Cell sheet manufacturing device and cell sheet

Abstract

To provide a cell sheet manufacturing apparatus that allows easy observation of both sides of a cell sheet in a state where a position of the cell sheet in a culture medium is stable in order to confirm the growth status of cells constituting the cell sheet, and also provide a cell sheet capable of ensuring its sufficient thickness and improving its strength.SOLUTION: A cell sheet manufacturing apparatus (100) comprises: a container (20); and a support unit (10) that has a mesh sheet (2) and a base (3) for holding the mesh sheet (2) so as to float above a bottom surface of the container (20), and is separably housed in the container (20). The support unit (10) is housed in the container (20) so that its position in a vertical direction and a horizontal direction is constant in a culture medium.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a cell sheet manufacturing apparatus and a cell sheet. [Background technology]

[0002] Cell sheets are currently being used as skin sheets for treating burn sites, etc. In the future, cell sheets may also be used as cardiomyocyte sheets to be transplanted into patients with heart failure or pancreatic islet cell sheets to be transplanted into patients with diabetes, and it is expected that they will be applied to many patients.

[0003] On the other hand, attempts have been made to reconstruct tissue structures similar to those in vivo using cell sheets consisting of multiple cell layers. Therefore, the importance of a method for producing a cell sheet in which cells are arranged three-dimensionally has been recognized. When produced by a cell sheet production method using an existing temperature-responsive culture dish, the cell sheet produced is often difficult to handle because of its fragility (see Non-Patent Documents 1 and 2). In addition, when attempting to produce a cell sheet consisting of multiple cell layers (dermis layer and epidermis layer) on a dish, there is a concern that the mature epidermis layer forms a kind of barrier called tight junction, which inhibits the supply of nutrients to the inside of the cell sheet.

[0004] Also, a method of preparing a cell sheet on a non-cellular sheet-like support has been proposed. However, if the support is not compatible with the cells of the affected area, this method does not allow the exposed surface of the support to be attached to the affected area. In other words, the surface of the cell sheet that is attached to the affected area is limited to one side. When attaching a cell sheet in which a keratinocyte layer (epidermis layer) is arranged on a fibroblast layer (dermis layer) to an affected area, it is extremely inconvenient that the surface to be attached is limited as described above. This is because, when transplanting a cell sheet, it is necessary to attach the surface on the dermis layer side to the affected area.

[0005] Another known method is to prepare a cell sheet consisting of multiple cell layers on a porous membrane (e.g., a porous membrane made of polycarbonate) (see Non-Patent Document 3). The cell sheet prepared by this method is expected to supply nutrients to cells through the pores of the porous membrane, and therefore is superior in supplying nutrients to cells compared to a multi-layered cell sheet prepared on a dish. However, even when a cell sheet is prepared on a porous membrane, the prepared cell sheet is difficult to use for transplantation because only one side is attached to the affected area. Therefore, the cell sheet prepared by the above method is mainly used in permeability tests of compounds, etc.

[0006] A method for producing a cell sheet using a nanofiber sheet made of gelatin, polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), etc. has been proposed (see Non-Patent Document 4). In this method, a sheet made of nanofibers is produced by electrospinning, and cells are cultured on the sheet. This method has the problem that it is difficult to precisely control the arrangement of each nanofiber by electrospinning. Furthermore, due to the problem of the irregularity of the nanofibers, it may be difficult to obtain highly reproducible results from the produced cell sheet.

[0007] In addition, the use of a micromesh sheet has been proposed to prepare a cell sheet. As a method using a micromesh sheet, for example, (i) a method of culturing cells on a micromesh sheet to prepare a two-dimensional cell sheet, and (ii) a method of culturing iPS cells on a micromesh sheet to prepare spherical trophoblast-like cells have been reported (see Patent Documents 1 and 2, and Non-Patent Document 5). As an application of the cell culture method using this micromesh sheet, a technology has been developed to culture an established hepatic cell line, HepG2 cells, on a micromesh sheet to prepare a three-dimensional cell sheet. It has been suggested that this three-dimensional cell sheet has some improved cell functions compared to three-dimensional spheroids, is excellent in cell observation and nutrient supply, and is compatible with fluid devices (see Non-Patent Document 6). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication WO2015 / 005349 Brochure (Published January 15, 2015) [Patent Document 2] JP 2019-50773 A (Published on April 4, 2019) [Non-patent literature]

[0009] [Non-Patent Document 1] Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci US A. 1979;76(11):5665-8. [Non-Patent Document 2] Yamada N, Okano T, Sakai H, Karikusa F, Sawasaki Y, Sakurai Y. Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells. Die Makromolekulare Chemie, Rapid Communications. 1990;11(11):571-6. [Non-Patent Document 3] Kojima H, Ishii I, Nakata S, Konishi H. Dose-response Evaluation Using an Epidermal Model, an Alternative to Skin Irritation Testing. Alternatives to Animal Testing and Experimentation. 2006;11(3):177-84. [Non-Patent Document 4] Li J, Minami I, Shiozaki M, Yu L, Yajima S, Miyagawa S, et al. Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium. Stem Cell Reports. 2017;9(5):1546-59. [Non-Patent Document 5] Okeyo KO, Kurosawa O, Yamazaki S, Oana H, Kotera H, Nakauchi H, et al. Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells. Tissue Eng Part C Methods. 2015;21(10):1105-15. [Non-Patent Document 6] Hori T, Kurosawa O. A Three-dimensional Cell Culture Method with a Micromesh Sheet and Its Application to Hepatic Cells. Tissue Eng Part C Methods. 2018. Summary of the Invention [Problem to be solved by the invention]

[0010] In a method for producing a cell sheet using a micromesh sheet, first, the mesh sheet is placed in a container while floating in a medium for culturing cells. Next, cells are seeded on the micromesh. Then, the cells seeded on the micromesh sheet are cultured in the medium. In such a method for producing a cell sheet, both sides of the cell sheet need to be examined under a microscope to check the growth status of the cells that compose the cell sheet.

[0011] For example, in the technology disclosed in Patent Document 1, a micromesh sheet for seeding cells is attached to a hanging member attached to a container. In such a configuration, it is difficult to (i) invert the cell sheet for culturing and (ii) invert the cell sheet for observation under a microscope (i.e., double-sided observation).

[0012] In addition, in the device disclosed in Patent Document 2, the micromesh sheet is held in a groove provided on the inner wall of the container that contains the culture medium. In this configuration, it is difficult to separate the micromesh sheet from the container and place it upside down. This makes it difficult to observe both sides of the cell sheet under a microscope.

[0013] The above-mentioned difficulties in observing cells can be particularly problematic, for example, when preparing a cell sheet consisting of multiple cell layers.

[0014] A first objective of one aspect of the present invention is to realize a cell sheet manufacturing apparatus that allows easy observation of both sides of a cell sheet while maintaining a stable position of the cell sheet in a culture medium in order to check the growth status of the cells that constitute the cell sheet.

[0015] A second object of one embodiment of the present invention is to provide a cell sheet that can ensure a sufficient thickness and improve strength. [Means for solving the problem]

[0016] In order to solve the above problems, a cell sheet manufacturing apparatus according to one embodiment of the present invention comprises a container for containing a culture medium for culturing cells, a mesh sheet which is a substrate to which the cells are attached and cultured, and a holding member for holding the mesh sheet so that it floats above the bottom surface of the container, and a support unit which is contained in the container so that it can be detached from the container, and is characterized in that the support unit is contained in the container so that its vertical and horizontal positions in the culture medium are constant.

[0017] A cell sheet according to one embodiment of the present invention is characterized in that it has at least two cell layers and a mesh sheet, which serves as a substrate for attaching and culturing cells, is disposed between the two cell layers. Effect of the Invention

[0018] According to one aspect of the present invention, a cell sheet manufacturing device can be realized that allows easy observation of both sides of a cell sheet with the cell sheet in a stable position in a culture medium in order to check the growth status of cells in the cell sheet. Furthermore, according to one aspect of the present invention, a cell sheet can be realized that can ensure a sufficient thickness and improve strength. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a diagram showing a schematic configuration of a cell sheet manufacturing device of Configuration Example 1. [Diagram 2] FIG. 2 is a diagram for explaining the effect of the cell sheet manufacturing apparatus shown in FIG. [Diagram 3] FIG. 1 is a diagram showing the configuration of a cell sheet manufacturing device according to a first modified example. [Figure 4] FIG. 11 is a diagram showing the configuration of a cell sheet manufacturing device according to Modification Example 2. [Diagram 5] FIG. 2 is a schematic diagram showing the cross-sectional structure of a skin sheet produced using a cell sheet manufacturing apparatus according to one embodiment of the present invention. [Figure 6] FIG. 13 is a diagram showing a schematic configuration of a support unit provided in a cell sheet manufacturing apparatus of Configuration Example 2. [Figure 7]FIG. 13 is a diagram showing a schematic configuration of a cell sheet manufacturing device of Configuration Example 2. [Figure 8] FIG. 13 is a diagram showing a schematic configuration of a cell sheet manufacturing device of Configuration Example 3. [Figure 9] FIG. 13 is a diagram showing a schematic configuration of a cell sheet manufacturing device of Configuration Example 4. [Figure 10] FIG. 1 shows the results of Example 1. [Figure 11] FIG. 1 shows the results of Example 1. [Figure 12] FIG. 1 shows the results of Example 2. [Figure 13] FIG. 1 shows the results of Example 3. [Figure 14] FIG. 1 shows the results of Example 4. [Figure 15] FIG. 1 shows the results of Example 5. [Figure 16] FIG. 1 shows the results of Example 5. [Figure 17] FIG. 1 shows the results of Example 5. [Figure 18] FIG. 1 shows the results of Example 6. [Figure 19] FIG. 1 shows the results of Example 7. [Figure 20] FIG. 13 is a diagram showing the configuration of a cell sheet manufacturing device according to Modification 3. [Figure 21] 13 is a diagram for explaining the effect of the cell sheet manufacturing device of Modification 3. FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] An embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to each of the configurations described below, and various modifications are possible within the scope of the claims. The embodiments and examples obtained by appropriately combining the technical means disclosed in the different embodiments and examples are also included in the technical scope of the present invention. In addition, all of the documents described in this specification are incorporated herein by reference. In this specification, when "A to B" is described with respect to a numerical range, the description intends "A or more and B or less."

[0021] 1. Cell sheet manufacturing equipment The cell sheet manufacturing apparatus according to this embodiment is an apparatus that is premised on manufacturing a cell sheet by placing a mesh sheet in a culture medium and growing cells seeded on the mesh sheet in the culture medium. By placing the mesh sheet in the culture medium (culture solution), cells can be grown according to the shape of the mesh sheet.

[0022] The cell sheet manufacturing apparatus according to this embodiment includes a container that contains a culture medium for culturing cells, and a support unit. The support unit is detachably housed in the container, and includes a mesh sheet and a holding member. The mesh sheet is a substrate for attaching and culturing cells. The holding member holds the mesh sheet so that it floats above the bottom surface of the container. The support unit is housed in the container so that its vertical and horizontal positions in the culture medium are constant.

