Method for producing cell structures, culture fixtures, and culture substrates

Abstract

To provide a method for producing a cell structure.SOLUTION: Provided is a method for producing a structure, comprising: seeding a plurality of cells inside a storage space formed by a bottom surface, and a wall surface at least a portion of which has cell adhesive properties; and culturing the plurality of cells to form a structure.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing cell structures, a culture jig, and a culture substrate. [Background technology]

[0002] In recent years, several three-dimensional culture methods have been proposed because two-dimensional culture methods, which involve culturing cells in a single layer on a plastic culture dish, do not reflect cellular metabolism within living tissues. Furthermore, in regenerative medicine, attempts have been made to embed cells in scaffolds such as MATRIGEL (registered trademark) for three-dimensional culture and transplant them into the body. However, since MATRIGEL is extracted from EHS (Engelbreth-Holm-Swarm) mouse sarcoma, challenges remain, such as the risk of inflammatory reactions and infection due to foreign body contamination, and the lack of sufficient therapeutic effect.

[0003] Therefore, efforts are underway to develop transplant tissues derived from pure cells without using scaffolds. Simple methods include culturing cells into spheroids, and further methods such as the "pin-printing" method (Non-Patent Literature 1), which involves stacking spheroids using a three-dimensional bioprinter, and the "net mold" method (Non-Patent Literature 2), which involves three-dimensional culture using a special net. In addition, a method has been devised in which cells are cultured in a single layer in a temperature-responsive culture dish to create a sheet-like structure, which is then stacked to create a three-dimensional structure (Non-Patent Literature 3).

[0004] The challenges of the spheroid piercing method include the need to insert spheroids into needles, the resulting temporary punctures after removal of the piercings, the slight cytotoxicity observed, the difficulty in creating thin structures such as sheets due to the need to use spheroids that are sufficiently thicker than the needles, the requirement for specialized equipment (a very expensive 3D bioprinter), the time required for layering, and the fact that approximately 50% shrinkage occurs during the maturation process, making it impossible to maintain the shape (Non-Patent Literature 1).

[0005] The challenges of the net molding method include the fact that while it is possible to create block-shaped cell structures, it is difficult to create thin sheet-shaped structures; it requires specialized equipment (special net kits); it can only create simple shapes such as predetermined rectangles; and it is not possible to observe the culture process under a microscope (Non-Patent Literature 2).

[0006] The challenges of temperature-responsive culture dishes include the fact that they basically involve a monolayer culture process, making them difficult to apply to cells and stem cells whose phenotypes change in monolayer culture; the fact that creating thick structures requires stacking multiple sheets, which is cumbersome; and the fact that shrinkage occurs after stacking, making it impossible to maintain the shape (Non-Patent Literature 3). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Zhang XY, Yanagi Y, Sheng Z, Nagata K, Nakayama K, Taguchi T. Regeneration of diaphragm with bio-3D cellular patch. Biomaterials. 2018 Jun;167:1-14. [Non-Patent Document 2] Yang B, Lui C, Yeung E, Matsushita H, Jeyaram A, Pitaktong I, Inoue T, Mohamed Z, Ong CS, DiSilvestre D, Jay SM, Tung L, Tomaselli G, Ma C, Hibino N. A Net Mold-Based Method of Biomaterial-Free Three-Dimensional Cardiac Tissue Creation. Tissue Eng Part C Methods. 2019 Apr;25(4):243-252. [Non-Patent Document 3] Mitani G, Sato M, Lee JI, Kaneshiro N, Ishihara M, Ota N, Kokubo M, Sakai H, Kikuchi T, Mochida J. The properties of bioengineered chondrocyte sheets for cartilage regeneration. BMC Biotechnol. 2009 Mar 6;9:17. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] There is a need for a simple method to manufacture cell structures without introducing foreign substances. [Means for solving the problem]

[0009] The inventors of the present invention have diligently conducted research to solve the above problems and, surprisingly, have discovered a method for producing a cell structure, as well as a culture jig and a culture substrate, which include the steps of seeding multiple cells inside a containment space formed by a bottom surface and a wall surface having at least a portion of cell adhesion properties, and culturing multiple cells to form a structure. Thus, the present invention has been completed. In other words, the present invention provides the following.

