Cell sheet manufacturing apparatus and cell sheet

Abstract

The present application ensures that both sides of a cell sheet are easily observed in a stable state in which the position of the cell sheet in a culture medium is stabilized. A cell sheet manufacturing device (100) includes a container (20) and a support unit (10) that is contactably and detachably housed in the container (20), the support unit (10) having a mesh sheet (2) and a pedestal (3) that holds the mesh sheet (2) so as to float from the bottom surface of the container (20); the support unit (10) is housed in the container (20) in such a manner that the position is fixed in both the vertical direction and the horizontal direction in the culture medium.

Description

Technical Field

[0001] This invention relates to a cell membrane manufacturing apparatus and cell membranes. Background Technology

[0002] Currently, cell sheets are actually used as skin grafts for treating burns. In the future, cell sheets will also be used as myocardial cell grafts for transplantation into patients with heart failure or as pancreatic islet cell grafts for transplantation into patients with diabetes, and are expected to be applied to more patients.

[0003] On the other hand, researchers are also exploring the use of cell sheets composed of multiple cell layers to reconstruct tissue structures similar to those in living organisms. This has led to the recognition of the importance of methods for fabricating cell sheets with three-dimensional cell configurations. When using existing methods for fabricating cell sheets in temperature-sensitive culture dishes, the resulting cell sheets are often brittle and difficult to handle (see Non-Patent Literature 1 and 2). Furthermore, when fabricating cell sheets composed of multiple cell layers (dermis and epidermis) on a culture dish, the mature epidermis forms a barrier called tight junctions, which may hinder nutrient supply to the interior of the cell sheet.

[0004] Additionally, methods for fabricating cell sheets on non-cellular membrane-like supports have been proposed. However, if the support does not fuse with the cells of the affected area, this method cannot adhere the exposed side of the support to the affected area. That is, the adhesion surface of the cell sheet to the affected area is limited to one side. When applying such cell sheets, which consist of a fibroblast layer (dermis) with a keratinocyte layer (epidermis) on top, to the affected area, the limited adhesion surface, as described above, makes it difficult to achieve the desired effect. This is because when transplanting cell sheets, the dermal side needs to be adhered to the affected area.

[0005] In addition to the above, other methods for preparing cell sheets include fabricating cell sheets consisting of multiple cell layers on a porous membrane (e.g., a porous membrane made of polycarbonate) (see Non-Patent Literature 3). Cell sheets prepared by this method are expected to supply nutrients to cells through the pores of the porous membrane, thus offering advantages over preparing multi-layered cell sheets on a culture dish. However, when preparing cell sheets on a porous membrane, the surface that adheres to the affected area is only one side, making them difficult to use for transplantation. Therefore, cell sheets prepared according to the above method are mainly used for applications such as compound permeability testing.

[0006] A proposed method for fabricating cell membranes using nanofiber sheets made of gelatin, polylactic acid (PLA), or lactoglycolic acid copolymer (PLGA) is described (see Non-Patent Literature 4). This method utilizes electrospinning to create membranes composed of nanofibers, on which cells are then cultured. A problem with this method is that it is difficult to accurately control the arrangement of each nanofiber through electrospinning. Furthermore, due to the irregularity of the nanofibers, the fabricated cell membranes may not yield highly reproducible results.

[0007] In addition, the use of micromesh membranes has been proposed for fabricating cell sheets. Methods for using micromesh membranes include, for example, (i) a method for culturing cells on micromesh membranes to fabricate two-dimensional cell sheets, and (ii) a method for culturing iPS cells on micromesh membranes to fabricate spherical feeder-like cells (see Patent Documents 1 and 2, and Non-Patent Document 5). As an application of this method of culturing cells using micromesh membranes, a technique for fabricating three-dimensional cell sheets by culturing HepG2 hepatocytes on micromesh membranes has been developed. Compared to three-dimensional spheres, this three-dimensional cell sheet improves some cell functions and facilitates cell observation and nutrient supply, demonstrating excellent compatibility with fluid devices (see Non-Patent Document 6).

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: International Publication No. WO 2015 / 005349 (published on January 15, 2015)

[0011] Patent Document 2: Japanese Patent Application Publication No. 2019-50773 (published on April 4, 2019)

[0012] Non-patent literature

[0013] Non-patent document 1: Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl AcadSci USA. 1979; 76(11): 5665-8.

[0014] 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.

[0015] 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

[0016] 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

[0017] 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

[0018] Non-patent document 6: Hori T, Kurosawa OA Three–dimensional Cell Culture Methodwith a Micromesh Sheet and Its Application to Hepatic Cells.Tissue Eng Part CMethods.2018 Summary of the Invention

[0019] The problem that the invention aims to solve

[0020] In a method for manufacturing cell membranes using micromesh sheets, the mesh sheet is first placed in a container, floating in the culture medium used for cell culture. Next, cells are seeded onto the micromesh sheet. Then, the cells seeded onto the micromesh sheet are cultured in the culture medium. In this method, to confirm the growth and development of the cells constituting the cell membrane, both sides of the cell membrane are examined using a microscope.

[0021] For example, according to the technology disclosed in Patent Document 1, a micromesh sheet for inoculating cells is disposed on a suspension member, which is disposed in a container. With such a configuration, it is difficult (i) to flip the cell sheet for culture and difficult (ii) to flip the cell sheet for microscopic observation (i.e., double-sided observation).

[0022] Furthermore, the device disclosed in Patent Document 2 holds the micromesh membrane within a groove set in the inner wall of the container holding the culture medium. With this configuration, it is difficult to detach the micromesh membrane from the container for reversible placement. Therefore, it is also difficult to confirm both sides of the cell membrane using a microscope.

[0023] The challenges faced in cell observation are particularly evident when preparing cell sheets composed of multiple cell layers.

[0024] The first objective of this invention is to provide a cell membrane manufacturing apparatus that allows for easy observation of both sides of a cell membrane when the cell membrane is in a stable position within a culture medium, in order to confirm the growth and development of the cells constituting the cell membrane.

[0025] Furthermore, a second objective of this invention is to realize a cell membrane sheet that can adequately ensure thickness and improve strength.

[0026] Technical solutions for solving the problem

[0027] To address the aforementioned issues, one aspect of the present invention relates to a cell membrane manufacturing apparatus characterized by comprising: a container holding a culture medium for culturing cells; and a support unit detachably housed within the container, having a substrate for attaching and culturing the cells, namely a mesh membrane, and a retaining member for holding the mesh membrane to float from the bottom of the container; the support unit is housed within the container in a manner that fixes its position in both the vertical and horizontal directions within the culture medium.

[0028] The cell membrane sheet involved in one aspect of the present invention is characterized in that it has at least two cell layers, and a substrate, namely a mesh membrane, for attaching and culturing cells is disposed between the two cell layers.

[0029] Invention Effects

[0030] According to one aspect of the present invention, a cell membrane manufacturing apparatus is available that allows for easy observation of both sides of a cell membrane when the cell membrane is in a stable position within a culture medium, in order to confirm the growth and development of cells within the cell membrane. Furthermore, according to another aspect of the present invention, a cell membrane that adequately ensures thickness and improves strength is available. Attached Figure Description

[0031] Figure 1 This is a diagram showing the simplified structure of the cell membrane fabrication apparatus that constitutes Example 1.

[0032] Figure 2 It is used for explanation Figure 1 A diagram showing the effect of the cell membrane fabrication device.

[0033] Figure 3 This is a diagram showing the configuration of the cell membrane fabrication apparatus of Improved Example 1.

[0034] Figure 4 This is a diagram showing the configuration of the cell membrane fabrication apparatus of Improved Example 2.

[0035] Figure 5 This is a schematic diagram showing the cross-sectional structure of a skin sheet manufactured using a cell sheet manufacturing apparatus according to an embodiment of the present invention.

[0036] Figure 6 This is a diagram illustrating the simplified structure of the support unit in the cell membrane fabrication apparatus of Example 2.

[0037] Figure 7 This is a diagram showing the simplified structure of the cell membrane fabrication apparatus that constitutes Example 2.

[0038] Figure 8 This is a diagram showing the simplified structure of the cell membrane fabrication apparatus that constitutes Example 3.

[0039] Figure 9 This is a diagram showing the simplified structure of the cell membrane fabrication apparatus that constitutes Example 4.

[0040] Figure 10 This is a graph showing the results of Example 1.

[0041] Figure 11 This is a graph showing the results of Example 1.

[0042] Figure 12 This is a graph showing the results of Example 2.

[0043] Figure 13 This is a graph showing the results of Example 3.

[0044] Figure 14 This is a graph showing the results of Example 4.

[0045] Figure 15 This is a graph showing the results of Example 5.

[0046] Figure 16 This is a graph showing the results of Example 5.

[0047] Figure 17 This is a graph showing the results of Example 5.

[0048] Figure 18 This is a graph showing the results of Example 6.

[0049] Figure 19 This is a graph showing the results of Example 7.

[0050] Figure 20 This is a diagram showing the configuration of the cell membrane fabrication apparatus of Improved Example 3.

[0051] Figure 21 This is a diagram used to illustrate the effect of the cell membrane fabrication apparatus of Improved Example 3. Detailed Implementation

[0052] The following is a description of one embodiment of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope shown in the claims. Embodiments and examples obtained by appropriately combining different embodiments and examples of the disclosed technical solutions are also included within the technical scope of the present invention. Furthermore, all documents described in this specification are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," this description means "A or more, B or less (inclusive of the values ​​of A and B)."

[0053] 1. Cell membrane fabrication device

[0054] The cell membrane manufacturing apparatus described in this embodiment is based on the premise that a mesh membrane is placed in a culture medium, and cells seeded on the mesh membrane are allowed to grow and develop in the culture medium, thereby manufacturing a cell membrane. By placing the mesh membrane in the culture medium (culture solution), cells can proliferate according to the shape of the mesh membrane.

[0055] The cell membrane manufacturing apparatus of this embodiment comprises a container and a support unit, wherein the container holds a culture medium for culturing cells. The support unit, detachably housed within the container, includes a mesh membrane and a retaining member. The mesh membrane serves as a substrate for cell attachment and culture. The retaining member holds the mesh membrane so that it floats above the bottom of the container. The support unit is housed within the container in a manner that fixes its position both vertically and horizontally within the culture medium.

[0056] Here, "keeping the mesh membrane floating from the bottom of the container" means keeping the mesh membrane in such a way that neither side of the mesh membrane is in contact with the bottom of the container (preferably the inner wall and the bottom of the container), but both sides of the mesh membrane are in full contact with the culture medium. The holding member is only required to be configured to keep the mesh membrane floating from the bottom of the container, and there are no restrictions on the specific configuration.

[0057] In the above-described operation of manufacturing cell sheets using a mesh membrane, the cell sheet manufacturing apparatus of this embodiment improves the convenience for users to observe cells. More specifically, in the above configuration, the support unit is detachably housed in a container. Therefore, the user can easily remove the support unit from the container, flip it over, and then put it back into the container, making it easy to flip the support unit. Flipping the support unit also flips the cell sheet formed on the mesh membrane held by the holding member of the support unit. Therefore, according to the above configuration, it is easy to observe both sides of the cell sheet formed on the mesh membrane using a microscope.