[0023] Here, "holding the mesh sheet so that it floats above the bottom surface of the container" means that both sides of the mesh sheet are not in contact with the bottom surface of the container (preferably the inner wall and bottom surface of the container) and are held so that both sides of the mesh sheet are in sufficient contact with the culture medium. The holding member may be configured to hold the mesh sheet so that it floats above the bottom surface of the container, and the specific configuration is not limited.

[0024] In cell sheet production using the above-mentioned mesh sheet, the cell sheet production apparatus according to this embodiment improves the convenience of cell observation by the user. More specifically, in the above configuration, the support unit is detachably housed in a container. Therefore, the user can easily turn the support unit upside down by removing the support unit from the container, turning it over, and then placing it in the container again. When the support unit is turned over, the cell sheet formed on the mesh sheet held by the holding member of the support unit is also turned over. Therefore, according to the above configuration, both sides of the cell sheet formed on the mesh sheet can be easily observed with a microscope.

[0025] Furthermore, since the support unit is accommodated in the container so that its vertical and horizontal positions in the culture medium are constant, the position of the mesh sheet in the container, in other words, the position of the cell sheet formed on the mesh sheet, does not change during observation of the cell sheet under a microscope. Therefore, with the above configuration, the cell sheet can be observed precisely.

[0026] In the cell sheet manufacturing apparatus according to this embodiment, the holding member preferably holds the mesh sheet in a removably manner. This allows the cell sheet formed on the mesh sheet to be easily separated from the support unit. This makes it easier to handle the cell sheet, for example, when transplanting the cell sheet.

[0027] The support unit may be stored in the container so that its vertical and horizontal positions in the culture medium are constant, and the specific configuration is not limited. For example, the support unit may be configured such that the holding member has a higher specific gravity than the culture medium. Since the holding member has a higher specific gravity than the culture medium, the holding member does not float in the culture medium but sinks, and the vertical position is particularly stable. The specific gravity of the holding member can be appropriately set according to the type of culture medium. For example, when the specific gravity of the culture medium is 1, the specific gravity of the holding member is preferably 1.1 or more, more preferably 1.5 or more, more preferably 2.0 or more, more preferably 5.0 or more, and most preferably 10.0 or more. Examples of materials constituting the holding member include polystyrene, polyester, polyacetal, polycarbonate, polyvinyl chloride, and the like.

[0028] The mesh sheet is not particularly limited as long as it has a structure that allows cells to be seeded and the cells to grow. The mesh sheet is preferably a planar structure in which openings of a predetermined shape are arranged regularly or irregularly. It is more preferable that the mesh sheet has a large number of polygonal openings in a planar view. The shape of the openings of the mesh sheet is typically a polygon such as a triangle, a rectangle, or a hexagon, but may also be a circle, an ellipse, or another polygon.

[0029] Here, the portion of the mesh sheet other than the openings is called the frame or frame portion. When the openings are of a micrometer size, the mesh sheet may be called a micromesh. The shape of the openings of the mesh sheet can be said to be the shape of the area surrounded by the frame that constitutes the mesh sheet.

[0030] The openings of the mesh sheet may be large enough to allow at least one cell to be cultured to pass through. The openings being large enough to allow at least one cell to be cultured to pass through means that the size of the openings and the size of the cells are in a relationship in which the cells can pass through the openings with or without deformation. The openings may be large enough to allow the cells to pass through without contacting the openings, or may be large enough to allow the cells to pass through while contacting the openings (in other words, while deforming).

[0031] Preferably, the shape of the opening of the mesh sheet is a shape elongated in one direction. The term "shape elongated in one direction" refers to a shape in which, among a plurality of axes defining the shape, there is one axis (long axis) that is longer than the other axes (short axes). Examples of such shapes include a rectangle, a rhombus, an ellipse, and the like. When the shape of the opening is a rectangle, the long side corresponds to the long axis, and the short side corresponds to the short axis. When the shape of the opening is a rhombus, the longer of the two diagonals corresponds to the long axis, and the shorter diagonal corresponds to the short axis. Examples of the "shape elongated in one direction" include a rectangle whose long axis (long side) is much longer than the short side (short axis), and a rhombus or ellipse whose long axis is much longer than the short axis. When it is a rectangle, the shape elongated in one direction has a short side:long side ratio of 1:2 to 1:5, preferably 1:2 to 1:10. In addition, in the case of a rhombus, the shape elongated in one direction has a minor axis:major axis ratio of 1:2 to 1:5, preferably 1:2 to 1:10. In the case of an ellipse, the shape elongated in one direction has a minor axis:major axis ratio of 1:2 to 1:5, preferably 1:2 to 1:10. Of course, the present invention is not limited to these configurations. Note that the "shape elongated in one direction" is not limited to a rectangle, a rhombus, or an ellipse, and may be any shape in which, among multiple axial lines defining the shape, one axis is far longer than the other axes.

[0032] When the opening of the mesh sheet is elongated in one direction as in the above configuration, the opening has a large wall surface that extends long in one direction. Since a large number of cells adhere to the wall surface and grow, and grow with the same orientation, the above configuration makes it possible to control the orientation of the growing cells in the elongated direction of the opening.

[0033] The material of the mesh sheet may be any material to which cells can attach and grow, such as photocurable resin, biocompatible material, or biodegradable material.

[0034] If a photocurable resin is used, the mesh sheet can be produced by a photolithography method. Examples of the photocurable resin include acrylate compounds, methacrylate compounds, epoxy compounds, isocyanate compounds, thiol compounds, and silicone compounds. Two or more of these may be used in combination. Specific examples of the photocurable resin include, but are not limited to, urethane acrylate, polyester acrylate, epoxy acrylate, poly(methyl meth)acrylate, ethoxylated bisphenol A acrylate, aliphatic urethane acrylate, polyester acrylate, polyethylene terephthalate, polystyrene, polycarbonate, acrylic modified alicyclic epoxide, bifunctional alcohol ether type epoxide, acrylic silicone, acrylic dimethyl siloxane, and polydimethyl siloxane (PDMS).

[0035] In addition, when the mesh sheet is produced by photolithography, a positive resist in which exposed portions are removed can be used to produce the micromesh sheet. For example, the positive resist may be a DNQ (diazonaphthoquinone) novolac resin positive resist, a deprotection reaction type such as t-butoxycarbonyl, tetrahydropyran, phenoxyethyl, trimethylsilyl, or t-butoxycarbonylmethyl, or a depolymerization reaction type such as polyphthalaldehyde, polycarbonate, or polysilyl ether. In addition, tetramethylammonium hydroxide (TMAH), dimethylsulfoxide (DMSO), MEK (methylethylketone), GBL (γ-butyrolactone), or EL (ethyl lactate) may be used as the developer.

[0036] Furthermore, when the mesh sheet is made of a biocompatible or biodegradable material, the resulting cell sheet can be transplanted directly into a living body together with the mesh sheet, and is therefore suitable for use in regenerative medicine or drug discovery.

[0037] Examples of the biocompatible material include, but are not limited to, silicone, polyether block amide (PEBAX), polyurethane, silicone-polyurethane copolymer, ceramics, collagen, hydroxyapatite, nylon, polyethylene terephthalate, ultra-high molecular weight polyethylene such as Gore-Tex (trademark), polyvinyl chloride, other bio-derived materials, etc. The mesh sheet may be formed of a material other than a biocompatible material, and the surface may be treated with a biocompatible material.

[0038] Examples of the biodegradable material include, but are not limited to, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), and copolymers thereof, PHB-PHV-based poly(alkanoic acids), polyesters, starch, cellulose, chitosan, and other natural polymers and their derivatives.

[0039] Among the above, polyester, particularly polyethylene terephthalate, is preferred as the material for the mesh sheet. Since polyethylene terephthalate has low toxicity to the living body, the obtained cell sheet can be transplanted into the living body. In addition, in order to suppress autofluorescence during observation under a fluorescent microscope, it is preferable that the polyethylene terephthalate is dyed black. Specific examples of polyester mesh sheets include fabrics AG001N0, AG00Z1N0, and AG00Z3N0 manufactured by Amaike Gosen Co., Ltd. Specific examples of polyester mesh sheets dyed black include fabrics AG001N1, AG00Z1N9, and AG00Z3N9 manufactured by Amaike Gosen Co., Ltd.

[0040] The mesh sheet may be coated in advance with a cell adhesion promoting material. The cell adhesion promoting material is used to allow cells to adhere to the culture support and facilitate spreading and proliferation, and examples of the cell adhesion promoting material include extracellular matrix proteins such as collagen, fibronectin, and laminin, and positively charged substances such as poly-L-lysine. A specific example of the cell adhesion promoting material is Matrigel (registered trademark).

[0041] In the cell sheet manufacturing apparatus according to this embodiment, the container may be any container capable of containing a medium for culturing cells, and its configuration (e.g., shape) is not particularly limited. Examples of the container include a dish for cell culture, a plate having multiple wells (e.g., a 6-hole plate, a 12-hole plate, etc.), and a column for cell culture.

[0042] 2. Configuration example 1 of the cell sheet manufacturing device according to this embodiment Configuration example 1 of the cell sheet manufacturing apparatus according to this embodiment will be described. FIG. 1 is a diagram showing an outline of the configuration of a cell sheet manufacturing apparatus 100 as configuration example 1. Reference numeral 1010 in FIG. 1 is a perspective view showing the configuration of a support unit 10 provided in the cell sheet manufacturing apparatus 100. Reference numerals 1020 to 1023 in FIG. 1 are diagrams showing the steps of a method for manufacturing a cell sheet using the cell sheet manufacturing apparatus 100. Reference numeral 1030 in FIG. 1 is a diagram showing the configuration of a cell sheet produced using the cell sheet manufacturing apparatus 100. Reference numeral 1040 in FIG. 1 is an image showing an example of a specific configuration of the cell sheet manufacturing apparatus 100.

[0043] 1, the cell sheet manufacturing apparatus 100 includes a support unit 10 and a container 20. The support unit 10 is accommodated in the container 20 so as to be detachable therefrom.

[0044] 1, the support unit 10 includes an annular member 1, a mesh sheet 2, and a base 3 (holding member). The base 3 includes a base main body 3a, a cylindrical mounting portion 3b, a bottom contact portion 3c, and a side wall contact portion 3d.

[0045] The base body 3a is disk-shaped. The tubular mounting portion 3b is bottomless and cylindrical, with both axial ends open. The tubular mounting portion 3b is formed to protrude vertically only from one side of the base body 3a. The tubular mounting portion 3b is configured to penetrate the inside of the base body 3a. In other words, the upper and lower sides of the base body 3a are connected via the tubular mounting portion 3b. The mesh sheet 2 is placed on the tubular mounting portion 3b. The mesh sheet 2 can be observed by a microscope or the like through the tubular mounting portion 3b from the opposite side of the base body 3a to the tubular mounting portion 3b.