[0010] [1] A step of seeding multiple cells inside a containment space formed by a bottom surface and a wall surface having at least a portion of its cell-adherent properties, A process of culturing multiple cells to form a structure, A method for producing cell structures, including [the specified element]. [2] The manufacturing method described in [1], wherein the adhesive force between the wall surface and the cells is stronger than the cohesive force between cells. [3] The manufacturing method according to [1] or [2], further comprising the step of separating the structure from the containment space. [4] A manufacturing method according to any one of [1] to [3], wherein the number of cells seeded is within the product of the value obtained by dividing the wall area by the square of the cell diameter and the value obtained by dividing the wall height by the cell diameter. [5] The production method according to any one of [1] to [3], wherein the seeded cells are spheroids. [6] The production method according to [5], wherein the number of seeded spheroids is not more than the product (cells) of the value obtained by dividing the inner wall area by the square of the spheroid diameter multiplied by the value obtained by dividing the wall height by the spheroid diameter. [7] The spheroids consist of 30 to 5×10 5 cells, the production method according to [5] or [6]. [8] The wall surface is composed of a cell-adhesive material, The production method according to any one of [1] to [7], wherein the cell-adhesive material comprises at least one component selected from the group consisting of components derived from extracellular matrix proteins, glycoproteins and mucopolysaccharides. [9] The wall surface is composed of a cell-adhesive material, The production method according to any one of [1] to [8], wherein the cell-adhesive material comprises at least one component selected from gelatin, collagen, fibrin, fibronectin, vitronectin, laminin, proteoglycan, glycosaminoglycan, chitin and chitosan.

[10] The wall surface is composed of a cell-adhesive material, The production method according to any one of [1] to [9], wherein the cell-adhesive material comprises at least one component selected from fibrin, collagen, laminin, gelatin, gelatin methacrylate (GelMA), or chitosan.

[11] The production method according to any one of [1] to

[10] , wherein the adhesive force between the bottom surface and cells is lower than the adhesive force between the wall surface and cells.

[12] The production method according to any one of [1] to

[11] , wherein the bottom surface is cell non-adhesive.

[13] The production method according to any one of [1] to

[12] , wherein the structure is a three-dimensional structure.

[14] The seeding step comprises seeding a plurality of cells into an accommodation space formed by a wall surface having at least partial cell adhesiveness and a bottom surface having lower cell adhesiveness than the wall surface, The forming step comprises culturing a plurality of spheroids to form a structure, and the seeded cells are spheroids, the production method according to [1] or [2].

[15] A step of seeding multiple spheroids inside a containment space formed by a bottom surface and a wall surface having at least a portion of cell adhesion, A process of culturing multiple spheroids to form a three-dimensional structure, A method for producing cell structures, including the following:

[16] The manufacturing method according to

[15] , wherein the number of spheroids sown is within the product of the value obtained by dividing the wall area by the square of the spheroid diameter and the value obtained by dividing the wall height by the spheroid diameter.

[17] Spheroids range from 30 to 5 × 10 5 A method for producing (individual) cells as described in

[15] or

[16] .

[18] The method of manufacture according to any one of

[15] to

[17] , wherein the adhesion strength to the cells on the bottom surface is lower than the adhesion strength to the cells on the wall surface.

[19] A step of seeding multiple cells inside a containment space formed by a wall surface having cell adhesion properties in at least part and a bottom surface having lower cell adhesion properties than the wall surface, A process of culturing multiple cells to form a structure, A method for producing cell structures, including the following:

[20] The manufacturing method according to

[19] , wherein the number of cells to be seeded is within the product of the value obtained by dividing the wall area by the square of the cell diameter and the value obtained by dividing the wall height by the cell diameter.