[0058] Furthermore, the support unit is housed in the container in a manner that fixes its position both vertically and horizontally within the culture medium. Therefore, during microscopic observation of cell membrane sheets, the position of the mesh membrane within the container—in other words, the position of the cell membrane sheets formed on the mesh membrane—remains unchanged. Thus, based on the above configuration, cell membrane sheets can be observed precisely.

[0059] Furthermore, in the cell sheet manufacturing apparatus according to this embodiment, the retaining member preferably retains the mesh sheet detachably. This facilitates the separation of the cell sheet formed on the mesh sheet from the support unit. Consequently, the handling of the cell sheet becomes easier, for example, during cell sheet transplantation.

[0060] Furthermore, the support unit can be housed in the container in a manner that fixes its position both vertically and horizontally within the culture medium; its specific configuration is not limited. For example, a configuration where the holding member of the support unit has a specific gravity greater than that of the culture medium can be cited. Because the specific gravity of the holding member is greater than that of the culture medium, the holding member will not float in the culture medium but will sink, and its position in the vertical direction will be 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 set to 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 particularly preferably 10.0 or more. Regarding the materials constituting the holding member, examples include polystyrene, polyester, polyacetal, polycarbonate, and polyvinyl chloride.

[0061] Furthermore, there are no particular restrictions on the type of mesh membrane, as long as it is a structure capable of seeding and proliferating cells. The mesh membrane is preferably a planar structure formed by the regular or irregular repetition of openings of a specific shape. More preferably, the mesh membrane has multiple polygonal openings when viewed from above. Typical shapes of the openings are triangles, quadrilaterals, hexagons, etc., but can also be circles, ellipses, or other polygons.

[0062] Here, the portion of the mesh membrane other than its opening is referred to as the frame or frame portion. Additionally, when the opening is micrometer-sized, the mesh membrane is also called a micro-mesh membrane, etc. The shape of the opening of the mesh membrane can also be described as the shape of the area surrounded by the frame constituting the mesh membrane.

[0063] The opening of the mesh membrane can be of a size sufficient to allow at least one cultured cell to pass through. An opening of such a size means that, regardless of whether the cell is deformable or not, the relationship between the opening size and the cell size ensures that the cell can pass through the opening. The opening can be of a size that allows the cell to pass through without contacting it, or it can be of a size that allows the cell to pass through the opening while simultaneously contacting it (in other words, while deforming).

[0064] The shape of the opening of the mesh membrane is preferably an elongation in one direction. "An elongation in one direction" refers to a shape in which, among the multiple axes defining this shape, one axis (major axis) is longer than the others (minor axes). Examples of such shapes include rectangles, rhombuses, and ellipses. When the opening shape is rectangular, the longer side corresponds to the major axis, and the shorter side corresponds to the minor axis. Furthermore, when the opening shape is rhombus, the longer diagonal corresponds to the major axis, and the shorter diagonal corresponds to the minor axis. Examples of "elongation in one direction" include rectangles where the major axis (long side) is much longer than the short side (minor axis), rhombuses where the major axis is much longer than the minor axis, and ellipses. If it is a rectangle, the elongation in one direction is a ratio of short side to long side of 1:2 to 1:5, preferably 1:2 to 1:10. Similarly, if it is a rhombus, the elongation in one direction is a ratio of minor axis to major axis of 1:2 to 1:5, preferably 1:2 to 1:10. If it is an ellipse, the shape that extends in one direction is a minor axis to 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. In addition, the "shape that extends in one direction" is not limited to rectangles, rhombuses, or ellipses, but can be any shape in which one of the multiple axes used to define the shape is longer than the other axes.

[0065] As described above, when the opening of the mesh membrane is elongated in one direction, the opening has a large wall surface that elongates in that direction. A large number of cells adhere to and proliferate on this wall surface, and their proliferation is directional. Therefore, based on the above configuration, the directionality of the proliferating cells can be controlled to the elongation direction of the opening.

[0066] Furthermore, the material of the mesh membrane can be any material that allows cells to attach and proliferate, such as photocurable resins, biocompatible materials, and biodegradable materials.

[0067] If a photocurable resin is used, the mesh film can be fabricated using photolithography. Examples of photocurable resins include acrylate compounds, methacrylate compounds, epoxy compounds, isocyanate compounds, thiol compounds, and organosilicon compounds. Combinations of two or more types can also be used. Specific examples of photocurable resins include polyurethane acrylates, polyester acrylates, epoxy acrylates, poly(meth)acrylate, ethoxylated bisphenol A acrylates, aliphatic polyurethane acrylates, polyester acrylates, polyethylene terephthalate, polystyrene, polycarbonate, acrylic-modified alicyclic epoxides, difunctional alcohol ether epoxides, acrylic silicones, dimethylsiloxane acrylate, polydimethylsiloxane (PDMS), etc., but are not limited to these.

[0068] Furthermore, when fabricating the mesh film using photolithography, the exposed portion of the positive resist can be removed to create the micro-mesh film. Examples of such positive resists include DNQ (diazonaphthoquinone) phenolic resin positive resists, deprotection-reactive positive resists such as tert-butoxycarbonyl, tetrahydrofuran, phenoxyethyl, trimethylsilyl, and tert-butoxycarbonylmethyl, and depolymerization-reactive positive resists such as polyphthalaldehyde, polycarbonate, and polysilierlutel. Additionally, the developing solution may include tetramethylammonium hydroxide (TMAH), dimethyl sulfoxide (DMSO), MEK (methyl ethyl ketone), GBL (γ-butyrolactone), and EL (ethyl lactate).

[0069] In addition, when the material of the mesh membrane is a biocompatible or biodegradable material, the obtained cell membrane can be directly transplanted into the body along with the mesh membrane, making it suitable for regenerative medicine or drug innovation.

[0070] Regarding the biocompatible materials mentioned, examples include organosilicon, block polyetheramide (PEBAX), polyurethane, organosilicon-polyurethane copolymers, ceramics, collagen, hydroxyapatite, nylon, polyethylene terephthalate, ultra-high molecular weight polyethylene such as GORE-TEX (trademark), polyvinyl chloride, and other biological materials, but they are not limited to these. Furthermore, the mesh membrane can also be formed from materials other than biocompatible materials, with the surface treated with a biocompatible material.

[0071] The biodegradable materials mentioned above include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), copolymers thereof, PHB-PHV poly(alkanoic acid) compounds, polyesters, starch, cellulose, chitosan and other natural polymers and their derivatives.

[0072] Regarding the material of the aforementioned mesh membrane, polyester is preferred, and polyethylene terephthalate (PET) is particularly preferred. PET has low biotoxicity, thus allowing the obtained cell membrane to be transplanted into organisms. Furthermore, it suppresses autofluorescence during fluorescence microscopy observation; therefore, PET is preferably dyed black. Specific examples of polyester mesh membranes include materials AG001N0, AG00Z1N0, and AG00Z3N0 manufactured by Tianchi Synthetic Fiber Co., Ltd. Additionally, specific examples of black-dyed polyester mesh membranes include materials AG001N1, AG00Z1N9, and AG00Z3N9 manufactured by Tianchi Synthetic Fiber Co., Ltd.

[0073] Furthermore, the mesh membrane can be pre-coated with a cell adhesion promoting material. This material helps cells adhere to the culture support, facilitating their extension and proliferation. Examples include extracellular matrix proteins such as collagen, fibronectin, and laminin, as well as positively charged substances like poly-L-lysine. Matrigel (registered trademark) is a specific example of a cell adhesion promoting material.

[0074] Furthermore, in the cell membrane manufacturing apparatus according to this embodiment, the container is not particularly limited in its configuration (e.g., shape) as long as it can hold the culture medium used for culturing cells. Examples of such containers include cell culture dishes, culture plates with multiple wells (6-well plates, 12-well plates, etc.), and cell culture columns.

[0075] 2. Example 1 of the configuration of the cell membrane sheet manufacturing apparatus according to this embodiment

[0076] The following describes Example 1 of the configuration of the cell membrane manufacturing apparatus according to this embodiment. Figure 1 This is a diagram showing a simplified configuration of the cell membrane manufacturing apparatus 100, which is a constituent example 1. Figure 1 1010 is a perspective view showing the configuration of the support unit 10 of the cell membrane manufacturing apparatus 100. Figure 1 Figures 1020 to 1023 are step diagrams illustrating the manufacturing method of manufacturing cell membrane sheets using the cell membrane sheet manufacturing apparatus 100. Figure 1 1030 is a diagram showing the structure of cell membrane sheets manufactured using the cell membrane sheet manufacturing apparatus 100. Furthermore, Figure 1 Image 1040 is an example of a specific configuration of a cell membrane manufacturing apparatus 100.

[0077] like Figure 1 As shown in Figure 1023, the cell membrane fabrication apparatus 100 includes a support unit 10 and a container 20. The support unit 10 is detachably housed in the container 20.

[0078] like Figure 1 As shown in Figure 1010, the support unit 10 includes an annular member 1, a mesh diaphragm 2, and a base 3 (holding member). The base 3 has a base body 3a, a cylindrical mounting portion 3b, a bottom abutment portion 3c, and a side wall abutment portion 3d.

[0079] The base body 3a is disc-shaped. The cylindrical mounting portion 3b is a bottomless cylinder with openings at both ends along the axial direction. The cylindrical mounting portion 3b protrudes vertically from only one surface of the base body 3a. The cylindrical mounting portion 3b extends through the interior of the base body 3a. In other words, the upper and lower sides of the base body 3a are connected via the cylindrical mounting portion 3b. A mesh membrane 2 is mounted on the cylindrical mounting portion 3b. From the opposite side of the cylindrical mounting portion 3b relative to the base body 3a, it is possible to observe the mesh membrane 2 through the cylindrical mounting portion 3b using a microscope or the like.

[0080] Viewed from above in a vertical direction relative to the base body 3a, the sidewall abutment portion 3d is a rectangular plate extending horizontally from the base body 3a. At least two sidewall abutment portions 3d are formed, and they are symmetrical with respect to the base body 3a. Figure 1 In the configuration shown in 1010, four sidewall abutment portions 3d are formed. The horizontally forward end portion of the sidewall abutment portion 3d abuts against the sidewall of the container 20. The sidewall abutment portion 3d serves as a spacer to maintain a fixed horizontal distance between the base body 3a and the sidewall of the container 20. Therefore, even if the container 20 vibrates in the horizontal direction, the horizontal position of the base body 3a will not change in the culture medium due to the contact between the forward end portion of the sidewall abutment portion 3d and the sidewall of the container 20, thus maintaining stability. In addition, the base 3 is configured such that all sidewall abutment portions 3d form a certain gap in size with the sidewall of the container 20 so as to allow contact / separation with the container 20.