[0046] In a plan view seen from a direction perpendicular to the base body 3a, the side wall abutment portion 3d has a rectangular plate shape extending horizontally from the base body 3a. At least two side wall abutment portions 3d are formed so as to be symmetrical with respect to the base body 3a. In the configuration shown in 1010 of FIG. 1, four side wall abutment portions 3d are formed. The horizontal tip portion of the side wall abutment portion 3d abuts on the side wall of the container 20. The side wall abutment portion 3d plays the role of a spacer that maintains a constant horizontal distance between the base body 3a and the side wall of the container 20. Therefore, even if the container 20 vibrates in the horizontal direction, the tip portion of the side wall abutment portion 3d abuts on the side wall of the container 20, so that the horizontal position of the base body 3a is stably maintained in the culture medium without fluctuating. The base 3 is configured such that all side wall abutment portions 3d form dimensional clearances with the side walls of the container 20 so that the base 3 can be attached to and detached from the container 20.

[0047] The bottom contact portion 3c has a rectangular plate shape and is erected with respect to each of the side wall contact portions 3d having a rectangular plate shape. The bottom contact portion 3c intersects with the side wall contact portion 3d. In the configuration shown in 1010 of FIG. 1, the bottom contact portion 3c intersects with the side wall contact portion 3d in the vertical direction (gravity direction). The vertical tip of the bottom contact portion 3c abuts against the bottom surface of the container 20. The bottom contact portion 3c plays the role of a spacer that maintains a constant vertical distance between the base body 3a and the bottom surface of the container 20. Therefore, when the base 3 is stored in the container 20, the bottom contact portion 3c abuts against the bottom surface of the container 20, so that the vertical position of the base body 3a is stably maintained in the culture medium without fluctuating. In addition, the bottom surface contact portion 3c intersects with the side wall contact portion 3d and is formed to protrude from both the upper and lower directions relative to the side wall contact portion 3d. Therefore, even if the base 3 is placed in the container 20 upside down, the vertical position of the base main body 3a is stably maintained.

[0048] The annular member 1 has a ring shape surrounding the outer periphery of the cylindrical mounting portion 3b, and is provided removably with respect to the base 3. The ring shape of the annular member 1 is not particularly limited, and may be any shape such as a circular ring, a square ring, etc. In the configuration shown in 1010 of Fig. 1, the annular member 1 has a ring shape.

[0049] The mesh sheet 2 is disposed between the annular member 1 and the cylindrical mounting portion 3b. The mesh sheet 2 is held by the base 3 with its periphery sandwiched between the inner surface of the annular member 1 and the outer surface of the cylindrical mounting portion 3b. When the annular member 1 has a circular ring shape and the cylindrical mounting portion 3b has a cylindrical shape, from the viewpoint of stably holding the mesh sheet 2, the closer the difference between the inner diameter of the annular member 1 and the outer diameter of the cylindrical mounting portion 3b is to the thickness of the mesh sheet 2, the better.

[0050] Next, we will explain the method for producing a cell sheet using the cell sheet producing apparatus 100. First, as shown in 1020 in Fig. 1, the mesh sheet 2 is placed on the cylindrical mounting portion 3b of the base 3. Then, with the mesh sheet 2 placed on the cylindrical mounting portion 3b, the annular member 1 is attached to the cylindrical mounting portion 3b.

[0051] At this time, the annular member 1 is attached so that its inner surface surrounds the outer surface of the cylindrical mounting portion 3b. Therefore, as shown in 1021 in Fig. 1, the periphery of the mesh sheet 2 is sandwiched between the inner surface of the annular member 1 and the outer surface of the cylindrical mounting portion 3b. In this way, a support unit 10 that holds the mesh sheet 2 is produced. In the produced support unit 10, of the openings at both ends in the axial direction of the cylindrical mounting portion 3b, the opening at one end is closed by the mesh sheet 2, and the opening at the other end is open.

[0052] Next, the support unit 10 is turned upside down so that the mesh sheet 2 is located below the cylindrical mounting portion 3b. As a result, the support unit 10 has a bottomed cylindrical portion 10a with the mesh sheet 2 as the bottom surface formed by the mesh sheet 2 and the inner surface of the cylindrical mounting portion 3b. As shown in 1022 in FIG. 1, a suspension in which cells 4 are suspended in a medium 6 is injected into the bottomed cylindrical portion 10a using a pipette 5 or the like. At this time, the suspension does not pass through the mesh sheet 2 due to the surface tension of the suspension relative to the inner surface of the cylindrical mounting portion 3b and the mesh sheet 2, and is retained on the mesh sheet 2. Then, the cells 4 in the suspension are attached to the mesh sheet 2, so that the cells 4 are seeded on the mesh sheet 2.

[0053] Thereafter, the support unit 10 is housed in a container 20. The container 20 is then filled with a culture medium 6, and the cells 4 attached to the mesh sheet 2 are cultured.

[0054] As shown in 1030 of FIG. 1, individual cells 4 spontaneously extend and adhere to each other toward the center of the openings in the mesh sheet 2 so as to fill the openings. When cells 4 that retain proliferation ability are cultured on the mesh sheet 2, the cells 4 proliferate not only horizontally but also vertically relative to the mesh sheet 2. As a result, a cell sheet 200 can be obtained in which a single or multiple cell layers made of cells 4 are formed so as to cover the openings in the mesh sheet 2. The thickness of the cell sheet 200 can be controlled by controlling the amount of cells 4 seeded on the mesh sheet 2 and / or the proliferation rate of the cells 4. The cell sheet 200 is sturdy and easy to handle because it is supported by the mesh sheet 2.

[0055] The cell sheet 200 has at least two cell layers made of cells 4, and a mesh sheet 2 is disposed between the two cell layers as a substrate to which the cells 4 are attached and cultured. That is, both sides of the cell sheet 200 are made of cells 4. Therefore, the surface of the cell sheet 200 that is attached to the affected area is not limited to one side. Furthermore, compared with the conventional cell sheet manufacturing method using a dish or a porous membrane as a scaffold, the cell sheet manufacturing method using the cell sheet manufacturing apparatus 100 is superior in terms of supplying nutrients to the cells 4 and observing the cells 4. Furthermore, by using a sheet having patterned openings like the mesh sheet 2, a cell sheet 200 having a similar structure (in other words, properties) can be obtained with high reproducibility.

[0056] 1, the container 20 may be a plate in which a plurality of wells 21 are formed. More specifically, the container 20 is a 12-well plate. In this case, the support unit 10 having the base 3 is configured to be accommodated in the wells 21 so as to be detachable therefrom.

[0057] 2 is a diagram for explaining the effect of the cell sheet manufacturing apparatus 100. As shown by 2010 and 2011 in FIG. 2, the support unit 10 is housed in a releasable manner in the container 20, so that the support unit 10 can be used even if it is turned upside down and the base 3 is housed in the container 20. Therefore, when the support unit 10 is turned upside down, the cell sheet formed on the mesh sheet 2 is also turned upside down, so that both sides of the cell sheet formed on the mesh sheet 2 can be easily observed with the microscope 7.

[0058] Also, as shown in 2020 to 2022 in FIG. 2, by using the cell sheet manufacturing apparatus 100, a cell sheet having a plurality of types of cell layers can be produced. More specifically, a cell sheet having a first cell layer made of cells 4a and a second cell layer made of cells 4b of a different type from the cells 4a can be produced. First, as shown in 2020 in FIG. 2, the support unit 10 is immersed in the medium 6 with the cells 4a seeded on the mesh sheet 2. Next, as shown in 2021 in FIG. 2, the support unit 10 is turned upside down. At this time, the surface of the mesh sheet 2 opposite to the cells 4a becomes the upper side. Therefore, if a suspension of cells 4b is dropped onto the mesh sheet 2 from above using a pipette 5 or the like, the cells 4b can also be seeded on the surface of the mesh sheet 2 opposite to the cells 4a (see 2022 in FIG. 2). In this way, according to the configuration of the cell sheet manufacturing apparatus 100, it is possible to seed additional cells on both the front and back surfaces of the cell sheet. From the viewpoint of easily seeding cells on both sides of the cell sheet, preferably, the annular member 1 protrudes from the cylindrical mounting portion 3b and is attached to the base 3, as shown in 2020 to 2022 in FIG. 2. According to this configuration, when the support unit 10 is turned over, the inner surface of the annular member 1 and the mesh sheet 2 form a bottomed cylindrical portion 10b with the mesh sheet 2 as the bottom surface. Therefore, by injecting a suspension of cells 4b into the bottomed cylindrical portion 10b, the suspension is stably held on the mesh sheet 2.

[0059] Furthermore, according to the configuration of the cell sheet manufacturing apparatus 100, the cell sheet 200 can be easily separated from the support unit 10. As shown in 2030 of FIG. 2, the cell 4 is cultured in a medium in the container 20 to produce a cell sheet 200 made of the cells 4. Thereafter, as shown in 2031 of FIG. 2, the support unit 10 is separated from the container 20. Next, as shown in 2032 of FIG. 2, the annular member 1 is removed from the cylindrical mounting portion 3b of the base 3. Thereby, the cell sheet 200 formed on the mesh sheet 2 can be removed from the cylindrical mounting portion 3b of the base 3 using, for example, tweezers 8. Such a cell sheet 200 that can be easily removed from the cell sheet manufacturing apparatus 100 is convenient, for example, when transplanting a cell sheet.

[0060] (Variation 1) Modified examples of the cell sheet manufacturing apparatus 100 will be described. FIG. 3 is a diagram showing the configuration of a cell sheet manufacturing apparatus 100A as modified example 1, and a cell sheet manufacturing method using the cell sheet manufacturing apparatus 100A. 3010 in FIG. 3 is a perspective view showing a configuration example of a base 3 provided in the cell sheet manufacturing apparatus 100A. Also, 3020 to 3024 in FIG. 3 are diagrams showing a cell sheet manufacturing method using the cell sheet manufacturing apparatus 100A. 3030 in FIG. 3 is a cross-sectional view showing the configuration of a lid 9 provided in the cell sheet manufacturing apparatus 100A. The cell sheet manufacturing apparatus 100A is capable of manufacturing a large-area cell sheet.

[0061] As shown in 3010 of Fig. 3, the base 3 can be designed for a 3.5 cm diameter dish (for a 6-hole plate), a 6 cm diameter dish, or a 10 cm diameter dish. That is, by increasing the diameter of the cylindrical mounting portion 3b of the base 3, the area of ​​the cell sheet to be produced can be made large. Accordingly, the area of ​​the mesh sheet 2 can also be increased.

[0062] The cell sheet manufacturing apparatus 100A of the first modified example is configured to hold a suspension on such a large-area mesh sheet 2 and to manufacture a large-area cell sheet. As shown in 3021 to 3024 in FIG. 3, the support unit 10A of the cell sheet manufacturing apparatus 100A includes a lid body 9. The lid body 9 is configured to cover one surface 2a of the mesh sheet 2. The lid body 9 is provided with an injection port 9i for injecting a suspension containing cells 4 between the mesh sheet 2 and the lid body 9. More specifically, the lid body 9 is disposed so as to cover the surface 2a of the mesh sheet 2 that contacts the cylindrical mounting portion 3b. The mesh sheet 2 and the lid body 9 are spaced apart. The cylindrical mounting portion 3b is disposed between the mesh sheet 2 and the lid body 9. The cylindrical mounting portion 3b maintains a constant distance between the mesh sheet 2 and the lid body 9.