[21] A culture fixture, It has a wall surface that is placed on the bottom of the culture vessel and forms a predetermined containment space, A culture jig in which at least a portion of the wall surface on the side facing the containment space has cell adhesion properties.

[22] The culture fixture described in

[21] , wherein the entire fixture is made of a cell adhesion material.

[23] The fixture according to

[21] , wherein the entire wall surface or the side of the wall surface facing the containment space is made of a cell-adhesive material.

[24] The fixture according to

[22] or

[23] , wherein the cell adhesion material comprises at least one component selected from the group consisting of components derived from extracellular matrix proteins, glycoproteins and mucopolysaccharides.

[25] The fixture according to any one of

[22] to

[24] , wherein the cell adhesion material comprises at least one component selected from gelatin, collagen, fibrin, fibronectin, vitronectin, laminin, proteoglycan, glycosaminoglycan, chitin, and chitosan.

[26] The fixture according to

[22] , wherein the cell adhesion material comprises at least one component selected from fibrin, collagen, laminin, gelatin, gelatin methacrylate (GelMA), or chitosan.

[27] Culture substrate, The base, and Wall surface surrounding the bottom surface to form a predetermined storage space It has, A culture substrate having cell adhesion properties in at least a portion of the wall surface on the side facing the containment space.

[28] The culture substrate according to

[27] , wherein the adhesion force of the bottom surface to cells is lower than the adhesion force of the wall surface to cells.

[29] The culture substrate according to

[27] or

[28] , wherein the entire wall surface or the side of the wall surface facing the containment space is made of a cell-adhering material.

[30] The culture substrate according to

[29] , wherein the cell adhesion material comprises at least one component selected from the group consisting of proteins, glycoproteins and mucopolysaccharides of the extracellular matrix.

[31] The culture substrate according to

[29] or

[30] , wherein the cell adhesion material comprises at least one component selected from gelatin, collagen, fibrin, fibronectin, vitronectin, laminin, proteoglycan, glycosaminoglycan, chitin, and chitosan.

[32] A culture substrate according to any one of

[29] to

[31] , wherein the cell adhesion material comprises at least one component selected from fibrin, collagen, laminin, gelatin, gelatin methacrylate (GelMA), or chitosan. [Effects of the Invention]

[0011] According to the present invention, cell structures can be easily manufactured without introducing foreign matter. For example, according to this disclosure, cells are not embedded using scaffolds such as MATRIGEL, thus preventing the introduction of foreign matter. Furthermore, according to this disclosure, cell structures can be easily manufactured without requiring specialized equipment or instruments, such as the pincushion method or net molding method. As a result, cell research in a microenvironment closer to that of living organisms can be conducted at a low cost. In addition, it becomes possible to manufacture cell structures using various cells in shapes desired by researchers, contributing to the development of transplantation and regenerative medicine for various tissues. [Brief explanation of the drawing]

[0012] [Figure 1] Image taken immediately after spheroid injection into the GelMA socket. [Figure 2] Changes over time in a three-dimensional sheet of porcine chondrocytes during culture. [Figure 3] Confirmation of differences in tissue thickness and cell viability due to differences in GelMA frame area. [Figure 4] Removal of the GelMA frame and handling of the fabricated cell structure. [Figure 5] Examples of three-dimensional structures created from human dermal fibroblasts (HDF). [Figure 6] Examples of three-dimensional structures created from human mesenchymal stem cells (MSCs). [Figure 7] Examination of frame material. The red arrowheads indicate that cells are detaching from the frame. [Figure 8] An example of fabricating a two-dimensional MSC sheet using a bio-3D printer. [Figure 9] A schematic diagram illustrating the case where the adhesion force between the wall and the cultured cells is equal to or stronger than the cohesive force between the cultured cells. [Figure 10] A schematic diagram of an example of a jig with a hollow frame. Figures 10A and 10B show a jig with a hollow frame. Figure 10C shows a cross-sectional view of the jig with a hollow frame when placed in a culture container. [Figure 11]A schematic diagram of an example of a jig with a base. Figures 11A and 11B show a jig that is a frame with a base. Figure 11C shows a cross-sectional view of the jig, which is a frame with a base, when placed in a culture container. [Modes for carrying out the invention]

[0013] (definition) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any similar or equivalent methods and materials as described herein may be used in carrying out or testing the present invention. Unless otherwise indicated, the present invention may be carried out using conventional methods in fields such as chemistry, biochemistry, molecular biology, cell biology, genetics, immunology, and pharmacology.