[0081] The bottom abutment portion 3c is a rectangular plate, erected corresponding to the rectangular plate-shaped sidewall abutment portions 3d. The bottom abutment portion 3c intersects with the sidewall abutment portions 3d. Figure 1 In the configuration shown in 1010, the bottom abutment portion 3c intersects the side wall abutment portion 3d in the vertical direction (gravity direction). Furthermore, the vertically forward end of the bottom abutment portion 3c abuts against the bottom surface of the container 20. The bottom abutment portion 3c acts as a spacer to maintain a fixed vertical distance between the base body 3a and the bottom surface of the container 20. Therefore, even when the base 3 is housed in the container 20, the vertical position of the base body 3a in the culture medium remains stable because it abuts against the bottom surface of the container 20 via the bottom abutment portion 3c. Moreover, the bottom abutment portion 3c intersects with the side wall abutment portion 3d and protrudes from both the top and bottom directions relative to the side wall abutment portion 3d. Therefore, even when the base 3 is housed in the container 20 with its top and bottom flipped, the vertical position of the base body 3a remains stable.

[0082] The annular member 1 has an annular shape surrounding the outer periphery of the cylindrical mounting portion 3b, and is designed to be detachable relative to the base 3. The annular shape of the annular member 1 is not particularly limited and can be any shape such as a circular ring or a four-cornered ring.Figure 1 In the configuration shown in 1010, the annular member 1 is in the shape of a ring.

[0083] A mesh diaphragm 2 is disposed between the annular member 1 and the cylindrical mounting portion 3b. The mesh diaphragm 2 is held by the base 3 in a state where its periphery is clamped by the inner surface of the annular member 1 and the outer surface of the cylindrical mounting portion 3b. When the annular member 1 is annular and the cylindrical mounting portion 3b is cylindrical, from the viewpoint of stably holding the mesh diaphragm 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 diaphragm 2, the better.

[0084] Next, the manufacturing method for cell membrane sheets using the cell membrane sheet manufacturing apparatus 100 will be described. First, as... Figure 1 As shown in 1020, the mesh diaphragm 2 is placed on the cylindrical mounting portion 3b of the base 3. Then, with the mesh diaphragm 2 placed on the cylindrical mounting portion 3b, the annular member 1 is installed onto the cylindrical mounting portion 3b.

[0085] At this time, the annular member 1 is installed such that its inner side surrounds the outer side of the cylindrical mounting portion 3b. Therefore, as Figure 1 As shown in 1021, the periphery of the mesh diaphragm 2 is held between the inner side of the annular member 1 and the outer side of the cylindrical mounting portion 3b. This creates a support unit 10 that holds the mesh diaphragm 2. In the created support unit 10, one end opening of the cylindrical mounting portion 3b is closed by the mesh diaphragm 2, while the other end opening is open.

[0086] Then, the support unit 10 is flipped upside down to ensure that the mesh diaphragm 2 is located below the cylindrical mounting portion 3b. As a result, a bottomed cylindrical portion 10a with the mesh diaphragm 2 as its bottom surface is formed in the support unit 10 through the mesh diaphragm 2 and the inner surface of the cylindrical mounting portion 3b. Figure 1 As shown in section 1022, a suspension of cells 4 in the culture medium 6 is injected into the bottomed cylindrical portion 10a using a pipette 5 or similar device. At this time, due to the surface tension of the suspension relative to the inner surface of the cylindrical mounting portion 3b and the mesh membrane 2, the suspension does not pass through the mesh membrane 2 and is held on the mesh membrane 2. Subsequently, by causing the cells 4 in the suspension to attach to the mesh membrane 2, the cells 4 are seeded onto the mesh membrane 2.

[0087] Next, the support unit 10 is housed in the container 20. Then, the container 20 is filled with culture medium 6, and the cells 4 attached to the mesh membrane 2 are cultured.

[0088] like Figure 1As shown in 1030, each cell 4 spontaneously extends and adheres to each other towards the center of the opening to block the opening of the mesh membrane 2. When proliferating cells 4 are cultured on the mesh membrane 2, the cells 4 proliferate not only horizontally relative to the mesh membrane 2 but also vertically. As a result, a cell membrane 200 can be obtained, forming a single or multiple cell layers composed of cells 4 to cover the opening of the mesh membrane 2. The thickness of the cell membrane 200 can be controlled by controlling the amount of cells 4 seeded on the mesh membrane 2 and / or the proliferation rate of the cells 4. The cell membrane 200 is supported by the mesh membrane 2, making it relatively strong and easy to handle.

[0089] Furthermore, the cell sheet 200 is configured to have at least two cell layers composed of cells 4, with a substrate, namely a mesh membrane 2, disposed between the two cell layers for attaching and culturing the cells 4. That is, both sides of the cell sheet 200 are composed of cells 4. Therefore, the surface of the cell sheet 200 that adheres to the affected area is not limited to one side. In addition, compared to existing cell sheet manufacturing methods that use culture dishes or porous membranes as scaffolds, the cell sheet manufacturing method using the cell sheet manufacturing apparatus 100 has the advantage of facilitating the supply of nutrients to the cells 4 and the observation of the cells 4. Furthermore, by using a membrane with patterned openings as shown in the mesh membrane 2, cell sheets 200 with the same structure (in other words, the same properties) can be obtained with high reproducibility.

[0090] like Figure 1 As shown in 1040, the container 20 can be a culture plate with a plurality of wells 21. More specifically, the container 20 is a 12-well culture plate. In this case, the support unit 10 with the base 3 is configured to be accessiblely / detachably housed in the wells 21.

[0091] Figure 2 This diagram illustrates the effect of the cell membrane fabrication apparatus 100. (For example...) Figure 2 As shown in 2010 and 2011, because the support unit 10 can be stored in contact with / separately from the container 20, the support unit 10 can still be used even if the base 3 is flipped upside down and stored in the container 20. Therefore, since the cell membranes formed on the mesh membrane 2 will also flip if the support unit 10 is flipped, it is easy to observe both sides of the cell membranes formed on the mesh membrane 2 with a microscope 7.

[0092] In addition, such as Figure 2 As shown in 2020-2022, if the cell membrane fabrication apparatus 100 is used, a cell membrane sheet having multiple cell layers can be fabricated. More specifically, a cell membrane sheet having two cell layers can be fabricated, namely, a first cell layer composed of cells 4a and a second cell layer composed of cells 4b of a different type than cells 4a. First, as...Figure 2 As shown in 2020, with cells 4a seeded on the mesh membrane 2, the support unit 10 was immersed in the culture medium 6. Then, as... Figure 2 As shown in 2021, the support unit 10 is flipped upside down. At this time, the side of the mesh sheet 2 opposite to the cell 4a becomes the upper side. Therefore, if a suspension of cell 4b is dropped onto the mesh sheet 2 from the upper side using a pipette 5 or the like, cells 4b can also be seeded on the side of the mesh sheet 2 opposite to the cell 4a (see reference 2021). Figure 2 (2022). In this way, according to the configuration of the cell sheet manufacturing apparatus 100, cells can be additionally seeded on either the surface or the back surface of the cell sheet. Furthermore, from the viewpoint of easily seeding cells on both sides of the cell sheet, such as... Figure 2 As shown in 2020-2022, the annular member 1 preferably protrudes from the cylindrical mounting portion 3b and is mounted on the base 3. According to this configuration, if the support unit 10 is flipped, a bottomed cylindrical portion 10b with the mesh membrane 2 as its base is formed by the inner surface of the annular member 1 and the mesh membrane 2. Therefore, by injecting the suspension of cells 4b into the bottomed cylindrical portion 10b, the suspension can be stably maintained on the mesh membrane 2.

[0093] Furthermore, based on the configuration of the cell membrane manufacturing apparatus 100, the cell membrane 200 can be easily separated from the support unit 10. For example... Figure 2 As shown in 2030, a cell membrane sheet 200 composed of cells 4 is prepared by culturing cells 4 in a culture medium within container 20. Then, as... Figure 2 As shown in 2031, the support unit 10 is separated from the container 20. Then, as... Figure 3 As shown in 2032, the annular member 1 is removed from the cylindrical mounting portion 3b of the base 3. This allows the cell membrane sheet 200 formed on the mesh membrane sheet 2 to be removed from the cylindrical mounting portion 3b of the base 3 using, for example, tweezers 8. This ease of removal of the cell membrane sheet 200 from the cell membrane sheet manufacturing apparatus 100 is convenient, for example, during cell membrane sheet transplantation.

[0094] (Improved Example 1)

[0095] An improved example of the cell membrane fabrication apparatus 100 will be described. Figure 3 This diagram illustrates the configuration of the cell membrane manufacturing apparatus 100A as an improved example 1, and the manufacturing method of manufacturing cell membranes using the cell membrane manufacturing apparatus 100A. Figure 3 3010 is a perspective view showing an example of the configuration of the base 3 included in the cell membrane fabrication apparatus 100A. Furthermore, Figure 3 Figures 3020 to 3024 are diagrams illustrating a manufacturing method for producing cell membrane sheets using a cell membrane sheet manufacturing apparatus 100A. Figure 33030 is a cross-sectional view showing the configuration of the cover 9 of the cell membrane manufacturing apparatus 100A. The cell membrane manufacturing apparatus 100A can manufacture large-area cell membranes.

[0096] like Figure 3 As shown in 3010, the base 3 can be designed for culture dishes with a diameter of 3.5 cm (for 6-well culture plates), a diameter of 6 cm, and a diameter of 10 cm. That is, by increasing the diameter of the cylindrical support portion 3b of the base 3, the cell membrane sheet produced can be made to have a large surface area. Correspondingly, the area of ​​the mesh membrane sheet 2 can also be increased.

[0097] The configuration of the cell membrane fabrication apparatus 100A in Improved Example 1 is suitable for maintaining a suspension on such a large-area mesh membrane 2, thereby fabricating a large-area cell membrane. Figure 3 As shown in Figures 3021 to 3024, the support unit 10A of the cell membrane manufacturing apparatus 100A includes a cover 9. The cover 9 is configured to cover one surface 2a of the mesh membrane 2. An injection port 9i is provided in the cover 9 for injecting a suspension containing cells 4 between the mesh membrane 2 and the cover 9. More specifically, the cover 9 is configured to cover the surface 2a of the mesh membrane 2 that contacts the cylindrical mounting portion 3b. The mesh membrane 2 is separated from the cover 9. The cylindrical mounting portion 3b is disposed between the mesh membrane 2 and the cover 9. The interval between the mesh membrane 2 and the cover 9 is maintained fixed by means of the cylindrical mounting portion 3b.

[0098] Here, the spacing between the mesh membrane 2 and the cover body 9 is set to ensure that the suspension of cells 4 is injected between the mesh membrane 2 and the cover body 9 while in contact with both sides. The spacing between the mesh membrane 2 and the cover body 9 is preferably set to 1 mm to 3 mm, more preferably 1 mm to 2 mm, and particularly preferably 1 mm to 1.5 mm.