[0063] Here, the gap between the mesh sheet 2 and the lid 9 is set so that the suspension of cells 4 is injected between the mesh sheet 2 and the lid 9 while being in contact with both the mesh sheet 2 and the lid 9. The gap between the mesh sheet 2 and the lid 9 is set preferably to 1 mm to 3 mm, more preferably to 1 mm to 2 mm, and particularly preferably to 1 mm to 1.5 mm.

[0064] In other words, the lid 9 is a member that closes the bottomed tubular portion 10a formed by the mesh sheet 2 and the tubular placement portion 3b. The lid 9 may have a laminated structure including at least a first layer 9a and a second layer 9b. The first layer 9a is the layer farthest from the mesh sheet 2, and the second layer 9b is the layer closest to the mesh sheet 2. In the configuration shown in 3030 of FIG. 3, the lid 9 has a two-layer structure consisting of the first layer 9a and the second layer 9b. Examples of materials that constitute the first layer 9a include silicone resin. Examples of materials that constitute the second layer 9b include polyethylene terephthalate (PET). The lid 9 may have a single-layer structure of either the first layer 9a or the second layer 9b.

[0065] In the cell sheet manufacturing method using the cell sheet manufacturing apparatus 100A, first, the support unit is placed in the dish 22. This support unit is not provided with a lid 9. The support unit is placed upside down so that the mesh sheet 2 is located below the cylindrical mounting portion 3b. By placing the support unit 10 in this manner, a bottomed cylindrical portion 10a is formed. This bottomed cylindrical portion 10a is formed by the mesh sheet 2 and the inner surface of the cylindrical mounting portion 3b, with the mesh sheet 2 serving as the bottom surface (see 3020 in FIG. 3). Next, the lid 9 is placed so as to close the open upper portion of the bottomed cylindrical portion 10a, thereby producing the support unit 10A (see 3021 in FIG. 3).

[0066] Next, a suspension of cells 4 suspended in a medium 6 is injected into the bottomed tube 10a from the injection port 9i using a pipette 5 or the like (see 3022 in FIG. 3). At this time, the suspension of cells 4 is injected into the bottomed tube 10a while contacting both the mesh sheet 2 and the lid 9. Therefore, the surface tension of the suspension relative to the lid 9 and the mesh sheet 2 prevents the suspension from passing through the mesh sheet 2 and dropping due to gravity. Therefore, even if the mesh sheet 2 has a large area, the suspension is held on the mesh sheet 2 due to the surface tension of the suspension relative to the lid 9 (see 3023 in FIG. 3). By attaching the cells 4 in the suspension to the mesh sheet 2 in this way, the cells 4 can be uniformly seeded on the mesh sheet 2 (for example, a mesh sheet 2 having a large area).

[0067] Thereafter, the entire dish 22 is filled with the medium 6, and the cells 4 attached to the mesh sheet 2 are cultured (see 3024 in FIG. 3).

[0068] In the above-described method, the lid 9 is placed on the bottomed tubular portion 10a, and then the suspension of cells 4 is injected. However, the lid 9 may be placed after the suspension of cells 4 is injected into the bottomed tubular portion 10a so that the suspension does not fall from the mesh sheet 2.

[0069] (Variation 2) Another modified example of the cell sheet manufacturing apparatus 100 will be described. Fig. 4 is a diagram showing the configuration of a cell sheet manufacturing apparatus 100B as modified example 2, and a method for manufacturing a cell sheet using the cell sheet manufacturing apparatus 100B. 4010 in Fig. 4 is an exploded perspective view showing the configuration of a support unit 10B provided in the cell sheet manufacturing apparatus 100B. 4020-4022 in Fig. 4 are diagrams showing the method for manufacturing a cell sheet using the cell sheet manufacturing apparatus 100B.

[0070] The cell sheet manufacturing apparatus 100B, like the first modified example, is provided with a lid 9 and is capable of manufacturing a large-area cell sheet. In the cell sheet manufacturing apparatus 100B, the configuration of the support unit 10B is different from that of the first modified example. As shown in 4010 of FIG. 4, the base 3 of the support unit 10B is not configured such that the cylindrical mounting portion 3b protrudes from the base main body 3a. The cylindrical mounting portion 3b is provided as an opening formed in the base main body 3a. The bottom surface abutment portion 3e is provided so as to protrude downward from the base main body 3a, and does not protrude upward. In addition, the side wall abutment portion 3f is provided so as to extend horizontally from the base main body 3a.

[0071] Further, the annular member 1 has an inner diameter substantially the same as the inner diameter of the cylindrical mounting portion 3b, so that the mesh sheet 2 is sandwiched between the lower surface of the annular member 1 and the upper surface of the base body 3a.

[0072] The lid body 9 is disposed so as to cover a surface 2b of the mesh sheet 2 that comes into contact with the annular member 1. The mesh sheet 2 and the lid body 9 are spaced apart. The annular member 1 is disposed between the mesh sheet 2 and the lid body 9. The annular member 1 keeps the distance between the mesh sheet 2 and the lid body 9 constant. The lid body 9 is a member that closes the bottomed tubular portion 10b formed by the mesh sheet 2 and the annular member 1.

[0073] In the cell sheet manufacturing method using the cell sheet manufacturing apparatus 100B, first, the support unit 10B is placed in the dish 22. The support unit 10B is placed so that the mesh sheet 2 is located below the annular member 1. By placing it in this manner, a bottomed tubular portion 10b is formed in the support unit 10B. This bottomed tubular portion 10b is formed by the mesh sheet 2 and the inner surface of the annular member 1, with the mesh sheet 2 serving as the bottom surface (see 4020 in FIG. 4).

[0074] Next, a suspension of cells 4 suspended in culture medium 6 is injected into bottomed cylinder 10a from injection port 9a using pipette 5 or the like, and cells 4 are seeded on mesh sheet 2 (see 4021 and 4022 in FIG. 4). Thereafter, dish 22 is filled with culture medium 6, and cell culture is performed.

[0075] The cell sheet manufacturing apparatus 100B is structured so that the produced cell sheet cannot be easily separated from the support unit 10B. However, it is possible to cut the cell sheet from the support unit 10B using scissors or a knife.

[0076] The cell sheet produced using the cell sheet production apparatus according to this embodiment can receive nutrition from both the top and bottom surfaces of the cell sheet in the culture medium. In the conventional method of producing a cell sheet on a dish, it is not possible to receive nutrition from the bottom surface of the cell sheet, so cells are cultured relying only on the supply of nutrition from the top surface. Therefore, in a situation where the supply of nutrition from the top surface of the cell sheet is limited, the supply of nutrition to the cells inside the cell sheet may be insufficient.

[0077] An example of a situation in which the supply of nutrients from the upper surface of a cell sheet is limited is when a skin sheet is produced. In the manufacture of skin sheets, a keratinocyte layer (epidermal layer) may be formed on a fibroblast layer (dermal layer) to produce a skin sheet. In this case, mature epidermal cells form tight junctions, which inhibit the supply of nutrients from the upper part of the skin sheet. Therefore, it is difficult to produce a skin sheet consisting of a fibroblast layer and a dermal layer using the conventional method of producing a cell sheet on a dish.

[0078] FIG. 5 is a schematic diagram showing the cross-sectional structure of a skin sheet produced using the cell sheet production apparatus according to this embodiment. 11 indicates dead keratinocytes. 12 indicates the above-mentioned tight junctions. Furthermore, 13 indicates keratinocytes. 14 indicates dermal fibroblasts. As shown in FIG. 5, in the skin sheet produced using the cell sheet production apparatus according to this embodiment, even if the supply of nutrients from the upper layer of keratinocytes 13 decreases, it is expected that the supply of nutrients from the lower layer of dermal fibroblasts 14 will be sufficient. Therefore, more nutrients can be supplied to the keratinocytes 13 and dermal fibroblasts 14 that constitute the skin sheet.

[0079] Another conventional technique is a method of producing a skin sheet consisting of multiple cell layers on a porous membrane. Since a porous membrane is used, it is believed that nutrients are supplied to a certain extent from the underside of the cell sheet. However, it is difficult to use a cell sheet produced by this method for transplantation as it is. This is because, when transplanting to an affected area, it is necessary to transplant the skin sheet while contacting the surface of the dermis layer with the affected area, rather than contacting the surface of the epidermis layer with the affected area, so a cell sheet in which the dermis layer is sandwiched between the porous membrane and the epidermis layer cannot be used for transplantation. On the other hand, in the skin sheet produced using the cell sheet manufacturing apparatus according to this embodiment, cells are present on the culture medium side not only on the surface of the epidermis layer but also on the surface of the dermis layer, that is, exposed to the culture medium. Therefore, it is possible to attach the surface of the dermis layer of the skin sheet to the affected area.

[0080] Furthermore, tight junctions 12 are also observed in intestinal cells, vascular endothelial cells, etc. Therefore, in addition to skin sheets, the cell sheet manufacturing apparatus according to this embodiment can also be used when culturing and using cell sheets that include intestinal cells, vascular endothelial cells, etc. that form tight junctions 12.

[0081] (Variation 3) A modified example of the cell sheet manufacturing apparatus will be described. Figures 20 and 21 are diagrams showing a support unit 60 of the cell sheet manufacturing apparatus as modified example 3. Note that in Figures 20 and 21, the containers constituting the cell sheet manufacturing apparatus are omitted.

[0082] In the cell sheet manufacturing apparatus of this embodiment, at least one of the base 3 (holding member) and the annular member 1 is formed with an engagement portion 67 that engages the base 3 and the annular member 1 so that they do not separate, and one of the base 3 and the annular member 1 is formed with a through hole 66 for passing a pressing pin 65 that presses the other, and is configured so that the engagement is released by pressing the other, and the base 3 and the annular member 1 are separated. Note that the cell sheet manufacturing apparatus of this embodiment may or may not be configured with the pressing pin 65. When the cell sheet manufacturing apparatus of this embodiment is configured without the pressing pin 65, the pressing pin 65 may be prepared as a separate component from the cell sheet manufacturing apparatus.

[0083] The locking portion 67 may have any configuration as long as the locking between the base 3 and the annular member 1 can be released by the pressing pin 65, and the specific configuration is not limited. Specific configurations of the locking portion 67 include, for example, (i) a friction force generating region formed on at least one surface of the base 3 and the annular member 1, which generates a large friction force between the base 3 and the annular member 1, (ii) a combination of a convex portion formed on the surface of the base 3 and a locking convex portion formed on the surface of the annular member 1, which locks the movement of the convex portion in the pressing direction, (iii) a combination of a convex portion formed on the surface of the annular member 1 and a locking convex portion formed on the surface of the base 3 and a locking convex portion formed on the surface of the base 3, which locks the movement of the convex portion in the pressing direction, and (iv) a combination of the above-mentioned (ii) and (iii). The friction generating region can be formed, for example, by attaching a material (e.g., rubber) capable of generating a desired friction force to at least one surface of the base 3 and the annular member 1.

[0084] The through-hole 66 may be formed in either the base 3 or the annular member 1, but from the viewpoint of ease of handling of the support unit 60, it is preferable that the through-hole 66 be formed in the base 3.