[0014] (Method for manufacturing cell structures) In one embodiment, the present disclosure provides a method for producing a cell structure (which may be referred to herein as "structure") that allows for the simple production of a cell structure without introducing foreign matter. The method for producing a structure according to the present disclosure includes the steps of seeding a plurality of cells inside a containment space formed by a bottom surface and a wall surface having at least a portion of cell adhesion (also referred to as the "first step"), and culturing the plurality of cells to form a structure (also referred to as the "second step").

[0015] In the first step of the method of this disclosure, the containment space refers to a region or space formed by a bottom surface and a wall surface covering the periphery of the bottom surface. The containment space may be of any shape, for example, cylindrical, oblique cylindrical, rectangular parallelepiped, or oblique prism. For example, the containment space may be formed by placing a jig, for example, shown in Figure 10, in a culture container such as a petri dish.

[0016] The jig is a hollow frame with openings on both the top and bottom sides, as shown in Figure 10 (Figures 10A and 10B). By placing the jig in the culture container as shown in Figure 10C, the area of ​​the bottom surface of the culture container enclosed by the hollow frame becomes the bottom surface forming the containment space, and the inner sides of the hollow frame become the walls forming the containment space.

[0017] The jig may be a frame with a bottom surface, as shown in Figure 11 (Figures 11A and 11B). In this case, the jig itself may serve as the containment space. Such a jig may be used by placing it in a culture container, as shown in Figure 11C, or it may be used on its own. The culture container itself, such as a petri dish or the wells of a microtiter plate, may also serve as the containment space. In this specification, jigs and culture containers that form a containment space on their own, such as the jig shown in Figure 11 or the wells of a microtiter plate, may be referred to as "culture substrates."

[0018] In the containment space of this disclosure, the wall surface is at least partially cell-adherent. The entire wall surface of this disclosure may also be cell-adherent. Cell adhesion, as used herein, refers to the property or characteristic of cells binding or adhering to it. For example, a wall surface that is at least partially cell-adherent is a wall surface to which cultured cells can bind or adhere so as to prevent the cultured cells from clumping together. For example, the adhesive force of a wall surface that is at least partially cell-adherent is the adhesive force that allows cultured cells to bind or adhere to the wall surface so as to prevent the cultured cells from clumping together. For example, the adhesive force between the wall surface and the cultured cells is equal to or stronger than the cohesive force between the cultured cells, as shown in Figure 9. The portion of the wall surface that is cell-adherent, its size, and shape are not particularly limited, but can be appropriately selected depending on, for example, the cell type, the number of cells to be seeded, the culture conditions, the cell-adherent material, etc.

[0019] In the containment space of this disclosure, at least a portion of the wall surface may be composed of a cell-adhesive material in order to make the wall surface cell-adhesive. Any material or substance that is cell-adhesive can be used as the cell-adhesive material, provided that the adhesive force between the wall surface and the cultured cells is equal to or stronger than the cohesive force between the cultured cells. The cell-adhesive material may include, for example, at least one component selected from the group consisting of cell-adhesive gels, plastics, resins, glass, rubber, metals, peptides, proteins, intercellular adhesion molecules, fusion proteins, lipid compounds, antibodies, extracellular matrix proteins, glycoproteins, and components derived from mucopolysaccharides. For example, the cell-adhesive material includes at least one component selected from the group consisting of extracellular matrix proteins, glycoproteins, and components derived from mucopolysaccharides. For example, the cell adhesion material contains at least one component selected from gelatin, collagen, fibrin, fibronectin, vitronectin, laminin, proteoglycan, glycosaminoglycan, chitin, and chitosan. Preferably, the cell adhesion material contains at least one component selected from gelatin, collagen, laminin, chitosan, and fibrin. The type, purity, quality, and concentration of the cell adhesion material can be selected as appropriate. For example, it is preferable that the cell adhesion material is a material with high cell (bio) compatibility.