[0099] In other words, the cover 9 is a component used to close the bottomed cylindrical portion 10a formed by the mesh diaphragm 2 and the cylindrical mounting portion 3b. The cover 9 can be a laminated structure comprising at least a first layer 9a and a second layer 9b. The first layer 9a is the layer furthest from the mesh diaphragm 2, and the second layer 9b is the layer closest to the mesh diaphragm 2. Figure 3 In the configuration shown in 3030, the cover 9 is a two-layer structure consisting of a first layer 9a and a second layer 9b. Examples of materials constituting the first layer 9a include silicone resin. Examples of materials constituting the second layer 9b include polyethylene terephthalate (PET). Alternatively, the cover 9 can be a single-layer structure consisting of either the first layer 9a or the second layer 9b.

[0100] In the method for manufacturing cell membrane sheets using the cell membrane sheet manufacturing apparatus 100A, firstly, a support unit is placed inside a culture dish 22. This support unit does not have a cover 9. Furthermore, the support unit is configured such that the mesh membrane 2 is located below the cylindrical support portion 3b, allowing it to be flipped up and down. In this way, by arranging the support unit 10, a bottomed cylindrical portion 10a can be formed. This bottomed cylindrical portion 10a is formed by the mesh membrane 2 and the inner surfaces of the cylindrical support portion 3b, with the mesh membrane 2 as the bottom surface (see reference). Figure 3 (refer to 3020). Next, the cover 9 is configured to close the open upper part with the bottom cylinder 10a, thereby creating the support unit 10A (see reference). Figure 3 (3021).

[0101] Then, using a pipette 5, the suspension of cells 4 in the culture medium 6 is injected from the injection port 9i into the bottom cylinder 10a (see reference). Figure 3 (3022). At this time, the suspension of cell 4 is injected into the bottom cylinder 10a while in contact with both the mesh membrane 2 and the cover 9. Therefore, under the action of the surface tension of the suspension relative to the cover 9 and the mesh membrane 2 respectively, the suspension can be prevented from dripping through the mesh membrane 2 due to gravity. Therefore, even if the mesh membrane 2 has a large area, the suspension is still held on the mesh membrane 2 under the action of the surface tension of the suspension relative to the cover 9 (see 3022). Figure 4 (3023). In this way, by attaching the cells 4 in the suspension to the mesh membrane 2, the cells 4 can be evenly seeded on the mesh membrane 2 (e.g., a large-area mesh membrane 2).

[0102] Subsequently, the entire culture dish 22 was filled with culture medium 6, and the cells 4 attached to the mesh membrane 2 were cultured (see reference). Figure 4 (3024).

[0103] In addition, in the above method, the suspension of cells 4 is injected after the cap 9 is placed on the bottom cylinder 10a. However, it is also possible to inject the suspension of cells 4 into the bottom cylinder 10a first, and then place the cap 9 to prevent the suspension from dripping from the mesh membrane 2.

[0104] (Improved Example 2)

[0105] Other improvements to the cell membrane fabrication apparatus 100 will be described. Figure 4 This diagram illustrates the configuration of the cell membrane manufacturing apparatus 100B as an improved example 2, and the manufacturing method of manufacturing cell membranes using the cell membrane manufacturing apparatus 100B. Figure 4 4010 is an exploded perspective view showing the structure of the support unit 10B included in the cell membrane manufacturing apparatus 100B. Figure 4Figures 4020 to 4022 are diagrams illustrating a manufacturing method for producing cell membrane sheets using a cell membrane sheet manufacturing apparatus 100B.

[0106] Similar to Improved Example 1, the cell membrane fabrication apparatus 100B includes a cover 9, enabling the fabrication of large-area cell membranes. However, the structure of the support unit 10B in the cell membrane fabrication apparatus 100B differs from that in Improved Example 1. For example... Figure 4 As shown in 4010, the base 3 of the support unit 10B is not a cylindrical mounting portion 3b protruding from the base body 3a. The cylindrical mounting portion 3b is configured as an opening formed in the base body 3a. The bottom abutment portion 3e is provided to protrude downward relative to the base body 3a, but not upward. Furthermore, the side wall abutment portion 3f extends horizontally from the base body 3a.

[0107] Furthermore, the annular member 1 has an inner diameter that is approximately the same as the inner diameter of the cylindrical mounting portion 3b. Therefore, the mesh diaphragm 2 is held between the lower surface of the annular member 1 and the upper surface of the base body 3a.

[0108] Furthermore, the cover 9 is configured to cover the surface 2b of the mesh diaphragm 2 that contacts the annular member 1. The mesh diaphragm 2 is separated from the cover 9. The annular member 1 is disposed between the mesh diaphragm 2 and the cover 9. The annular member 1 is used to maintain a fixed distance between the mesh diaphragm 2 and the cover 9. The cover 9 is a component used to close the bottomed cylindrical portion 10b formed by the mesh diaphragm 2 and the annular member 1.

[0109] In the method for manufacturing cell membrane sheets using the cell membrane sheet manufacturing apparatus 100B, firstly, a support unit 10B is placed inside a culture dish 22. The support unit 10B is arranged such that a mesh membrane 2 is located below an annular member 1. With this arrangement, a bottom cylindrical portion 10b is formed in the support unit 10B. This bottom cylindrical portion 10b is formed through the mesh membrane 2 and the inner surface of the annular member 1, with the mesh membrane 2 as the bottom surface (see reference). Figure 5 (4020).

[0110] Next, using a pipette 5, the suspension of cells 4 in the culture medium 6 is injected into the bottom cylinder 10a through the injection port 9a, and cells 4 are seeded on the mesh membrane 2 (refer to...). Figure 5 (4021 and 4022). Subsequently, culture dish 22 was filled with culture medium 6 for cell culture.

[0111] The structure of the cell membrane fabrication apparatus 100B makes it difficult to separate the fabricated cell membrane from the support unit 10B. However, the cell membrane can be cut from the support unit 10B using scissors or a knife.

[0112] Regarding cell sheets manufactured using the cell sheet manufacturing apparatus according to this embodiment, nutrients can be supplied from both the upper and lower surfaces of the cell sheet in the culture medium. In conventional methods for manufacturing cell sheets on culture dishes, since nutrients cannot be supplied from the lower surface of the cell sheet, cell culture relies solely on the nutrient supply from the upper surface. Therefore, the limited nutrient supply from the upper surface of the cell sheet leads to insufficient nutrient supply to the cells inside the cell sheet.

[0113] Regarding situations where nutrient supply to the upper surface of cell membranes is limited, such as in the fabrication of skin sheets, a common occurrence is the formation of a keratinocyte layer (epidermis) on top of the fibroblast layer (dermis) to create a skin sheet. In such cases, nutrient supply to the upper part of the skin sheet is hindered because mature epidermal cells form tight junctions. Therefore, it is difficult to fabricate skin sheets composed of a fibroblast layer and a dermis using existing methods for fabricating cell membranes on culture dishes.

[0114] Figure 20 This is a schematic diagram showing the cross-sectional structure of a skin sheet manufactured using the cell sheet manufacturing apparatus according to this embodiment. 11 represents dead keratinocytes. 12 represents the aforementioned tight junctions. Furthermore, 13 represents keratinocytes. 14 represents skin fibroblasts. Figure 21 As shown, for a skin sheet manufactured using the cell sheet manufacturing apparatus according to this embodiment, even if the nutrient supply from the upper keratinocyte layer 13 is reduced, it is still possible to supply nutrients from the lower skin fibroblast layer 14. Therefore, more nutrients can be supplied by the keratinocytes 13 and skin fibroblasts 14 constituting the skin sheet.

[0115] Furthermore, regarding other existing technologies, methods for fabricating skin sheets composed of multiple cell layers on porous membranes are known. Because of the use of porous membranes, it is possible to supply a certain amount of nutrition from the lower surface of the cell sheet. However, cell sheets fabricated using this method are difficult to use directly for transplantation. This is because, when transplanting to the affected area, the epidermal layer is not brought into contact with the affected area; instead, the dermal layer must be brought into contact with the affected area while the skin sheet is being transplanted. This results in cell sheets with the dermal layer sandwiched between the porous membrane and the epidermis being unsuitable for transplantation. On the other hand, the skin sheet fabricated using the cell sheet fabrication apparatus according to this embodiment has cells present on the culture medium side, i.e., exposed in the culture medium, not only at the epidermal layer but also at the dermal layer. Therefore, the dermal layer of the skin sheet can be adhered to the affected area.

[0116] Furthermore, intestinal cells, vascular endothelial cells, and the like can also be observed at the tight junctions 12. 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 containing intestinal cells, vascular endothelial cells, and the like to which tight junctions 12 are to be formed.

[0117] (Improved Example 3)

[0118] An improved example of a cell membrane fabrication apparatus will be described. Figure 20 and Figure 21 This is a diagram showing the support unit 60 of the cell membrane sheet manufacturing apparatus as an improved example 3. Additionally, in Figure 20 and Figure 20 The container constituting the cell membrane manufacturing device is omitted from the description.

[0119] The cell membrane manufacturing apparatus of this embodiment is configured such that at least one of the base 3 (holding member) and the annular member 1 has a locking portion 67 that locks the base 3 and the annular member 1 in a non-separating manner, and either the base 3 or the annular member 1 has a through hole 66 through which a pressing pin 65 for pressing the other is passed. By pressing the other, the locking is released, and the base 3 and the annular member 1 are separated. Alternatively, the cell membrane manufacturing apparatus of this embodiment may have a configuration with or without the pressing pin 65. When the cell membrane manufacturing apparatus of this embodiment does not have the pressing pin 65, the pressing pin 65 can be prepared as an additional component of the cell membrane manufacturing apparatus.

[0120] The locking portion 67 can be configured to release the locking of the base 3 and the annular member 1 by pressing the pin 65, and the specific configuration is not limited. Regarding the specific configuration of the locking portion 67, examples include (i) a friction-generating region formed on at least one surface of the base 3 and the annular member 1, creating a large frictional force between the base 3 and the annular member 1; (ii) a combination of a protrusion formed on the surface of the base 3 and a locking protrusion formed on the surface of the annular member 1 that locks the protrusion in the pressing direction; (iii) a combination of a protrusion formed on the surface of the annular member 1 and a locking protrusion formed on the surface of the base 3 that locks the protrusion in the pressing direction; and (iv) a combination of (ii) and (iii) above. For example, a material capable of generating the desired frictional force (e.g., rubber) can be adhered to the surface of at least one surface of the base 3 and the annular member 1 to form a friction-generating region.

[0121] The through hole 66 can be formed on either the base 3 or the annular member 1, but from the viewpoint that the support unit 60 is easy to handle, it is preferred to be formed on the base 3.

[0122] Focusing on a specific through hole 66, when the through hole 66 is formed on the base 3, it can be configured such that no through hole is formed in the region of the annular member 1 facing the through hole 66; when the through hole 66 is formed on the annular member 1, it can be configured such that no through hole is formed in the region of the base 3 facing the through hole 66. With this configuration, by passing the pressing pin 65 through the through hole 66, the pressing pin 65 can effectively press the component (base 3 or annular member 1) without a through hole.