[0085] Focusing on a particular through hole 66, when the through hole 66 is formed in the base 3, the configuration may be such that no through hole is formed in the region of the annular member 1 facing the through hole 66, and when the through hole 66 is formed in the annular member 1, the configuration may be such that no through hole is formed in the region of the base 3 facing the through hole 66. In this configuration, if a pressing pin 65 is inserted through the through hole 66, the pressing pin 65 can effectively press the component (base 3 or annular member 1) in which the through hole is not formed.

[0086] The shape and number of the through holes 66 are not limited. Examples of the shape of the through holes 66 include a cylindrical shape and a rectangular column shape. The number of through holes 66 formed in one holding member or one annular member may be an even number or an odd number, but an even number is preferable from the viewpoint of pressing the entire base 3 or the annular member 1 approximately evenly. The number of through holes 66 may be, for example, 1 to 10 or 1 to 20, and the number of through holes 66 may be appropriately set based on the size of the base 3 and the annular member 1.

[0087] More specifically, in 20010 and 20011 of Fig. 20, the diagrams on the left side of the arrows are perspective views of the support unit 60. As shown in detail in Fig. 21 described later, the base 3 is formed with a cylindrical mounting portion 3b on which the mesh sheet 2 is placed, and the periphery of the mesh sheet 2 is sandwiched between the cylindrical mounting portion 3b and the annular member 1. The support unit 60 may be provided with a lid (not shown) on the base 3 or the annular member 1.

[0088] In 20010 of FIG. 20 and 20011 of FIG. 20, the diagrams on the right side of the arrows are exploded perspective views of the support unit 60 after the lock between the base 3 and the annular member 1 is released by penetrating the pressing pin 65 into the through hole 66 and pressing the annular member 1. In these drawings, the base 3 is formed with the through hole 66, and no through hole is formed in the area of ​​the annular member 1 facing the through hole 66. Therefore, when the pressing pin 65 is inserted into the through hole 66, the pressing pin 65 effectively presses the annular member 1, and the lock between the base 3 and the annular member 1 is released. In these drawings, four through holes 66 are formed in the base 3 as an example. When the pressing pin 65 is inserted into each of these through holes 66, the entire annular member 1 can be pressed almost evenly.

[0089] 21010 in Fig. 21 is a top view of the support unit 60. 21011 in Fig. 21 is a cross-sectional view at the position "AA" of the support unit 60, showing the change over time of each component when the pressing pin 65 is inserted into the through hole 66. First, as shown on the left side of 21011 in Fig. 21, the pressing pin 65 is inserted into the through hole 66 along the direction of the arrow. Next, as shown in the center of 21011 in Fig. 21, when the pressing pin 65 penetrates the through hole 66, the pressing pin 65 presses the annular member 1 downward. Finally, as shown on the right side of 21011 in Figure 21, the pressing pin 65 further presses the annular member 1 downward, thereby releasing the engagement between the base 3 and the annular member 1 and separating the base 3 and the annular member 1, and as a result, the mesh sheet 2 (specifically, the peripheral edge of the mesh sheet 2) clamped between the tubular mounting portion 3b and the annular member 1 is removed.

[0090] 3. Configuration Example 2 of the Cell Sheet Manufacturing Apparatus According to the Present Embodiment Configuration example 2 of the cell sheet manufacturing apparatus according to this embodiment will be described. Fig. 6 is a diagram showing an outline of the configuration of the support unit 30 provided in the cell sheet manufacturing apparatus of configuration example 2. Reference numerals 6010 to 6013 in Fig. 6 are perspective views showing the assembly procedure of the support unit 30. Reference numeral 6020 in Fig. 6 is a cross-sectional view showing the outline of the configuration of the support unit 30.

[0091] 6, the support unit 30 includes a mesh sheet 2, a pair of frame bodies 31 and 32, a clip 33 (clamping member), a holding member 34, and a spacer 35. The holding member 34 constitutes the main body of the support unit 30. The mesh sheet 2, the pair of frame bodies 31 and 32, and the clip 33 constitute a mesh assembly A.

[0092] The frame bodies 31 and 32 are members that sandwich the mesh sheet 2 to reinforce the mesh sheet 2. The frame bodies 31 and 32 are arranged to sandwich the peripheral edge of the mesh sheet 2. The frame bodies 31 and 32 are provided removably with respect to the holding member 34.

[0093] 6, clip 33 is a member that clamps the pair of frame bodies 31 and 32 to integrate mesh sheet 2 with the pair of frame bodies 31 and 32. Clamping by clip 33 prevents frame body 31 and frame body 32 from separating and fixes them in a tight contact state.

[0094] The mesh assembly A is constructed by disposing the mesh sheet 2 between a pair of frames 31 and 32, and fixing the frames 31 and 32 with clips 33 (see 6010 to 6012 in FIG. 6).

[0095] The holding member 34 has a bottomless cylindrical shape. The inner diameter of the holding member 34 is larger than the outer diameter of the pair of frames 31 and 32. The holding member 34 has mounting portions 34a for mounting the mesh assembly A in two opposing portions of the inner wall of the holding member 34. The mounting portions 34a have flat plate portions 34b and 34c. The flat plate portions 34b and 34c are spaced apart from each other and protrude horizontally inward from the inner wall of the holding member 34. The mesh assembly A is mounted on the holding member 34 by inserting the peripheral portion of the mesh assembly A between the flat plate portions 34b and 34c. In addition, the holding member 34 has a flange 34d protruding horizontally at the upper end thereof.

[0096] When the mesh assembly A is attached to the holding member 34, there is a gap between the mesh assembly A and the flat plate portion 34b (see 6013 in FIG. 6). The spacer 35 is a member that fills the gap between the mesh assembly A and the flat plate portion 34b. The spacer 35 prevents the mesh assembly A from moving within the attachment portion 34a of the holding member 34. Therefore, the mesh assembly A is stably held within the holding member 34.

[0097] Further, the spacer 35 is provided with a tab 35a that extends upward. When separating the mesh assembly A from the holding member 34, the user holds the tab 35a of the spacer 35 and removes the spacer 35 from the holding member 34. Next, the mesh assembly A is separated from the holding member 34.

[0098] Fig. 7 is a diagram showing the configuration of a cell sheet manufacturing apparatus 100C as configuration example 2. Reference numerals 7010 to 7012 in Fig. 7 are diagrams showing a cell sheet manufacturing method using the cell sheet manufacturing apparatus 100C. Reference numerals 7020 to 7022, 7030, and 7031 in Fig. 7 are diagrams for explaining the effects of the cell sheet manufacturing apparatus 100C.

[0099] In the cell sheet manufacturing method using the cell sheet manufacturing apparatus 100C, first, the support unit 30 is placed in the container 23. At this time, the support unit 30 is positioned so that the mesh assembly A is closest to the bottom surface of the container 23 (see 7010 in FIG. 7).

[0100] Moreover, the lower surface of the flange 34d of the holding member 34 abuts against the entire upper end surface of the container 23. The outer diameter of the holding member 34 is close to the inner diameter of the container 23. A clearance is formed between the outer peripheral surface of the holding member 34 and the inner surface of the container 23 to such an extent that the holding member 34 can be removed from the container 23. With this configuration, the support unit 30 is housed in the container 23 so that its vertical and horizontal positions in the culture medium are constant.

[0101] Next, cells 4 are seeded onto the mesh sheet 2 (see 7011 in FIG. 7). The method for seeding the cells 4 is similar to the method using the cell sheet manufacturing apparatus 100, and therefore a description thereof will be omitted.

[0102] Next, the entire container 23 is filled with the culture medium 6, and the cells 4 attached to the mesh sheet 2 are cultured. In this way, a cell sheet 200 is produced.

[0103] Next, the effect of the cell sheet manufacturing apparatus 100C will be described. As described above, the mesh assembly A is separated from the holding member 34 by removing the spacer 35 from the holding member 34. The separated mesh assembly A can be easily attached to the holding member 34 upside down. Therefore, as shown in 7020 and 7021 in FIG. 7, different types of cells 4a and 4b can be seeded on the upper and lower surfaces of the mesh sheet 2, respectively. Furthermore, after the cell sheet 200 is formed, the cell sheet 200 can be easily separated from the assembly A. The frame bodies 31 and 32 can be easily separated from each other by simply removing the clip 33. Therefore, if the frame bodies 31 and 32 are separated, the cell sheet 200 can be easily taken out. In addition, as shown in 7030 and 7031 in FIG. 7, both sides of the mesh sheet 2 can be observed by the microscope 7.

[0104] Furthermore, in the cell sheet manufacturing apparatus 100C, the support unit 30 is held in the container 23 by the abutment between the flange 34d of the holding member 34 and the upper end surface of the container 23. The flange 34d is a part that does not contact the culture medium 6. Furthermore, the knob 35a of the spacer 35 is also a part that does not contact the culture medium 6. Therefore, when the support unit 30 is moved to another container using tweezers, the support unit 30 can be moved without the tweezers touching the culture medium 6 by moving the spacer 35 upward with the tweezers. Furthermore, when the cells 4 are cultured at a position relatively close to the bottom of the container 23, the cells 4 do not come into contact with the container 23 even when the support unit 30 is moved.

[0105] Even if cells different from the cells 4 are cultured on the bottom surface of the container 23, these cells do not come into contact with the support unit 30. In other words, in the cell sheet manufacturing apparatus 100C, when different cells are co-cultured (i) on the mesh sheet 2 of the support unit 30 and (ii) on the bottom surface of the container 23, the support unit 30 does not come into contact with the cells cultured on the bottom surface of the container 23. Furthermore, since the support unit 30 is structured to be inserted from the upper side of the container 23, the position of the support unit 30 relative to the container 23 is stable.

[0106] 4. Configuration Example 3 of the Cell Sheet Manufacturing Apparatus According to the Present Embodiment Configuration example 3 of the cell sheet manufacturing apparatus according to this embodiment will be described. Fig. 8 is a diagram showing the configuration of a cell sheet manufacturing apparatus 100D of configuration example 3. Reference numerals 8010 and 8020 in Fig. 8 are images showing the configuration of the support unit 40 provided in the cell sheet manufacturing apparatus 100D. Reference numerals 8030 to 8033 in Fig. 8 are diagrams showing a method for manufacturing a cell sheet using the cell sheet manufacturing apparatus 100D.

[0107] The support unit 40 is in the form of a column that is detachably accommodated in the well 24 (container). The support unit 40 includes a mesh sheet 2 and a column body 41. The lower surface of the column body 41 is formed of the mesh sheet 2. By placing the brim of the column body 41 on the well 24, the column body 41 is accommodated in the well 24 so that its vertical and horizontal positions in the culture medium 6 are constant (see 8030 in FIG. 8). The support unit 40 can be produced, for example, by removing the porous membrane from a commercially available Transwell (registered trademark) column and attaching the mesh sheet 2.

[0108] By using the cell sheet manufacturing apparatus 100D, it becomes easy to perform liquid-gas phase culture on the produced cell sheet. For example, in producing a skin sheet, first, as shown in 8031 ​​in FIG. 8, dermal fibroblasts 14 are seeded on a mesh sheet 2 and cultured in a medium 6. Next, as shown in 8032 in FIG. 8, keratinocytes 13 are further seeded on a layer of grown dermal fibroblasts 14 and cultured in the medium 6 to produce a skin sheet. As shown in 8033 in FIG. 8, the produced skin sheet can be cultured in a state where the dermal fibroblasts 14 are in contact with the medium and part of the keratinocytes 13 is in contact with gas, that is, liquid-gas phase culture can be performed.