[0020] More specifically, a cell-adhesive material may be used as an ink, and a hollow frame may be fabricated using a three-dimensional printer as a jig to form the walls of the containment space of this disclosure. In such cases, a bioink containing at least one component selected from laminin, fibrin, chitosan, collagen, gelatin, and gelatin methacrylate (GelMA) may be used.

[0021] Alternatively, a hollow, non-adhesive material may be used as a jig, and a cell-adhesive material may be applied to the inside of this jig to form the wall surface of the containment space of this disclosure. Furthermore, a cell-adhesive material may be applied to the wall surface of a culture container such as a petri dish to form the wall surface of the containment space of this disclosure.

[0022] The size of the accommodation space, the size of the bottom surface (bottom area), the height of the wall, etc. can be appropriately selected depending on the size of a desired structure, the number and type of cells, and the shape and size of the culture vessel, etc. The size of the bottom surface (bottom area) is not particularly limited, and is, for example, 0.1 mm 2 or more, 1.0 mm 2 or more, 4.0 mm 2 or more, 9.0 mm 2 or more, 10.0 mm 2 or more, 16.0 mm 2 or more, 20.0 mm 2 or more, 30.0 mm 2 or more, 32.0 mm 2 or more, 40.0 mm 2 or more, 50.0 mm 2 or more, 100.0 mm 2 or more, 500.0 mm 2 or more, or 1000.0 mm 2 or more. The size of the bottom surface (bottom area) is not particularly limited, and is, for example, 10000.0 mm 2 or less, 5000.0 mm 2 or less, 1000.0 mm 2 or less, 500.0 mm 2 or less, 100.0 mm 2 or less, 50.0 mm 2 or less, 40.0 mm 2 or less, 32.0 mm 2 or less, 30.0 mm 2 or less, 20.0 mm 2 or less, 16.0 mm 2 or less, or 10.0 mm 2The following are also possible. The wall height is not particularly limited, but may be, for example, 0.1 mm or more, 0.5 mm or more, 1.0 mm or more, 1.5 mm or more, 2.0 mm or more, 2.5 mm or more, 3.0 mm or more, 4.0 mm or more, 5.0 mm or more, 10.0 mm or more, 50.0 mm or more, or 100.0 mm or more. The wall height is not particularly limited, but may be, for example, 500.0 mm or less, 100.0 mm or less, 50.0 mm or less, 10.0 mm or less, 5.0 mm or less, 4.0 mm or less, 3.0 mm or less, 2.0 mm or less, or 1.0 mm or less. The wall thickness and bottom thickness can also be appropriately selected according to the desired size of the structure, the number and type of cells, and the shape and size of the culture vessel.

[0023] In the containment space of this disclosure, the bottom surface may be cell-adherent or cell-non-adherent (very low cell adhesion). If the bottom surface is cell-adherent, it is preferable that the adhesion force of the bottom surface to cells is lower than the adhesion force of the walls to cells. For example, when a containment space is formed by placing a jig, which is a hollow frame as shown in Figure 10, in a culture container, the area of ​​the bottom surface of the culture container surrounded by the jig becomes the bottom surface that forms the containment space. In this case, if a cell-non-adherent or very low cell adhesion culture container is used, it may be, for example, a very low adhesion container with a Corning treatment on its surface, and if a cell-adherent culture container is used, it may be, for example, a container with a Nunclon Delta treatment on its surface. Furthermore, it is known that the adhesion strength of the bottom surface of an adhesive culture vessel treated with Nunclon Delta to cells is lower than that of type I collagen, poly-L-lysine hydrochloride, or fibronectin (Chemistry & Biology 16, 773-782, July 31, 2009, Sayumi Yamazoe et al., A Dumbbell-Shaped Small Molecule that Promotes Cell Adhesion and Growth, see Figure 6 in particular).