[0123] There are no limitations on the shape and number of through holes 66. Examples of possible shapes for the through holes 66 include cylindrical and rectangular prism shapes. The number of through holes 66 formed in each retaining member or each annular member can be even or odd, but from the viewpoint of pressing the base 3 or annular member 1 as a whole in a substantially uniform manner, an even number is preferred. Examples of possible numbers for the through holes 66 include 1 to 10 or 1 to 20, and the number of through holes 66 can be appropriately set according to the size of the base 3 and the annular member 1.

[0124] More specifically, in Figure 21 20010 and Figure 20 In diagram 20011, the figure to the left of the arrow is a three-dimensional view of the support unit 60. (As described later...) Figure 20 As also shown in detail, the base 3 has a cylindrical mounting portion 3b for mounting the mesh diaphragm 2, and the periphery of the mesh diaphragm 2 is held by the cylindrical mounting portion 3b and the annular member 1. Alternatively, the support unit 60 may also have a cover (not shown) on the base 3 or the annular member 1.

[0125] exist Figure 21 20010 and Figure 21 In Figure 20011, the diagram to the right of the arrow is an exploded perspective view of the support unit 60 after pressing the annular member 1 by passing a pressing pin 65 through the through hole 66, thereby releasing the locking of the base 3 and the annular member 1. In these figures, the base 3 has a through hole 66, and no through hole is formed in the region of the annular member 1 opposite to the through hole 66. Therefore, if the pressing pin 65 passes through the through hole 66, the annular member 1 can be effectively pressed by the pressing pin 65, releasing the locking of the base 3 and the annular member 1. In these figures, as an example, four through holes 66 are formed relative to the base 3. If the pressing pin 65 passes through each of these through holes 66, the annular member 1 can be pressed approximately evenly as a whole.

[0126] Figure 21 21010 is a top view of the support unit 60. Figure 2121011 is a cross-sectional view of the support unit 60 at position "AA", showing the changes in each component over time when the pressing pin 65 passes through the through hole 66. First, as... Figure 21 As shown on the left side of 21011, insert the pressing pin 65 into the through hole 66 in the direction of the arrow. Then, as... Figure 6 As shown in the center of 21011, if the pressing pin 65 passes through the through hole 66, the pressing pin 65 will press the annular member 1 downwards. Finally, as Figure 6 As shown on the right side of 21011, pressing the pin 65 further presses the annular member 1 downwards, thereby releasing the locking between the base 3 and the annular member 1, and separating the base 3 and the annular member 1. As a result, the mesh diaphragm 2 (specifically the periphery of the mesh diaphragm 2) held by the cylindrical mounting part 3b and the annular member 1 is removed.

[0127] 3. Example 2 of the configuration of the cell membrane fabrication apparatus according to this embodiment

[0128] Example 2 of the configuration of the cell membrane manufacturing apparatus according to this embodiment will be described. Figure 6 This is a diagram showing a simplified configuration of the support unit 30 included in the cell membrane fabrication apparatus of Example 2. Figure 6 6010 to 6013 are perspective views showing the assembly steps of the support unit 30. Figure 6 6020 is a cross-sectional view showing the simplified structure of the support unit 30.

[0129] like Figure 6 As shown in 6010-6013 and 6020, the support unit 30 includes a mesh diaphragm 2, a pair of frames 31 and 32, a clamp 33 (clamping member), a retaining member 34, and a spacer 35. The retaining member 34 constitutes the main body of the support unit 30. The mesh diaphragm 2, the pair of frames 31 and 32, and the clamp 33 constitute a mesh assembly A.

[0130] Frames 31 and 32 are components that reinforce the mesh diaphragm 2 by clamping it. Frames 31 and 32 are configured to clamp the periphery of the mesh diaphragm 2. Furthermore, frames 31 and 32 are detachably provided relative to the retaining member 34.

[0131] In addition, such as Figure 6 As shown in 6011, the clamp 33 integrates the mesh diaphragm 2 and the pair of frames 31 and 32 into a single component by clamping a pair of frames 31 and 32. Through the clamping of the clamp 33, the frames 31 and 32 are fixed in a tightly fitted state without separating from each other.

[0132] A mesh membrane 2 is disposed between a pair of frames 31 and 32, and frames 31 and 32 are fixed by a clamp 33, thereby constructing a mesh assembly A (see reference). Figure 7 (6010~6012).

[0133] The retaining member 34 has a bottomless cylindrical shape. The inner diameter of the retaining member 34 is larger than the outer diameter of the pair of frames 31 and 32. Two opposing portions of the inner wall of the retaining member 34 each have mounting portions 34a for mounting the mesh assembly A. The mounting portions 34a have flat plates 34b and 34c. The flat plates 34b and 34c are separate from each other and protrude horizontally inward from the inner wall of the retaining member 34. The peripheral portion of the mesh assembly A is inserted between the flat plates 34b and 34c, thereby mounting the mesh assembly A onto the retaining member 34. Additionally, a flange 34d protruding horizontally is formed at the upper end of the retaining member 34.

[0134] With the mesh assembly A mounted on the retaining member 34, there is a gap between the mesh assembly A and the flat plate portion 34b (see reference). Figure 7 (6013). The spacer 35 is a component 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 mounting portion 34a of the retaining member 34. Therefore, the mesh assembly A is stably held within the retaining member 34.

[0135] Furthermore, the spacer 35 is provided with an upwardly protruding and extending gripper 35a. To separate the mesh assembly A from the retaining member 34, the user should grasp the gripper 35a of the spacer 35 and remove the spacer 35 from the retaining member 34. Then, the mesh assembly A is separated from the retaining member 34.

[0136] Figure 7 This is a diagram showing the structure of the cell membrane manufacturing apparatus 100C, which is a constituent example 2. Figure 7 Figures 7010 to 7012 are diagrams illustrating a manufacturing method for producing cell membrane sheets using a cell membrane sheet manufacturing apparatus 100C. Furthermore, Figure 7 Figures 7020-7022, 7030, and 7031 are used to illustrate the effect of the cell membrane manufacturing apparatus 100C.

[0137] In the method for manufacturing cell membrane sheets using the cell membrane sheet manufacturing apparatus 100C, firstly, the support unit 30 is housed within the container 23. At this time, the support unit 30 is arranged such that the mesh assembly A is closest to the bottom surface of the container 23 (see reference). Figure 7 (of 7010).

[0138] Furthermore, the lower surface of the flange 34d of the retaining member 34 abuts against the entire upper end face of the container 23. Also, the outer diameter of the retaining member 34 is close to the inner diameter of the container 23. Furthermore, a gap is formed between the outer peripheral surface of the retaining member 34 and the inner surface of the container 23, ensuring that the retaining member 34 can be disassembled relative to the container 23. With this configuration, the support unit 30 is housed within the container 23 in a manner that fixes its position both vertically and horizontally within the culture medium.

[0139] Next, cells 4 were seeded onto the mesh patch 2 (refer to...). Figure 7 (7011). The seeding method for cells 4 is the same as that using the cell sheet manufacturing apparatus 100, so the description is omitted.

[0140] Then, the entire container 23 is filled with culture medium 6, and the cells 4 attached to the mesh membrane 2 are cultured. This produces the cell membrane 200.

[0141] Next, the effects of the cell membrane fabrication apparatus 100C will be explained. 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 flipped up and down and mounted onto the holding member 34. Therefore, as Figure 8 As shown in 7020 and 7021, different types of cells 4a and 4b can be seeded on the upper and lower surfaces of the mesh membrane 2, respectively. Furthermore, after forming the cell membrane 200, the cell membrane 200 can be easily separated from the aggregate A. Simply by removing the clamp 33, the frames 31 and 32 can be easily separated from each other. Therefore, if the frames 31 and 32 are separated, the cell membrane 200 can be easily removed. Additionally, as... Figure 8 As shown in 7030 and 7031, it is possible to observe both sides of the mesh membrane 2 using microscope 7.

[0142] Furthermore, in the cell sheet manufacturing apparatus 100C, the support unit 30 is held within the container 23 by the contact between the flange 34d of the retaining member 34 and the upper end face of the container 23. The flange 34d is the part that does not contact the culture medium 6. Additionally, the gripper 35a of the spacer 35 is also the part that does not contact the culture medium 6. Therefore, when moving the support unit 30 to another container using tweezers, the spacer 35 is moved upwards using the tweezers, allowing the support unit 30 to be moved without contacting the culture medium 6. Furthermore, when culturing cells 4 near the bottom of the container 23, the cells 4 will not contact the container 23 even if the support unit 30 is moved.

[0143] Furthermore, even when cells different from cells 4 are cultured on the bottom surface of container 23, these cells will not come into contact with the support unit 30. That is, in the cell sheet manufacturing apparatus 100C, when different cells are co-cultured on (i) the mesh sheet 2 of the support unit 30 and (ii) the bottom surface of container 23, the support unit 30 will not come into contact with the cells cultured on the bottom surface of container 23. Moreover, since the support unit 30 is a structure inserted from the upper side of container 23, the position of the support unit 30 relative to container 23 is stable.

[0144] 4. Example 3 of the configuration of the cell membrane fabrication apparatus according to this embodiment

[0145] Example 3 of the configuration of the cell membrane manufacturing apparatus according to this embodiment will be described. Figure 8 This is a diagram showing the configuration of the cell membrane fabrication apparatus 100D constituting Example 3. Figure 8 Images 8010 and 8020 are images illustrating the configuration of the support unit 40 included in the cell membrane fabrication apparatus 100D. Furthermore, Figure 8 Figures 8030 to 8033 are diagrams illustrating a manufacturing method for producing cell membrane sheets using a cell membrane sheet manufacturing apparatus 100D.

[0146] The support unit 40 is a columnar form that can be accessibly / detachably housed relative to the hole 24 (container). The support unit 40 includes a mesh membrane 2 and a column body 41. The lower surface of the column body 41 is formed by the mesh membrane 2. Furthermore, by placing the flange portion of the column body 41 onto the hole 24, the column body 41 is housed within the hole 24 in a manner that fixes its position both vertically and horizontally within the culture medium 6 (see reference). Figure 8 (8030). In addition, the support unit 40 can be made, for example, by removing the porous membrane from the column of a commercially available Transwell (registered trademark) and attaching the mesh membrane 2.

[0147] By using the cell sheet fabrication apparatus 100D, liquid-gas phase culture is easily performed on the fabricated cell sheets. For example, in the fabrication of skin sheets, firstly... Figure 8 As shown in 8031, skin fibroblasts 14 were seeded on the mesh sheet 2 and cultured in culture medium 6. Then, as... Figure 9 As shown in 8032, keratinocytes 13 are then seeded onto a layer composed of growing and developing skin fibroblasts 14 and cultured in culture medium 6 to produce a skin sheet. Figure 9 As shown in 8033, the prepared skin sheet can ensure that skin fibroblasts 14 are in contact with the culture medium and a portion of the keratinocytes 13 are in contact with the gas, that is, liquid-gas phase culture can be performed.

[0148] 5. Example 4 of the configuration of the cell membrane sheet manufacturing apparatus according to this embodiment.