[0109] 5. Configuration Example 4 of the Cell Sheet Manufacturing Apparatus According to the Present Embodiment Configuration example 4 of the cell sheet manufacturing apparatus according to this embodiment will be described. Fig. 9 is a diagram showing the configuration of a cell sheet manufacturing apparatus 100E of configuration example 3. 9010 in Fig. 9 is a perspective view showing the configuration of the support unit 50 provided in the cell sheet manufacturing apparatus 100E. Also, 9011 in Fig. 9 is an image showing the configuration of the cell sheet manufacturing apparatus 100E.

[0110] As shown in 9011 of Fig. 9, in the cell sheet manufacturing apparatus 100E, a well plate 26 in which a plurality of wells 25a are formed is used as a container. In the configuration shown in 9011 of Fig. 9, the well plate 26 is a 12-hole plate. The support unit 50 is accommodated in such a well plate 26 so as to be detachable from it. The support unit 50 includes a mesh sheet 2 and a column body 51.

[0111] The column body 51 has a first annular portion 51a, a foot portion 51b, and a second annular portion 51c. The foot portion 51b is a portion that connects the first annular portion 51a and the second annular portion 51c. The first annular portion 51a and the second annular portion 51c are arranged so that their central axes coincide with each other.

[0112] The outer diameter of the first annular portion 51a is larger than the inner diameter of the well 25a of the well plate 25. The inner diameter of the second annular portion 51c is smaller than the inner diameter of the first annular portion 51a. The bottom of the second annular portion 51c is closed by the mesh sheet 2.

[0113] In the cell sheet manufacturing apparatus 100E, the first circular ring portion 51a of the column body 51 is placed on the well 25a, whereby the column body 51 is accommodated in the well 25a so that its vertical and horizontal positions are constant in the culture medium 6. The cell suspension is injected into the space formed by the mesh sheet 2 and the inner surfaces of the second circular ring portion 51c and foot portion 51b.

[0114] In the cell sheet manufacturing apparatus 100E, the support unit 50 is held in the well 25a by placing the first circular portion 51a of the column body 51 on the well 25a. The first circular portion 51a is a portion that does not contact the culture medium 6. Therefore, when moving the support unit 50 to another well 25a using tweezers, the support unit 50 can be moved without the tweezers touching the culture medium 6 by pinching the first circular portion 51a with the tweezers. Furthermore, when cells are cultured at a position relatively close to the bottom of the well 25a, the cells 4 do not come into contact with the well 25a even when the support unit 50 is moved. In the cell sheet manufacturing apparatus 100E, when different cells are co-cultured (i) on the mesh sheet 2 of the support unit 50 and (ii) on the bottom surface of the well 25a, the support unit 50 does not come into contact with the cells cultured on the bottom surface of the well 25a.

[0115] Furthermore, in the cell sheet manufacturing apparatus 100E, the position of the mesh sheet 2 is stably held by the first annular portion 51a and the foot portions 51b. Therefore, according to the cell sheet manufacturing apparatus 100E, the vertical and horizontal positions of the support unit 50 within the well 25a are more stably held.

[0116] 〔summary〕 In order to solve the above problems, a cell sheet manufacturing apparatus according to one embodiment of the present invention comprises a container for containing a culture medium for culturing cells, a mesh sheet which is a substrate to which the cells are attached and cultured, and a holding member for holding the mesh sheet so that it floats above the bottom surface of the container, and a support unit which is contained in the container so that it can be detached from the container, and is characterized in that the support unit is contained in the container so that its vertical and horizontal positions in the culture medium are constant.

[0117] According to the above configuration, the support unit is housed in a container so as to be detachable from the container. Therefore, a user can easily turn the support unit upside down by removing the support unit from the container, turning it upside down, and then placing it in the container again. When the support unit is turned upside down, the cell sheet formed on the mesh sheet held by the holding member of the support unit is also turned upside down. Therefore, according to the above configuration, both sides of the cell sheet formed on the mesh sheet can be easily observed under a microscope.

[0118] Furthermore, since the support unit is accommodated in the container so that its vertical and horizontal positions in the culture medium are constant, the position of the mesh sheet in the container, in other words, the position of the cell sheet formed on the mesh sheet, does not change during observation of the cell sheet under a microscope. Therefore, with the above configuration, the cell sheet can be observed precisely.

[0119] In the cell sheet manufacturing apparatus according to one aspect of the present invention, it is preferable that the holding member removably holds the mesh sheet.

[0120] According to the above configuration, the cell sheet formed on the mesh sheet can be easily separated from the support unit.

[0121] In a cell sheet manufacturing apparatus according to one embodiment of the present invention, the holding member is formed with a tubular mounting portion on which the mesh sheet is placed, the support unit is provided with a ring-shaped member having a shape that surrounds the outer periphery of the tubular mounting portion and that is removably provided on the holding member, and it is preferable that the periphery of the mesh sheet is clamped between the tubular mounting portion and the ring-shaped member.

[0122] According to the above configuration, by attaching the annular member to the holding member, the periphery of the mesh sheet is clamped between the cylindrical mounting portion and the annular member, thereby allowing the mesh sheet to be stably mounted on the cylindrical mounting portion. Meanwhile, by removing the annular member from the holding member, the periphery of the mesh sheet clamped between the cylindrical mounting portion and the annular member is released, allowing the mesh sheet placed on the cylindrical mounting portion to be easily removed.

[0123] In a cell sheet manufacturing apparatus according to one embodiment of the present invention, at least one of the holding member and the annular member is formed with a locking portion that locks the holding member and the annular member so that they do not separate, and one of the holding member and the annular member is formed with a through hole for passing a pressing pin that presses the other, and it is preferable that the lock is released by pressing the other, and the holding member and the annular member are separated.

[0124] According to the above configuration, by inserting a pressing pin into the through hole and pressing the retaining member or the annular member with the pressing pin, the engagement between the retaining member and the annular member is released, thereby making it possible to easily remove the mesh sheet placed on the cylindrical mounting portion.

[0125] In a cell sheet manufacturing apparatus according to one embodiment of the present invention, it is preferable that the support unit is removably provided with respect to the holding member and comprises a pair of frame bodies arranged to sandwich the periphery of the mesh sheet, and a clamping member that clamps the pair of frame bodies to integrate the mesh sheet and the pair of frame bodies.

[0126] According to the above configuration, a pair of frame bodies arranged to sandwich the peripheral edge of the mesh sheet are removably provided with respect to the holding member, so that the mesh sheet held by the pair of frame bodies can be easily removed by separating the pair of frame bodies from the holding member.

[0127] In the cell sheet manufacturing apparatus according to one aspect of the present invention, it is preferable that the holding member has a specific gravity greater than that of the culture medium.

[0128] According to the above configuration, since the holding member has a greater specific gravity than the culture medium, the vertical and horizontal positions of the holding member and the cell sheet formed on the mesh sheet held by the holding member are stably maintained in the culture medium.

[0129] In a cell sheet manufacturing apparatus according to one embodiment of the present invention, the support unit is a lid body covering one side of the mesh sheet, and is provided with an injection port for injecting a suspension containing the cells between the mesh sheet and the lid body, and it is preferable that the distance between the lid body and the mesh sheet is set so that the suspension is injected between the mesh sheet and the lid body while in contact with both the mesh sheet and the lid body.

[0130] According to the above configuration, the gap between the lid and the mesh sheet is set so that the suspension is injected between the mesh sheet and the lid while contacting both. In this case, the suspension containing cells can be spread laterally while preventing the suspension from passing through the mesh sheet and moving to the underside of the mesh sheet. Therefore, even if the area of ​​the mesh sheet is increased, the suspension containing cells can be spread evenly on the mesh sheet, and cell culture can be easily performed using a large-area mesh sheet.

[0131] In the cell sheet manufacturing apparatus according to one aspect of the present invention, the openings of the mesh sheet preferably have a shape elongated in one direction.

[0132] When the opening of the mesh sheet is elongated in one direction as in the above configuration, the opening has a large wall surface that extends long in one direction. Since a large number of cells adhere to the wall surface and grow, and grow with the same orientation, the above configuration makes it possible to control the orientation of the growing cells in the elongated direction of the opening.

[0133] A cell sheet according to one embodiment of the present invention is characterized in that it has at least two cell layers and a mesh sheet, which serves as a substrate for attaching and culturing cells, is disposed between the two cell layers.

[0134] According to the above configuration, it is possible to ensure a sufficient thickness of the cell sheet and improve the strength of the cell sheet. Also, according to the above configuration, it is possible to provide a cell sheet having multiple types of cell layers. EXAMPLES

[0135] Example 1: Cell culture using the cell sheet manufacturing device of Configuration Example 1 <Preparation of Support Unit 10> A design drawing for the base 3 to be housed in the 12-hole plate was created, and the base 3 was produced based on the design drawing using a 3D printer AGILISTA-3200 (Keyence). The 3D printed object (base 3) was covered with parylene (DPXC, CAS No. 28804-46-8) (Japan Parylene) with excellent biocompatibility using a Labocoater PDS-2010 (Japan Parylene). A polyester micromesh sheet (AG00Z3N9) (Tenchi Gosen) was used for the mesh sheet 2.

[0136] The annular member 1 was made using a 2 mm plate made of PDMS (polydimethylsiloxane) (product name: SILPOT 184, Toray Dow Corning). The 2 mm PDMS plate was made as follows. First, a PDMS solution made by mixing the base agent and the curing agent in a ratio of 10:1 was poured into a dish to a thickness of 2 mm, and then heated to harden. A ring (outer diameter 8 mm, inner diameter 6 mm) was made from the completed 2 mm thick PDMS plate using two biopsy trepans (Kai Industries) with diameters of 8 mm and 6 mm.

[0137] The annular member 1, mesh sheet 2, and base 3 prepared as described above were washed with 70% ethanol and air-dried, and then assembled in a clean bench to complete the support unit 10. The completed support unit 10 was subjected to UV treatment for 30 minutes, and then the support unit 10 was placed in each well of a 12-well plate.

[0138] <Cell culture conditions> Tig-1-20 cells (product number JCRB0501), which are normal human lung fibroblasts, were obtained from the JCRB Cell Bank (Osaka). Tig-1-20 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). Tig-1-20 cells were maintained at 37°C and 5% carbon dioxide (CO 2 ) and cultured in an incubator under conditions.

[0139] HepG2 cells (product number RCB1886) were obtained from the RIKEN BioResource Center (RIKEN BRC). HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). HepG2 cells (product number RCB1886) were incubated at 37°C in 5% carbon dioxide (CO 2 ) and cultured in an incubator under conditions.

[0140] Human adipose tissue-derived mesenchymal stem cells were purchased from Promo Cell. Human adipose tissue-derived mesenchymal stem cells were cultured using Mesenchymal Stem Cell Growth Medium 2 (Promo Cell). Human adipose tissue-derived mesenchymal stem cells were cultured at 37°C and 5% carbon dioxide (CO 2 ) and cultured in an incubator under conditions.