[0024] Furthermore, when using a jig that is a frame with a bottom surface as shown in Figure 11 as the housing space, for example, a cell-adhesive material may be applied to at least a portion of the inner wall surface of the frame, which is constructed in the same shape as in Figure 11 using a non-adhesive material such as silicone, while the cell-adhesive material may not be applied to the bottom surface.

[0025] The properties, size, and shape of the base are not particularly limited, but can be appropriately selected depending on, for example, the cell type, the number of cells seeded, the culture conditions, and the cell adhesion material. If the base is non-adhesive, it becomes easier to separate the cultured structure from the containment space. If the base is adhesive, the area on which cells adhere becomes wider than that on the walls, making it easier to maintain the structure. Also, if the base is adhesive, the adhesion force between the base and the cells can compensate for the adhesion force between the walls and the cells.

[0026] When a jig is placed in a culture container to form a containment space, the walls of the containment space may be made of a material or have a shape that allows the solution and nutrients required for culture to pass through. If the walls are made of the aforementioned material or have the aforementioned shape, the solution and nutrients can be added to the outside of the walls, which can lead to a reduction in cell loss and an improvement in cell viability. Furthermore, when a jig with a bottom is placed in a culture container to form a containment space, the bottom of the jig may be made of a material or have a shape that allows the solution and nutrients required for culture to pass through.

[0027] The cells seeded in the containment space may be any type of cell. The seeded cells may be in the form of suspended cells or cell spheroids, which are aggregates of cells. The types of cells seeded may be, for example, so-called stem cells derived from mesenchymal stem cells, embryonic stem cells, or induced pluripotent stem cells. The types of cells seeded may also be cells derived from any tissue. More specifically, the types of cells seeded may be, for example, chondrocytes or dermal fibroblasts. The seeded cells may be cells derived from healthy cells or unhealthy cells. The seeded cells may be cells derived from humans or from animals other than humans. Animals other than humans may be, for example, insects, fish, amphibians, reptiles, birds, or mammals, and mammals may be, for example, monkeys, pigs, horses, cattle, sheep, dogs, cats, rats, mice, or rabbits.

[0028] A spheroid generally refers to a mass formed by the aggregation of cells, or a simple cluster (aggregate) of cells. Spheroids are typically formed by the aggregation of thousands of cells into a spherical structure. Compared to two-dimensional cell cultures, they can maintain high levels of functional expression for longer periods, but they are not as developed as organoids because they cannot self-assemble or self-regenerate. Spheroids are composed of, for example, 2 or more, 30 or more, 500 or more, 1000 or more, 5000 or more, 10000 or more, 20000 or more, 30000 or more, 40000 or more, or 50000 or more cells. A spheroid is composed of, for example, 50 or fewer, 100 or fewer, 500 or fewer, 1000 or fewer, 5000 or fewer, 10000 or fewer, 20000 or fewer, 30000 or fewer, 40000 or fewer, 50000 or fewer, 100000 or fewer, 500000 or fewer, or 1000000 or fewer cells.

[0029] The number of cells or spheroids to be seeded is, for example, within the product of the value obtained by dividing the wall area by the square of the cell or spheroid diameter and the wall height by the cell or spheroid diameter. The diameter of a cell or spheroid is the average of its short and long diameters. More specifically, the diameter of a single cell is the average of its short and long diameters, and the diameter of a single spheroid is the average of its short and long diameters. When determining the diameter of multiple cells, the diameter of each arbitrarily selected cell may be determined, and the average of these diameters may be used as the cell diameter. Similarly, when determining the diameter of multiple spheroids, the diameter of each arbitrarily selected spheroid may be determined, and the average of these diameters may be used as the spheroid diameter.