[0149] Example 4 describes the configuration of the cell membrane manufacturing apparatus according to this embodiment. Figure 9 This is a diagram showing the configuration of the cell membrane fabrication apparatus 100E constituting Example 3. Figure 9 9010 is a perspective view showing the configuration of the support unit 50 included in the cell membrane fabrication apparatus 100E. Furthermore, Figure 9 Image 9011 is an image showing the configuration of cell membrane manufacturing apparatus 100E.

[0150] like Figure 10 As shown in 9011, in the cell membrane fabrication apparatus 100E, a well plate 26 with multiple pores 25a is used as a container. Figure 10 In the configuration shown in 9011, the well plate 26 is a 12-well culture plate. The support unit 50 is housed in contact with / separable from such well plate 26. The support unit 50 includes a mesh membrane 2 and a column body 51.

[0151] The column body 51 has a first annular portion 51a, a support portion 51b, and a second annular portion 51c. The support portion 51b is the part 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 with their central axes aligned.

[0152] The outer diameter of the first annular portion 51a is larger than the inner diameter of the hole 25a in the perforated plate 25. Furthermore, 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 sealed by the mesh membrane 2.

[0153] In the cell membrane manufacturing apparatus 100E, the column body 51 is housed in the orifice 25a by placing the first annular portion 51a on the orifice 25a, thereby fixing the column body 51 in both its vertical and horizontal positions within the culture medium 6. Furthermore, a cell suspension is injected into the space formed by the inner surface of the second annular portion 51c, the inner surface of the support portion 51b, and the mesh membrane 2.

[0154] Furthermore, in the cell sheet manufacturing apparatus 100E, the support unit 50 is held within the hole 25a by placing the first annular portion 51a of the column body 51 onto the hole 25a. The first annular portion 51a is the part that does not contact the culture medium 6. Therefore, when moving the support unit 50 to other holes 25a using tweezers, the first annular portion 51a is held by the tweezers, allowing the support unit 50 to be moved without contacting the culture medium 6. Furthermore, when culturing cells near the bottom of the hole 25a, the cells 4 will not contact the hole 25a even when the support unit 50 is moved. Additionally, in the cell sheet manufacturing apparatus 100E, when different cells are co-cultured on (i) the mesh sheet 2 of the support unit 50 and (ii) the bottom surface of the hole 25a, the support unit 50 will not contact the cells cultured on the bottom surface of the hole 25a.

[0155] Furthermore, in the cell membrane manufacturing apparatus 100E, the position of the mesh membrane 2 is stably maintained by the first annular portion 51a and the support portion 51b. Therefore, according to the cell membrane manufacturing apparatus 100E, the position of the support unit 50 within the aperture 25a is more stably maintained in both the vertical and horizontal directions.

[0156] 〔Summarize〕

[0157] To address the aforementioned issues, one aspect of the present invention relates to a cell membrane manufacturing apparatus characterized by comprising: a container holding a culture medium for culturing cells; and a support unit detachably housed within the container, having a substrate for attaching and culturing the cells, namely a mesh membrane, and a retaining member for holding the mesh membrane to float from the bottom of the container; the support unit is housed within the container in a manner that fixes its position in both the vertical and horizontal directions within the culture medium.

[0158] According to the above configuration, the support unit can be detachably stored in the container. Therefore, the user can easily remove the support unit from the container, flip it over, and then store it back in the container. When the support unit is flipped, the cell membrane sheets formed on the mesh membrane held by the holding member of the support unit also flip over. Therefore, according to the above configuration, both sides of the cell membrane sheets formed on the mesh membrane can be easily observed under a microscope.

[0159] Furthermore, the support unit is housed in the container in a manner that fixes its position both vertically and horizontally within the culture medium. Therefore, during microscopic observation of cell membrane sheets, the position of the mesh membrane sheets within the container—in other words, the position of the cell membrane sheets formed on the mesh membrane sheets—remains unchanged. Thus, based on this configuration, precise observation of cell membrane sheets is possible.

[0160] In a cell membrane manufacturing apparatus according to one aspect of the present invention, the retaining member preferably retains the mesh membrane detachably.

[0161] Based on the above configuration, the cell membrane sheets formed on the mesh membrane can be easily separated from the support unit.

[0162] In a cell membrane manufacturing apparatus according to one aspect of the present invention, preferably: a cylindrical mounting portion for mounting the mesh membrane is formed on the holding member, the support unit has an annular member having a shape surrounding the outer periphery of the cylindrical mounting portion and is detachably provided relative to the holding member, and the periphery of the mesh membrane is held by the cylindrical mounting portion and the annular member.

[0163] According to the above configuration, the annular member is mounted on the retaining member, and the periphery of the mesh diaphragm is clamped by the cylindrical mounting portion and the annular member, thereby stably mounting the mesh diaphragm on the cylindrical mounting portion. On the other hand, by removing the annular member from the retaining member, the periphery of the mesh diaphragm clamped by the cylindrical mounting portion and the annular member is released, thereby easily removing the mesh diaphragm mounted on the cylindrical mounting portion.

[0164] In the cell membrane manufacturing apparatus according to one aspect of the present invention, it is preferable that: at least one of the holding member and the annular member is provided with a locking portion that locks the holding member and the annular member in a non-separable manner, and a through hole is provided in either the holding member and the annular member for a pressing pin to pass through, thereby releasing the locking by pressing the other member and separating the holding member and the annular member.

[0165] According to the above configuration, by inserting the pressing pin into the through hole and pressing the retaining member or the annular member with the pressing pin, the retaining member and the annular member can be released from their locking, thereby making it easy to disassemble the mesh diaphragm placed on the cylindrical mounting portion.

[0166] In a cell membrane manufacturing apparatus according to one aspect of the present invention, preferably: the support unit includes a pair of frames and a clamping member, the pair of frames being detachably disposed relative to the holding member and configured to clamp the periphery of the mesh membrane; the clamping member integrates the mesh membrane and the pair of frames by clamping the pair of frames.

[0167] According to the above configuration, since the pair of frames configured to clamp the periphery of the mesh diaphragm are detachably provided relative to the retaining member, the mesh diaphragm held by the pair of frames can be easily disassembled by separating the pair of frames from the retaining member.

[0168] In the cell membrane manufacturing apparatus according to one aspect of the present invention, it is preferable that the specific gravity of the holding member is greater than that of the culture medium.

[0169] Based on the above configuration, since the specific gravity of the retaining component is greater than that of the culture medium, the positions of the retaining component in the culture medium and the cell membrane sheets formed on the mesh membrane sheets held by the retaining component are stably maintained in the vertical and horizontal directions.

[0170] In the cell membrane manufacturing apparatus according to one aspect of the present invention, preferably: the support unit includes a cover that covers one side of the mesh membrane and is provided with an injection port for injecting a suspension containing the cells between the mesh membrane and the cover, and the distance between the cover and the mesh membrane is set so that the suspension is in contact with both the mesh membrane and the cover while being injected between the mesh membrane and the cover.

[0171] Based on the above configuration, the gap between the cap and the mesh membrane is set so that the suspension is injected between the mesh membrane and the cap while in contact with both sides. In this case, the cell-containing suspension can be prevented from moving under the mesh membrane, and the suspension can be spread out laterally. Therefore, even if the area of ​​the mesh membrane is increased, the cell-containing suspension can still be spread evenly on the mesh membrane, and cell culture can be easily performed using a large-area mesh membrane.

[0172] In the cell membrane manufacturing apparatus according to one aspect of the present invention, it is preferable that the opening of the mesh membrane is elongated in one direction.

[0173] As described above, when the opening of the mesh membrane is elongated in one direction, the opening has a large wall surface that elongates in that direction. A large number of cells adhere to and proliferate on this wall surface, and their proliferation exhibits the same directionality. Therefore, according to the above configuration, the directionality of the proliferating cells can be controlled to the elongation direction of the opening.

[0174] The cell membrane sheet involved in one aspect of the present invention is characterized in that it has at least two cell layers, and a substrate, namely a mesh membrane, for attaching and culturing cells is disposed between the two cell layers.

[0175] Based on the above configuration, the thickness of the cell membrane sheet can be sufficiently ensured and the strength of the cell membrane sheet can be improved. Furthermore, based on the above configuration, a cell membrane sheet having multiple cell layers can be provided.

[0176] Example

[0177] [Example 1: Cell culture using the cell sheet manufacturing apparatus of Example 1]

[0178] <Fabrication of Support Unit 10>

[0179] A design drawing for the base 3, which is housed in a 12-well culture plate, was created. Based on this design, the base 3 was fabricated using an AGILISTA-3200 (Keyence) 3D printer. The 3D-printed model (base 3) was covered with biocompatible Parylene (DPXC, CAS No. 28804-46-8) (Parylene, Japan) using a PDS-2010 laboratory coater. The mesh membrane 2 was made of polyester micromesh membrane (AG00Z3N9) (Tianchi Synthetic Fiber).

[0180] Furthermore, the annular component 1 was fabricated using a 2mm plate made of PDMS (polydimethylsiloxane) (trade name: SILPOT 184, Toray Dow Corning). The fabrication method for the 2mm PDMS plate is as follows: First, the base agent and curing agent were mixed at a ratio of 10:1. The mixed PDMS solution was poured into a petri dish to ensure a thickness of 2mm, and then heated to cure. For the completed 2mm thick PDMS plate, a ring (8mm outer diameter, 6mm inner diameter) was fabricated using two biopsy puncture needles (Kai Kogyo) with diameters of 8mm and 6mm.

[0181] The annular component 1, mesh membrane 2, and base 3, prepared as described above, were cleaned and air-dried with 70% ethanol, 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 into each well of a 12-well culture plate.

[0182] Cell Culture Conditions

[0183] Tig-1-20 cells (product number JCRB0501) derived from normal human lung fibroblasts were obtained from the JCRB Cell Bank (Osaka). Tig-1-20 cells were cultured in DMEM (Dulbecco Modified Eagle Medium) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). The cells were cultured at 37°C in a 5% carbon dioxide (CO2) incubator.

[0184] HepG2 cells (product number RCB1886) were obtained from the RIKEN Bioresource Center (RIKEN BRC). HepG2 cells were cultured in DMEM (Dulbecco modified Eagle medium) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). The HepG2 cells (product number RCB1886) were cultured in an incubator at RIKEN Bioresource Center (37°C, 5% CO2) for further processing.

[0185] Mesenchymal stem cells (MSCs) from human adipose tissue were obtained from Promo Cell. The MSCs were cultured using mesenchymal stem cell proliferation medium 2 (Promo Cell). The MSCs were cultured in an incubator at 37°C and 5% carbon dioxide (CO2).

[0186] <Cell Seeding and Microscopic Observation>

[0187] 0.5 × 10⁻⁶ seeds were seeded on the mesh membrane 2 (circular region with a diameter of 4 mm) of the support unit 10. 5 (in 1×10) 6 Seeds were inoculated with 50 μL of TiG-1-20 cells, HepG2 cells, or mesenchymal stem cells. Five hours after inoculation, 3 mL of culture medium was added to each well containing 10 support units. The culture medium was changed every 3 days (3 mL / well). Cell images were taken using a digital microscope or an inverted phase-contrast microscope.

