelial cells or iris pigment epithelial cells.

22. The cell sheet according to claim 19, wherein The cells contained in the cell sheet express at least one molecular marker selected from the group consisting of the following substances: yolk maculopathy protein-1, RPE-65, pan-spectrum cytokeratin and any subtype of cytokeratin, occludin, ZO-1, elastin, actin, type 1 collagen, type 2 collagen and type 4 collagen.

23. The cell sheet according to claim 19, wherein The cells contained in the cell sheet secrete soluble Flt-1 (VEGF receptor 1) and / or TIMP-3.

24. A method for producing a multilayered cell sheet, comprising the following steps (1) to (4): Step (1), preparing one or more mammal-derived cells; Step (2), seeding the prepared cells on the porous membrane of a first cell culture container having a porous membrane on the bottom surface; Step (3), placing the first cell culture container further into a second cell culture container, culturing cells, and preparing a cell sheet on the porous membrane; and Step (4), repeating the following steps (a) and (b) once or more, Step (a), seeding one or more mammal-derived cells on a cell sheet formed in a first cell culture container, Step (b) is to place the first cell culture container in a second cell culture container, culture cells, and prepare a new cell sheet on the cell sheet.

25. A multilayer cell sheet produced by the method of claim 24.

26. A multilayer cell sheet disposed on a porous membrane, wherein an extracellular matrix exists between cells in each cell sheet included in the multilayer cell sheet, and a membrane containing the extracellular matrix is ​​formed on the side opposite to the side in contact with the porous membrane.

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