[0030] Cell or spheroid seeding can be carried out using known means and methods. For example, a cell suspension or spheroid may be placed in the containment space using a pipette.

[0031] The cell or spheroid culture in the second step of the method disclosed herein can be carried out using known means and methods. Culture conditions such as culture medium composition, culture temperature, and culture time can be appropriately selected by those skilled in the art, taking into consideration the type of cell, the size of the target structure, etc.

[0032] The method for producing the structure of the present disclosure may further include a step of preparing spheroids. For example, the step of preparing spheroids includes harvesting expanded cultured cells, reseeding and culturing them. Reagents, such as trypsin-EDTA solution, may be used to harvest expanded cultured cells.

[0033] The method for manufacturing the structure of the present disclosure may further include a step of confirming that the seeded cells are adhering to the wall surface. For example, the method for manufacturing the structure of the present invention may further include a step of confirming that the cells seeded in the containment space are not clumping together. The step of confirming that the seeded cells are adhering to the wall surface may be done visually or by actually touching them with an instrument.

[0034] The method for producing the structure of this disclosure may further include a step of confirming the viability of cells in culture, cultured cells, or cells in the structure. For example, a Live / Dead cell viability assay may be used.

[0035] The method for manufacturing the structure of the present disclosure may further include a step of separating the structure from the containment space. When a jig is placed on the bottom surface of a culture vessel to form the containment space, the manufactured structure can be separated from the containment space by grasping and lifting the jig with an instrument such as tweezers. In this case, for example, the wall surface of the containment space is touched with an instrument such as tweezers, but the structure is not directly touched. Since the structure is not touched by the instrument, the risk of contamination by foreign matter can be reduced. In addition, the risk of unintended mechanical stress on cells caused by grasping or pinching with an instrument can be reduced. This makes it possible to avoid affecting cell metabolism, such as changes in gene expression, or to improve cell viability. The height of the wall surface of the jig may be set higher than the height of the structure to be manufactured, or a handle may be attached to the outer wall surface of the jig so that it can be grasped with an instrument. When a cell-adhesive material is applied to the wall surface of a culture vessel to form the wall surface of the containment space, the manufactured structure can be separated from the containment space by lifting the structure with an instrument such as a spatula. Furthermore, when separating the manufactured structure from the containment space, it is preferable that the bottom or walls be made of a material or shape that facilitates the separation of the cultured structure.

[0036] The structures produced may be two-dimensional or three-dimensional. For example, a structure is a collection of cells containing cultured cells and extracellular matrix secreted from these cells. The structure is scaffold-free (i.e., does not contain scaffolds) and does not contain foreign substances such as heterogeneous extracellular matrix like MATRIGEL. Scaffold-free structures can reduce the risk of foreign substance contamination, thereby reducing the risk of inflammatory reactions and infections caused by foreign substance contamination and improving cell viability. Examples of three-dimensional structures include, but are not limited to, three-dimensional sheets, organ-like, tissue-like, spherical, or block-like shapes.

[0037] (Culture jig) The culture fixture of this disclosure has a wall surface that is placed on the bottom surface of a culture container to form a predetermined containment space, and at least a portion of the wall surface on the containment space side is cell-adherent. That is, the culture fixture may be a hollow frame, for example, as shown in Figure 10. Here, the culture fixture may be a frame constructed in the same shape as in Figure 10 using a three-dimensional printer with a cell-adherent material as ink. Alternatively, the culture fixture may be a frame constructed in the same shape as in Figure 10 using a non-adherent material such as silicone, with a cell-adherent material applied to at least a portion of the inner wall surface.

[0038] (Culture substrate) The culture substrate of this disclosure has a bottom surface and a wall surface surrounding the bottom surface to form a predetermined containment space, and at least a portion of the wall surface on the containment space side is cell-adherent. That is, the culture substrate may be a frame having a bottom surface, as shown in Figure 11, for example. In this case, the bottom surface of the frame constitutes the bottom surface of the containment space. Alternatively, the culture substrate may be a culture container on which a frame, as shown in Figure 10, with at least a portion of its inner wall surface being cell-adherent, is placed. Furthermore, the culture substrate may be a culture container on which at least a portion of the inner wall surface is coated with a cell-adherent material.