[0188] The membranes of Tig-1-20 cells, HepG2 cells, and mesenchymal stem cells cultured for 16 days were analyzed using the Cell3imager Estier (SCREEN Holdings) optical coherence tomography system.

[0189] For Tig-1-20 cells, mesh sheet 2 was covered with collagen solution before cell seeding. More specifically, the acidic collagen solution I-AC 5 mg / mL (Gaoyan) was diluted 15-fold with 1 mM HCl solution, and 50 μL of the diluted collagen solution was placed on mesh sheet 2. The membrane was incubated at 37°C for 30 minutes, and then the diluted collagen solution was removed. The cells were then washed twice with PBS(-) and stored in a 37°C incubator before cell seeding.

[0190] <Results>

[0191] Figure 11 Image 1010 shows a microscope image of support unit 10 one day after cell seeding. 50 μL of cell suspension was firmly held on the mesh membrane 2 (left image). Culture medium was added to the wells, ensuring no air bubbles formed in the gaps (center image). Furthermore, support unit 10 could be flipped upside down (right image).

[0192] It was confirmed that Tig-1-20 cells, HepG2 cells, and mesenchymal stem cells could be cultured on the mesh patch 2. Figure 11 (1011). Tig-1-20 cells showed weak adhesion to mesh patch 2, so mesh patch 2 was covered with a coating solution. By coating with collagen solution, it was confirmed that the cell membrane did not detach from the mesh patch even after adding culture medium 5 hours later.

[0193] In addition, the results of analysis using an optical coherence tomography system on Tig-1-20 cell sheets, HepG2 cell sheets, and mesenchymal stem cell sheets cultured for 16 days are shown below. Figure 11 .like Figure 12 As shown, the surfaces of the Tig-1-20 cell sheet and the mesenchymal stem cell sheet are flat, while the HepG2 cell sheet is not. Analysis using a unilateral optical coherence tomography (OCT) system reveals that both the HepG2 cell sheet and the mesenchymal stem cell sheet are flat near the plane of the reticular membrane 2. Because the Tig-1-20 cell sheet is thinner than the other cell sheets, its surface can still be analyzed through unilateral observation. Figure 12 [Example 2: Fabrication of a cell sheet composed of three types of cells using the cell sheet fabrication apparatus of Example 1] <Cell Culture Conditions>

[0194] The cell culture conditions were the same as in Example 1.

[0195] <Seed cells on the mesh membrane 2 of the support unit 10>

[0196] Collagen was applied to the support unit 10 (for 12-well culture plates) of the mesh membrane 2. Then, 4 × 10⁴ Tig-1-20 cells stained with the CellBrite Green Cytoplasmic Membrane-Labeling Kit (80-fold dilution; Biotium, Inc.) were seeded onto the mesh membrane 2. 5 (in 1×10) 7Cells / mL were seeded at 40 μL. Five hours later, 3 mL of DMEM medium containing FBS was added to the wells. Two days later, 4 × 102 mesenchymal stem cells (MSCs) stained with the CellBrite Red Cytoplasmic Membrane-Labeling Kit (diluted 120-fold; Biotium, Inc.) were seeded onto Tig-1-20 cells. 5 (at 1.6 × 10) 7 Seeds were generated using 25 μL of the medium per 100 cells / mL. The medium was then replaced with mesenchymal stem cell medium. The next day, support unit 10 was inverted (set to reverse) and 4 × 10⁴ HepG2 cells stained with 0.025 mg / mL Dil (1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were seeded onto Tig-1-20 cells. 5 (at 1.6 × 10) 7 Cells were seeded at 25 μL per 100 mL. The next day, cells were washed with PBS (-) and then immersed in 4% PFA, incubated at room temperature for 50 minutes. After washing with PBS (-), cell sheets were fixed between a coverslip and a slide using VECTASHIELD (VECTOR) as a mounting medium. Specifically, to prevent the coverslip from crushing the cell sheets, three layers of approximately 100 μm thick Kapton tape were placed between the coverslip and the slide. The fixed cell sheets were analyzed using a confocal microscope LSM 800 (ZEISS).

[0197] <Results>

[0198] Figure 11 1210 to 1212 represent the results of this embodiment. For example... Figure 13 As shown in 1210-1212, mesenchymal stem cell sheets were fabricated on top of the Tig-1-20 cell sheet. Furthermore, HepG2 cell sheets were fabricated below the Tig-1-20 cell sheet. The overall thickness of the cell sheets was approximately 140-150 μm. The results indicate that (i) cells can be seeded from both the upper and lower sides of the support unit 10, and (ii) cell sheets composed of various cell types can be fabricated.

[0199] [Example 3: Culture of human skin fibroblasts using the cell sheet manufacturing apparatus of Example 2]

[0200] <Fabrication of Support Unit 30>

[0201] All components in the support unit 30, except for the mesh membrane 2, were fabricated using a 3D printer, AGILISTA-3200. These components were covered with Perylene (DPXC, CAS No. 28804-46-8). The mesh membrane 2 was made of polyester micro-mesh membrane (AG00Z3N9) (Tianchi synthetic fiber). As a control group, a support unit 30 was also fabricated by replacing the mesh membrane 2 with a porous membrane (Transwell polycarbonate membrane, 0.4 μm pore size, Corning Incorporated) used by Transwell (registered trademark) and installing it.

[0202] Cell Culture Conditions

[0203] Normal human skin fibroblasts (NHDF) were purchased from Promo Cell and cultured using fibroblast proliferation medium 2 (Promo Cell). The NHDF cells were cultured in an incubator at 37°C and 5% carbon dioxide (CO2).

[0204] <Cell Seeding and Microscopic Observation>

[0205] 1.65 × 10⁻⁶ seeds were inoculated onto the mesh membrane 2 of the support unit 30. 6 NHDF (550 μL / sheet, 3 × 0.6 cells / mL). Add culture medium approximately 18 hours later. Change the medium every 3 days and culture for 14 days (6 mL / well). Take inverted phase-contrast microscope images of the cells. Figure 13 Additionally, HE-stained sections were prepared (New Histology), and cross-sectional images of cell membrane slides were taken using a BZ-X710 All-in-One (Keyence) camera. Figure 14 (of 1311).

[0206] <Results>

[0207] The results are as follows Figure 14 As shown in 1310 and 1311, NHDF exists in the form of blocking the mesh of the mesh sheet 2 and covering the grid lines of the mesh sheet 2. Regarding both the cell sheet made of the mesh sheet 2 and the cell sheet made of the porous membrane, the surface of the cell sheet is uniform and smooth. Unlike the cell sheet made of the porous membrane, the cell sheet made of the mesh sheet 2 has its lower surface with the cells exposed. Therefore, it facilitates nutrient supply and cell observation, and also facilitates adhesion of the cell sheet during medical transplantation.

[0208] [Example 4: Culturing Tig-1-20 cells using the cell sheet manufacturing apparatus of Example 2]

[0209] <Fabrication of the support unit 30>

[0210] The method for fabricating the support unit 30 in this embodiment is the same as that in Embodiment 3. However, the structure of the support unit 30 in this embodiment is partially different from that in Embodiment 3 only in the holding member 34. In this embodiment, the holding member 34 has no voids for facilitating the inflow of the culture medium from the outside to the inside (refer to Figure 14 1410).

[0211] In addition, the culture conditions (composition of the culture medium) for Tig-1-20 cells are the same as those in Embodiment 1. However, in this embodiment, the reticular membrane 2 is not coated.

[0212] <Inoculation of Tig-1-20 cells and microscopic observation>

[0213] 4×10 6 Tig-1-20 cells stained or unstained with CellBrite Green Cytoplasmic Membrane-Labeling Kit (diluted 100-fold; Biotium, Inc.) (500 μL / mesh used at 8×10 6 cells / mL) were inoculated onto the reticular membrane 2 of the support unit 30 fabricated according to <Fabrication of the support unit 30>. Then, 6 mL of the culture medium was added to the wells after culturing for17 hours. The culture medium was changed once after 3 days. On the 6th day after the start of culturing, the fabricated cell membrane sheet was washed, the cells were fixed with 4% paraformaldehyde (PFA), and after washing, it was fixed in a mounting medium VECTA containing DAPI (4',6-diamidino-2-phenylindole). The fixed cell membrane sheet was analyzed using a confocal microscope LSM 800 (ZEISS). On the other hand, HE-stained sections (New Tissue Science) of cell membrane sheets fixed with 4% PFA and 1% formaldehyde and not fluorescently stained were prepared, and cross-sectional views were taken with BZ-X710 All-in-one (Keyence).

[0214] <Inoculation of cardiomyocytes derived from human iPS cells and microscopic observation>

[0215] In the above experiment, to supplement the Tig-1-20 cell sheets (cultured for 6 days) that had not been used for analysis with cardiomyocytes derived from human iPS cells, the culture medium in the wells was replaced with 4 mL of MiraCell CM Culture Medium. 1.8 × 10⁻⁶ cells stained with 0.025 mg / mL DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were then seeded. 5 Cardiomyocytes derived from human iPS cells were cultured. Approximately 16 hours later, 2 mL of culture medium was added. Subsequently, the cells were co-cultured with Tig-1-20 cells for 3 days, fixed in the same manner as above, and analyzed using a confocal microscope.

[0216] <Results>

[0217] The results are as follows Figure 14 As shown in 1420-1423. Figure 14 As shown in Figure 1420, Tig-1-20 cells are oriented according to the mesh shape of the micromembrane sheet. That is, they are oriented approximately parallel to the long sides of the rectangular mesh. Cells located outside the plane of the micromembrane sheet, i.e., cells located above or below the plane of the micromembrane sheet, are also oriented in the same direction as cells located within the plane of the micromembrane sheet. The thickness of the cell membrane sheet is approximately 50 μm. Figure 15 (1421). Furthermore, when cardiomyocytes derived from human iPS cells were seeded onto the Tig-1-20 cell sheet, the cardiomyocytes also oriented in the same direction as the Tig-1-20 cells. These results demonstrate that the orientation of three-dimensional cell sheets can be controlled by culturing cells using micromesh sheets. Figure 15 (1422 and 1423).

[0218] [Example 5: Culturing Tig-1-20 cells using the cell sheet manufacturing apparatus of Improved Example 2]

[0219] <Method>

[0220] <Fabrication of Support Unit 10B and Cell Culture Conditions>

[0221] The annular member 1 and the base 3 are fabricated using a 3D printer AGILISTA-3200. Subsequently, the fabricated annular member 1 and base 3 are coated with parylene (DPXC, CAS No. 28804-46-8). The mesh diaphragm 2 uses a polyester micro mesh diaphragm (AG00Z3N9) (Tianchi Synthetic Fiber). The mesh diaphragm 2 is sandwiched between the annular member 1 and the base 3 and adhered using a PDMS solution. After subjecting the completed support unit 10B to ethanol treatment and UV treatment, the support unit 10B is placed in a 10-cm petri dish 22. In the support unit 10B used in the experiment, a 1-mm PDMS plate is used to adjust the distance from the bottom surface of the petri dish 22 to the mesh diaphragm 2 to be 2.5 mm ( Figure 15 of 1510).