[0141] <Cell seeding and microscopic observation> On the mesh sheet 2 (circular area with a diameter of 4 mm) of the support unit 10, 0.5×10 5 pieces (1×10 6 Tig-1-20 cells, HepG2 cells, or mesenchymal stem cells were seeded at 50 μL (cells / mL). Five hours after seeding, 3 mL of medium was added to the well containing the support unit 10. The medium was replaced once every three days (3 mL / well). Photographs of the cells were taken using a digital microscope or an inverted phase contrast microscope.

[0142] Tig-1-20 cell sheets, HepG2 cell sheets, and mesenchymal stem cell sheets cultured for 16 days were analyzed using the optical coherence tomography imaging system Cell3imager Estier (SCREEN Holdings).

[0143] For Tig-1-20 cells, the mesh sheet 2 was covered with a collagen solution before cell seeding. More specifically, a collagen acidic solution I-AC 5 mg / mL (Koken) was diluted 15-fold with 1 mM HCl solution, and 50 μL of the diluted collagen solution was placed on the mesh sheet 2. Then, after leaving it to stand at 37°C for 30 minutes, the diluted collagen solution was removed. Then, it was washed twice with PBS(-) and stored in a 37°C incubator until cell seeding.

[0144] <Result> 1010 in FIG. 10 is a microscope image of the support unit 10 one day after cell seeding. 50 μL of cell suspension was firmly held on the mesh sheet 2 (left image). Culture medium could be added to the well without any trouble such as bubbles forming in the gaps (center image). Furthermore, the support unit 10 could be turned upside down (right image).

[0145] It was confirmed that Tig-1-20 cells, HepG2 cells, and mesenchymal stem cells could be cultured on the mesh sheet 2 (1011 in FIG. 10). Because Tig-1-20 cells have weak adhesion to the mesh sheet 2, the mesh sheet 2 was covered with a coating solution. It was confirmed that by coating with the collagen solution, the cell sheet did not come off the mesh even when a medium was added after 5 hours.

[0146] In addition, the results of analyzing the Tig-1-20 cell sheet, HepG2 cell sheet, and mesenchymal stem cell sheet cultured for 16 days using an optical coherence tomography system are shown in Figure 11. As shown in Figure 11, the surfaces of the Tig-1-20 cell sheet and mesenchymal stem cell sheet were flat, whereas the HepG2 cell sheet was not flat. In the optical coherence tomography system analysis from one side, the HepG2 cell sheet and mesenchymal stem cell sheet could be clearly analyzed up to about the second plane of the mesh sheet. Since the Tig-1-20 cell sheet was thinner than those cell sheets, the cell sheet surface could be analyzed by observation from one side (Figure 11).

[0147] [Example 2: Production of cell sheets consisting of three types of cells using the cell sheet production device of Configuration Example 1] <Cell culture conditions> The cell culture conditions were the same as in Example 1.

[0148] <Seeding of cells onto the mesh sheet 2 of the support unit 10> The mesh sheet 2 of the support unit 10 (for 12-well) was coated with collagen. Then, 4 × 10 Tig-1-20 cells stained with CellBrite Green Cytoplasmic Membrane-Labeling Kit (80-fold dilution; Biotium, Inc.) were placed on the mesh sheet 2. 5 pieces (1x10 7After 5 hours, 3 mL of FBS-containing DMEM medium was added to the wells. After 2 days, 4 × 10 mesenchymal stem cells (MSCs) stained with CellBrite Red Cytoplasmic Membrane-Labeling Kit (120-fold dilution; Biotium, Inc.) were added on top of the Tig-1-20 cells. 5 pieces (1.6×10 7 Then, the medium was replaced with a medium for mesenchymal stem cells. The next day, the support unit 10 was turned upside down (installed upside down), and 4 × 10 HepG2 cells stained with 0.025 mg / mL DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were seeded on top of the Tig-1-20 cells. 5 pieces (1.6×10 7 The cells were seeded at 25 μL (100 cells / mL). The next day, the cells were washed with PBS(-) and immersed in 4% PFA and left to stand at room temperature for 50 minutes. After washing with PBS(-), the cell sheet was fixed between a cover glass and a slide glass together with the mounting medium VECTASHIELD (VECTOR). In detail, to prevent the cell sheet from being crushed by the cover glass, three layers of Kapton tape with a thickness of approximately 100 microns were placed between the cover glass and the slide glass. The fixed cell sheet was analyzed using a confocal microscope LSM 800 (ZEISS).

[0149] <Result> 1210-1212 in FIG. 12 show the results of this example. As shown in 1210-1212 in FIG. 12, a mesenchymal stem cell sheet could be produced on the Tig-1-20 cell sheet. Furthermore, a HepG2 cell sheet could be produced under the Tig-1-20 cell sheet. The overall thickness of the cell sheet was about 140-150 μm. As a result, it was shown that (i) cells could be seeded from both the top and bottom of the support unit 10, and (ii) a cell sheet consisting of multiple types of cells could be produced.

[0150] Example 3: Cultivation of human skin fibroblasts using the cell sheet manufacturing device of Configuration Example 2 <Preparation of Support Unit 30> The various members of the support unit 30 other than the mesh sheet 2 were produced using a 3D printer AGILISTA-3200. The above-mentioned various members produced were covered with parylene (DPXC, CAS No. 28804-46-8). The mesh sheet 2 was a polyester micromesh sheet (AG00Z3N9) (Tenchigosen). As a control group, a support unit 30 was also produced in which a porous membrane (Transwell polycarbonate membrane, 0.4 μm pore size, Corning) used in Transwell (registered trademark) was attached instead of the mesh sheet 2.

[0151] <Cell culture conditions> Normal human adult dermal fibroblasts (NHDF) were purchased from Promo Cell and cultured using Fibroblast Growth Medium 2 (Promo Cell). Normal human adult dermal fibroblasts were cultured in an incubator at 37°C and 5% carbon dioxide (CO2).

[0152] <Cell seeding and microscopic observation> NHDF is 1.65 x 10 6 pieces (3×10 6 The cells were seeded onto the mesh sheet 2 of the support unit 30 at 550 μL / sheet (cells / mL). After about 18 hours, the medium was added. The medium was changed once every three days, and the cells were cultured for 14 days (6 mL / well). An inverted phase contrast microscope image of the cells was taken (Figure 11A). In addition, HE stained sections were prepared (Shin Histopathology), and a cross-sectional view of the cell sheet was photographed using a BZ-X710 All-in-one (Keyence) (1311 in Figure 13).

[0153] <Result> The results are shown in 1310 and 1311 of FIG. 13. NHDF existed in a form that filled the mesh openings of the mesh sheet 2 and covered the mesh threads of the mesh sheet 2. The surfaces of both the cell sheet fabricated with the mesh sheet 2 and the cell sheet fabricated with the porous membrane were uniform and smooth. Unlike the cell sheet fabricated with the porous membrane, the lower surface of the cell sheet fabricated with the mesh sheet 2 had cells exposed. For this reason, it was considered to be excellent in nutrient supply and cell observation, and also excellent in attaching the cell sheet during transplantation medicine.

[0154] 〔Example 4: Cultivation of Tig-1-20 cells using the cell sheet manufacturing apparatus of Configuration Example 2〕 <Fabrication of the support unit 30> The method for fabricating the support unit 30 of this example is the same as that of Example 3. However, only the structure of the holding member 34 of the support unit 30 of this example is partially different from that of Example 3. In this example, the holding member 34 has no voids for facilitating the flow of the culture medium from the outside to the inside (see 1410 in FIG. 14).

[0155] Also, the culture conditions (composition of the culture medium) for the Tig-1-20 cells are the same as those in Example 1. However, in this example, the mesh sheet 2 is not coated.

[0156] <Seeding and microscopic observation of Tig-1-20 cells> 4×10 stained with CellBrite Green Cytoplasmic Membrane-Labeling Kit (100-fold dilution; Biotium, Inc.) or not stained 6 pieces (8×10 6Tig-1-20 cells (cells / mL, 500μL / mesh) were seeded on the mesh sheet 2 of the support unit 30 prepared in <Preparation of support unit 30>. Then, 17 hours later, 6mL of medium was added to the well. Three days later, the medium was replaced once. On the sixth day after the start of culture, the prepared cell sheet was washed, and the cells were fixed with 4% paraformaldehyde (PFA), and after washing, they were fixed in a mounting medium VECTA containing DAPI (4',6-diamidino-2-phenylindole). The fixed cell sheet was analyzed using a confocal microscope LSM 800 (ZEISS). Meanwhile, HE-stained sections of the cell sheet that was fixed with 4% PFA and 1% formaldehyde and not fluorescently stained were prepared (Shin Histoscience), and the cross-sections were photographed with a BZ-X710 All-in-one (Keyence).

[0157] <Seeding of human iPS cell-derived cardiomyocytes and microscopic observation> In order to seed additional human iPS cell-derived cardiomyocytes on top of the Tig-1-20 cell sheet (cultured for 6 days) that was not used in the above experiment, the medium in the well was replaced with 4 mL of MiraCell CM Culture Medium. 5 Human iPS cell-derived cardiomyocytes were seeded. After about 16 hours, 2 mL of medium was added. After that, the cells were cultured with Tig-1-20 cells for 3 days, and the cells were fixed and analyzed by confocal microscopy as described above.

[0158] <Result> The results are shown in 1420 to 1423 in FIG. 14. As shown in 1420 in FIG. 14, the Tig-1-20 cells were oriented according to the shape of the openings in the micromesh sheet. In other words, they were oriented almost parallel to the long side of the rectangular openings. Cells in locations other than the plane of the micromesh sheet, i.e., cells in the plane above or below the plane of the micromesh sheet, were also oriented in the same direction as the cells in the plane of the micromesh sheet. The thickness of the cell sheet was about 50 μm (1421 in FIG. 14). In addition, as a result of additionally seeding human iPS cell-derived cardiomyocytes on top of the Tig-1-20 cell sheet, the cardiomyocytes were also oriented in the same direction as the Tig-1-20 cells. These results showed that the orientation of a three-dimensional cell sheet can be controlled in some cases by performing cell culture using a micromesh sheet (1422 and 1423 in FIG. 14).

[0159] Example 5: Cultivation of Tig-1-20 cells using the cell sheet manufacturing device of Modification 2 <Method> <Preparation of Support Unit 10B and Cell Culture Conditions> The annular member 1 and the base 3 were produced using a 3D printer AGILISTA-3200. The produced annular member 1 and the base 3 were then covered with parylene (DPXC, CAS No. 28804-46-8). The mesh sheet 2 was a polyester micromesh sheet (AG00Z3N9) (Tenchigosen). The mesh sheet 2 was sandwiched between the annular member 1 and the base 3 and bonded using a PDMS solution. After the completed support unit 10B was subjected to ethanol treatment and UV treatment, the support unit 10B was placed in a 10 cm dish 22. In the support unit 10B used in the experiment, the distance from the bottom surface of the dish 22 to the mesh sheet 2 was adjusted to 2.5 mm using a 1 mm PDMS plate (1510 in FIG. 15).

[0160] The culture conditions (medium composition) for the Tig-1-20 cells were the same as in Example 1, but the mesh sheet 2 was not coated.

[0161] <Seeding of Tig-1-20 cells and microscopic observation> First, 3 mL of Tig-1-20 cells (6x10 5 cells / mL) were placed on mesh sheet 2. Then, lid 9 was placed on top. Next, 5 mL of cell suspension was added through the inlet 9i of lid 9, and the gap between lid 9 and mesh sheet 2 was filled with the cell suspension. Three days after cell seeding, 25 mL of medium was added to a 10-cm dish 22 containing support unit 10B, and then lid 9 was removed. The medium was changed once every three days (35 mL of medium / dish), and the culture was terminated 14 days after cell seeding.

[0162] As shown in 1511 of FIG. 15, the cell sheet was cut from support unit 10B using a knife. A part of the cut cell sheet was washed with phosphate buffered saline (PBS)(-), treated with 4% PFA for 20 minutes, and stained with DiI (37 °C, 1 hour). After washing with PBS(-), the cell sheet was fixed between a cover glass and a slide glass together with the mounting medium VECTASHIELD (VECTOR) containing DAPI. The fixed cell sheet was analyzed using a confocal microscope LSM 800 (ZEISS).

[0163] A part of the cell sheet prepared by support unit 10B was washed with PBS(-), fixed by treatment with 4% PFA for 20 minutes. Next, it was treated with 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, approximately 100% ethanol for 5 minutes each, and then air-dried. Then, the cell sheet was observed using a low-vacuum scanning electron microscope Miniscope TM3030Plus (Hitachi).

[0164] The viability of the cells constituting the cell sheet cultured on the support unit 10B was examined. Approximately half of the cell sheet cut from the support unit 10B was washed with PBS(-) and treated with 0.05% trypsin-EDTA for 7 minutes (37°C). After removing the trypsin, the cells were suspended in 10 mL of medium. The viability of the cells was measured using a Countess II Automated Cell Counter (Thermo Fisher Scientific).

[0165] <Result> The results are shown in 1512 of FIG. 15, 1611 and 1612 of FIG. 16, and 1711 and 1712 of FIG. 17. Tig-1-20 cells were cultured for 14 days using the support unit 10B (1512 of FIG. 15). The cross section of the cell sheet was analyzed by confocal microscopy and found to have a thickness of approximately 40-45 μm (1611 and 1612 of FIG. 16). The Tig-1-20 cells were oriented according to the shape of the openings of the mesh sheet 2. That is, they were oriented approximately parallel to the long side of the rectangular openings. Cells that were not on the plane of the mesh sheet 2 were also oriented in the same direction.

[0166] The cell sheet was treated with ethanol in order to observe it under an electron microscope. This treatment may have caused the cell sheet to shrink and become thinner, but it was confirmed that the cells filled the openings of the mesh sheet 2 without any gaps (1711 in Figure 17). Using a trypsin-EDAT solution and a trypan blue solution, the survival rate of the cells constituting the 3D cell sheet created with the support unit 10B was examined, and it was found that approximately 92% of the cells were alive (1712 in Figure 17). In other words, many cells were alive even in the 3D culture.

[0167] Example 6: Cultivation of HepG2 cells using the cell sheet manufacturing device of Configuration Example 4 <Method> <Preparation of Support Unit 50> The column body 51 of the support unit 50 was produced using a 3D printer AGILISTA-3200. The produced column body 51 was then covered with parylene (DPXC, CAS No. 28804-46-8). The mesh sheet 2 was a polyester micromesh sheet (AG00Z3N9) (Tenchigosen). The mesh sheet 2 was bonded to the column body 51 using a PDMS solution. The completed support unit 50 was treated with ethanol and air-dried, and then placed in the well 25a of the well plate 25 (12-hole plate) and subjected to UV treatment.

[0168] <Cell culture conditions> HepG2 cells (product number RCB1886) were obtained from the RIKEN BioResource Center (RIKEN BRC). HepG2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). The cells were maintained at 37°C and 5% carbon dioxide (CO 2 ) and cultured in an incubator under conditions.

[0169] <Cell seeding and cell observation> 1×10 5 pieces (2×x10 6 HepG2 cells (1×10 cells / mL×50 μL / mesh) were seeded on the mesh sheet 2 (4 mm diameter circle) of the support unit 50. Six hours after cell seeding, 2 mL of medium was added to the well 25a. The medium was replaced every two days (2 mL / well). As a control, 1×10 5 HepG2 cells were seeded into well 25a of well plate 25 (12-well plate).

[0170] <Gene expression analysis> RNA was extracted from HepG2 cells on day 10 after the start of culture. RNA was extracted using TRIzol RNA Isolation Reagents (Thermo Fisher Scientific), and cDNA was synthesized using the extracted RNA and ReverTra Ace qPCR RT Kit (Toyobo). Gene expression analysis was performed using the obtained cDNA sample, DNA primers, PowerUp SYBR Green Master Mix (APPLIED BIOSYSTEMS), and QuantStudio 5 Real-Time PCR System (APPLIED BIOSYSTEMS). The expression levels of albumin, known as a maturation marker of human liver, and the drug metabolizing enzyme CYP1A2 were analyzed. The expression levels of each gene were normalized using the expression level of 18SrRNA, known as a housekeeping gene. Values ​​are mean ± SE (N = 3).

[0171] <Result> 1810 in FIG. 18 shows a microscope image of HepG2 cells. The HepG2 cells adhered to the mesh sheet 2 within 6 hours. 1811 in FIG. 18 shows the results of gene expression analysis. Compared to when HepG2 cells were cultured on a normal well (2D), the expression levels of albumin and CYP1A2 genes were higher when the cells were cultured on the mesh sheet 2 (Mesh) of the support unit 50.

[0172] [Example 7: Cultivation of human iPS cell-derived cardiomyocytes using the cell sheet manufacturing device of Configuration Example 4] <Method> <Cell culture conditions> Human iPS cell-derived cardiomyocytes (MiraCell Cardiomyocytes from ChiPSC12, Takara) were cultured in MiraCell CM Culture Medium according to the product instructions at 37°C and 5% CO. 2 The cells were cultured in the presence of human fibronectin on dishes coated with human fibronectin.

[0173] <Cell seeding and cell observation> Before cell seeding, the mesh sheet 2 of the support unit 50 was coated with human fibronectin. A fibronectin solution (final concentration: 0.05 mg / mL) was prepared by diluting 20 times with PBS(+). 50 μL of the diluted fibronectin solution was placed on the mesh sheet 2 and allowed to stand at 37° C. for 1 hour or more.

[0174] Remove the fibronectin solution and add 2 x 10 4 pieces (4×10 5 Cardiomyocytes (cells / mL×50 μL / mesh) were seeded on the mesh sheet 2 (circle of φ4 mm) of the support unit 50. After 6 hours, 2 mL of medium was placed in well 25a to culture the cardiomyocytes. The medium was replaced once every two days (2 mL / well).

[0175] <Result> The results are shown in Figure 19. It was confirmed that cardiomyocytes adhered and proliferated on the mesh sheet 2 of the support unit 50. The cardiomyocytes maintained their pulsating ability even after being cultured for 8 days. [Industrial Applicability]

[0176] The present invention can be used to prepare cell sheets for use in regenerative medicine, transplantation, and the like. [Explanation of symbols]

[0177] 1 Annular member 2 Mesh sheet 3 Base (holding member) 3b Cylindrical placement part 4, 4a, 4b cells 9 Lid 9a Inlet 10, 10A, 10B, 30, 40, 50, 60 Support unit 20, 23 containers 21, 24, 25a Wells (containers) 22 Dish (container) 25, 26 Well plate (container) 31, 32 Frame 33 Clip (clamping member) 41, 51 Column body (holding member) 65 Pressing pin 66 Through hole 67 Locking part 100, 100A, 100B, 100C, 100D, 100E Cell sheet manufacturing equipment 200 cell sheets

Claims

1. (1) Preparing a cell sheet manufacturing apparatus, the cell sheet manufacturing apparatus comprising: A container for containing a medium for culturing cells; A support unit including a mesh sheet that is a substrate on which the cells are attached and cultured, and a holding member that holds the mesh sheet so that it floats above the bottom surface of the container, the support unit being detachably accommodated in the container; The support unit includes a lid that covers one side of the mesh sheet and has an injection port for injecting a suspension containing the cells between the mesh sheet and the lid, a gap between the lid and the mesh sheet is set so that the suspension is injected between the mesh sheet and the lid while being in contact with both the mesh sheet and the lid; (2) placing the lid body so as to cover the mesh sheet from above, and injecting the suspension from the injection port between the mesh sheet and the lid body while contacting both the mesh sheet and the lid body, thereby seeding the cells on the surface of the mesh sheet; (3) culturing the cells seeded on the surface of the mesh sheet by immersing the support unit in a medium in the container; The cell sheet production method includes the steps of:

2. (4) turning the support unit upside down; and (5) seeding additional cells on a surface of the mesh sheet opposite the surface; and (6) culturing the additional cells by immersing the support unit in a medium in the container; The method for producing a cell sheet according to claim 1 ,

3. The above (2) is (2-1) seeding the cells on the surface of the mesh sheet; (2-2) immersing the support unit in the medium in the container with the cells seeded on the surface of the mesh sheet; The method for producing a cell sheet according to claim 1 or 2, comprising:

4. 4. The cell sheet manufacturing method according to claim 1, wherein the openings of the mesh sheet are elongated in one direction.

5. The cell sheet manufacturing method according to claim 4, wherein the opening is rectangular, and the shape extended in one direction has a short side:long side ratio of 1:2 to 1:

10.

6. The cell sheet manufacturing method according to any one of claims 1 to 5, characterized in that the holding member removably holds the mesh sheet.

7. A cell sheet manufacturing method described in any one of claims 1 to 6, wherein the distance between the lid body and the mesh sheet is 1 to 3 mm.

8. A cell sheet manufacturing apparatus, comprising: A container for containing a medium for culturing cells; A support unit including a mesh sheet that is a substrate on which the cells are attached and cultured, and a holding member that holds the mesh sheet so that it floats above the bottom surface of the container, the support unit being detachably accommodated in the container; Equipped with The support unit is accommodated in the container so that its vertical and horizontal positions in the culture medium are constant; The support unit includes a lid covering one side of the mesh sheet, the lid being provided with an injection port for injecting a suspension containing the cells between the mesh sheet and the lid, and a distance between the lid and the mesh sheet is set so that the suspension is injected between the mesh sheet and the lid from the injection port of the lid arranged to cover the mesh sheet from above while contacting both the mesh sheet and the lid, Furthermore, the cell sheet manufacturing apparatus is characterized in that even when the support unit is turned upside down, the holding member holds the mesh sheet so that it floats above the bottom of the container, and the support unit is contained in the container so that its vertical and horizontal positions in the culture medium are constant.

9. The cell sheet manufacturing apparatus according to claim 8 , wherein the openings of the mesh sheet are elongated in one direction.

10. The cell sheet manufacturing apparatus according to claim 9, wherein the opening is rectangular, and the shape extended in one direction has a short side:long side ratio of 1:2 to 1:

10.

11. A cell sheet manufacturing apparatus described in any one of claims 8 to 10, wherein the distance between the lid body and the mesh sheet is 1 to 3 mm.

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