[0222] The culture conditions (composition of the culture medium) of Tig-1-20 cells are the same as those in Example 1, but the mesh diaphragm 2 is not coated.

[0223] <Tig-1-20 Cell Inoculation and Microscopic Observation>

[0224] First, 3 mL of Tig-1-20 cells (6×10 5 cells / mL) are placed on top of the mesh diaphragm 2. Then, a cover 9 is placed thereon. Subsequently, 5 mL of cell suspension is added through the injection port 9i of the cover 9 to fill the gap between the cover 9 and the mesh diaphragm 2 with the cell suspension. Three days after cell inoculation, 25 mL of culture medium is added to the 10-cm petri dish 22 containing the support unit 10B, and then the cover 9 is removed. The culture medium is changed once every three days (35 mL of culture medium / dish), and the culture is terminated 14 days after cell inoculation.

[0225] As Figure 16 shown in 1511, a cell membrane sheet is cut from the support unit 10B using a cutter. After washing a part of the cut cell membrane sheet with phosphate buffered saline (PBS)(-), it is treated with 4% PFA for 20 minutes and stained with DiI (37 °C, 1 hour). After washing with PBS(-), the cell membrane sheet is fixed between a cover glass and a slide glass together with a mounting medium VECTASHIELD (VECTOR) containing DAPI. The fixed cell membrane sheet is analyzed using a confocal microscope LSM 800 (ZEISS).

[0226] After washing a portion of the cell sheet fabricated from support unit 10B with PBS(-), it was treated with 4% PFA for 20 minutes and fixed. Next, it was treated with 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and approximately 100% ethanol for 5 minutes each, and then air-dried. The cell sheet was then observed using a low-vacuum scanning electron microscope, Miniscope™ 3030Plus (Hitachi).

[0227] The viability of cells forming cell sheets cultured in support unit 10B was investigated. Approximately half of the cell sheet cut from support unit 10B was washed with PBS(-), treated with 0.05% trypsin-EDTA for 7 minutes (37°C), and after removing the trypsin, it was resuspended in 10 mL of culture medium. Cell viability was measured using a Countess II Automated Cell Counter (Thermo Fisher Scientific).

[0228] <Results>

[0229] The results are as follows Figure 17 1512, Figure 15 1611 and 1612, and Figure 16 As shown in 1711 and 1712. Tig-1-20 cells were cultured for 14 days using support unit 10B. Figure 17 (1512). Analysis of the cell membrane section using confocal microscopy showed a thickness of approximately 40-45 μm. Figure 17 (1611 and 1612). Tig-1-20 cells are oriented according to the mesh shape of the reticular membrane 2. That is, they are oriented approximately parallel to the long side of the rectangular mesh. Cells not present in the plane of the reticular membrane 2 are also oriented in the same direction.

[0230] To allow observation of cell membrane sheets using an electron microscope, the cell membrane sheets were treated with ethanol. This treatment may have caused the cell membrane sheets to shrink and thin, but it confirmed that the cells completely blocked the opening of the reticular membrane sheet 2. Figure 18 (1711). Using trypsin-EDAT solution and trypan blue solution, the cell viability of cells constituting a three-dimensional cell sheet made of support unit 10B was investigated, and it was found that approximately 92% of the cells survived. Figure 18 (1712). That is, even in three-dimensional culture, a large number of cells survive.

[0231] [Example 6: Culturing HepG2 cells using the cell sheet manufacturing apparatus of Example 4]

[0232] <Method>

[0233] <Fabrication of Support Unit 50>

[0234] The column body 51 of the support unit 50 was fabricated using a 3D printer, AGILISTA-3200. The fabricated column body 51 was then covered with Perylene (DPXC, CAS No. 28804-46-8). The mesh membrane 2 was made of polyester micro-mesh membrane (AG00Z3N9) (Tianchi Synthetic Fiber). The mesh membrane 2 was adhered to the column body 51 using PDMS solution. After the completed support unit 50 was treated with ethanol and air-dried, it was placed in the wells 25a of a 12-well culture plate for UV treatment.

[0235] Cell Culture Conditions

[0236] HepG2 cells (product number RCB1886) were obtained from the RIKEN Bioresource Center (RIKEN BRC). HepG2 cells were cultured in DMEM (Dulbecco Modified Eagle Medium) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin (GIBCO). The cells were cultured in an incubator at 37°C and 5% carbon dioxide (CO2).

[0237] <Cell Seeding and Cell Observation>

[0238] 1×10 5 (2X×10) 6 HepG2 cells (cells / mL × 50 μL / mesh) were seeded onto the mesh membrane 2 of support unit 50. (Circular shape). Six hours after cell seeding, 2 mL of culture medium was added to well 25a. The culture medium was changed every 2 days (2 mL / well). As a control, 1×10⁻⁶ cells were added to well 25a. 5 One HepG2 cell was seeded into well 25a of a 25-well plate (12-well culture plate).

[0239] <Gene Expression Analysis>

[0240] RNA was extracted from HepG2 cells on day 10 of culture. RNA extraction was performed using TRIzol RNAIsolation Reagents (Thermo Fisher Scientific), and cDNA was synthesized using the extracted RNA and the ReverTraAce qPCR RT Kit (Toyobo). Gene expression analysis was performed using the obtained cDNA samples, DNA primers, PowerUp SYBRGreen Master Mix (applied biosystems), and the QuantStudio 5 Real-Time PCR System (applied biosystems). The gene expression levels of albumin and the drug-metabolizing enzyme CYP1A2, well-known markers of human liver maturation, were analyzed. The expression levels of each gene were normalized using the expression levels of 18S rRNA, a well-known housekeeping gene. Values ​​are expressed as mean ± SE (N=3).

[0241] <Results>

[0242] Figure 19 Image 1810 represents a microscopic image of HepG2 cells. HepG2 cells adhered to the mesh membrane 2 within 6 hours. ​ 1811 indicates the results of gene expression analysis. Compared with culturing HepG2 cells in conventional wells (2D), the gene expression levels of albumin and CYP1A2 were higher when HepG2 cells were cultured on the mesh sheet 2 of the support unit 50.

[0243] [Example 7: Culture of cardiomyocytes derived from human iPS cells using the cell sheet manufacturing apparatus of Example 4] <Method>

[0244] Cell Culture Conditions

[0245] Cardiomyocytes derived from human iPS cells (MiraCell Cardiomyocytes from ChiPSC12, TaKaRa) were cultured using MiraCell CM Culture Medium at 37°C and in the presence of 5% CO2, according to the product instructions. The cells were cultured on culture dishes coated with human fibronectin.

[0246] <Cell Seeding and Cell Observation>

[0247] Before cell seeding, the mesh sheet 2 of support unit 50 was coated with human fibronectin. The fibronectin solution diluted 20-fold (final concentration 0.05 mg / mL) was adjusted with PBS(+). 50 μL of the diluted fibronectin solution was placed on the mesh sheet 2 and incubated at 37°C for at least 1 hour.

[0248] Remove the fibronectin solution and add 2×10 4 (4×10) 5 Cardiac cells (cells / mL × 50 μL / mesh) were seeded onto the mesh sheet 2 of the support unit 50. (Circular shape). After 6 hours, 2 mL of culture medium was added to well 25a to culture cardiomyocytes. The culture medium was changed every 2 days (2 mL / well).

[0249] <Results>

[0250] The results are as follows ​ As shown, cardiomyocytes adhered to and proliferated on the mesh sheet 2 of the support unit 50. The cardiomyocytes retained their beating function even after 8 days of culture.

[0251] Industrial utilization potential

[0252] This invention can be used to manufacture cell membrane sheets for use in fields such as regenerative medicine and transplantation.

[0253] Symbol Explanation

[0254] 1. Ring-shaped component

[0255] 2. Mesh membrane 3. Base (Retaining Member)

[0256] 3b Cylindrical mounting section

[0257] Cells 4, 4a, and 4b

[0258] 9. Cover

[0259] 9a Injection Entry

[0260] Support units 10, 10A, 10B, 30, 40, 50, 60

[0261] Containers 20 and 23 Holes 21, 24, and 25a (containers)

[0262] 22. Petri dish (container)

[0263] 25- and 26-well plates (containers)

[0264] Frames 31 and 32 33. Fixtures (clamping components) Columns 41 and 51 (main structure, retaining members) 65 Pressing Pin

[0265] 66 Through holes

[0266] 67. Locking part

[0267] 100, 100A, 100B, 100C, 100D, 100E Cell membrane fabrication apparatus 200 cell membrane sheets

Claims

1. A cell sheet manufacturing apparatus characterized by comprising: The cell sheet manufacturing apparatus includes: a container that houses a culture medium for culturing cells; and a support unit that is contactably / separably housed in the container, has a substrate, i.e., a mesh sheet, on which the cells are attached and cultured, and a holding member that holds the mesh sheet so as to float from a bottom surface of the container; the support unit is housed in the container in a manner that its position in the vertical direction and the horizontal direction in the culture medium is fixed, the holding member detachably holds the mesh sheet, the holding member has a cylindrical placement portion on which the mesh sheet is placed, the support unit has a ring-shaped member that has a shape surrounding an outer periphery of the cylindrical placement portion, is detachably provided with respect to the holding member, a periphery of the mesh sheet is sandwiched by the cylindrical placement portion and the ring-shaped member; and at least one of the holding member and the ring-shaped member includes a locking portion that locks the holding member and the ring-shaped member so that the holding member and the ring-shaped member are not separated from each other. at least one of the holding member and the ring-shaped member includes a through hole through which a pressing pin of the other of the holding member and the ring-shaped member is pushed, and pressing the other of the holding member and the ring-shaped member releases the locking of the holding member and the ring-shaped member so that the holding member and the ring-shaped member are separated from each other.

2. The cell sheet manufacturing device according to claim 1, wherein the ring member has a surface which is substantially perpendicular to an axis of the ring member and which is capable of contacting the mesh cell sheet. the holding member has a surface that is substantially perpendicular to the cylindrical placement portion and is in contact with the surface; the through hole extends toward a surface of the ring-shaped member or a surface of the holding member.

3. The cell sheet manufacturing apparatus according to claim 2, comprising a protrusion formed on a surface of the holding member or the ring-shaped member, and a locking protrusion that locks the protrusion on the surface of the holding member or the ring-shaped member from moving in a pressing direction.

4. The cell sheet manufacturing apparatus according to any one of claims 1 to 3, wherein a specific gravity of the holding member is greater than that of the culture medium.

5. The cell sheet manufacturing apparatus according to any one of claims 1 to 3, wherein the support unit has a lid that covers one surface of the mesh sheet and is provided with an injection port through which a suspension containing the cells is injected 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 the suspension is in contact with both the mesh sheet and the lid.

6. The cell sheet manufacturing apparatus according to any one of claims 1 to 3, wherein an opening portion of the mesh sheet has an elongated shape in one direction.

7. The cell sheet manufacturing apparatus according to claim 6, wherein the shape is a rectangular shape, and a ratio of a short side to a long side of the elongated shape in one direction is 1:2 to 1:10.

Images