[0039] (modified version) The above-described embodiment can be modified as appropriate as follows.

[0040] For example, the bottom and walls do not need to completely cover the containment space, as long as cells do not leak out of the containment space. Furthermore, the containment space may be covered by a lid that covers the upper opening. For example, the lid may be attached after the cells have been seeded in the containment space.

[0041] Furthermore, in addition to allowing nutrients to pass through the walls of the containment space, nutritional factors and growth factors may be incorporated into the jigs such as the walls. This may promote cell differentiation and growth, and improve cell viability.

[0042] The present invention will be described in more specific and detailed terms below with reference to examples, but the scope of the present invention should not be limited to these examples. [Examples]

[0043] Example 1: Cell expansion culture and cell spheroid preparation (1) Pig cartilage cells Cells were cultured in GibcoDulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin mixture on a culture dish (CELLSTAR 145x20mm Petri dish [Greiner Japan]). The cultured cells were harvested with trypsin-EDTA solution and placed on a PrimeSurface® plate 96U in 1x10⁶ cells. 4 The cells were re-seed in a cell / well and cultured for 1 day to form cell spheroids. (2) Human dermal fibroblasts (HDF) FGM TM -2 Fibroblast Growth Medium-2 Bullet Kit TMThe cells were cultured on a culture dish using Lonza (a product of Lonza). The cultured cells were harvested in trypsin-EDTA solution and placed in a PrimeSurface® plate 96U in a 3 × 10⁶ layer. 4 The cells were re-seed in a cell / well and cultured for 1 day to form cell spheroids. (3) Human mesenchymal stem cells (MSCs) The cells were cultured in a culture dish using MSC NutriStem® XF Basal Medium + Supplement Mix (Sartorius). The cultured cells were harvested in trypsin-EDTA solution and 1 × 10⁶ cells were placed on the lid of the culture dish using the hanging drop method. 4 The cells were re-seed in a cell / drop container and cultured for one day to form cell spheroids.

[0044] Example 2: Fabrication of wall and frame (1) Silicone wall A 4mm x 4mm window was cut in the center of a 2mm thick silicone plate, and it was placed inside a 35mm diameter non-adhesive culture dish (PrimeSurface® Petri Dish [manufactured by Sumitomo Bakelite Co., Ltd.]). (2) Gelatin methacrylate (GelMA) slot 10-12.5% ​​GelMA was mixed with 0.1% photopolymerization initiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate), and a GelMA frame approximately 2 mm high and with an inner frame size of approximately 4 mm x 8 mm or 4 mm x 4 mm was fabricated using a bio-3D printer (Cellink) in a 35 mm diameter non-adhesive culture dish or a standard adhesive cell culture dish, and cured with 405 nm visible light. (3) Alginate gel frame, RGD peptide modified alginate gel frame Using alginate gel (CELLINK A [Cellink]) and alginate gel modified with the cell adhesion active sequence L-arginine-Glycine-L-aspartic Acid (RGD) peptide (CELLINK A-RGD [Cellink]), frames approximately 2 mm high and 4 mm x 4 mm inside were fabricated in non-adhesive culture dishes using a bio-3D printer, and cured with calcium chloride solution as a crosslinking agent. (4) Chitosan Gel Slot Using chitosan-based bioink (manufactured by Cellink), a chitosan gel frame approximately 2 mm high and 4 mm x 4 mm inside was fabricated in a standard adhesive cell culture dish using a bio-3D printer, and cured with tripolyphospate as a crosslinking agent. (5) Matrigel Wall A 2mm thick, 4mm x 4mm wide silicone plate was placed in the center of each well of a 96-well cell culture plate. Matrigel® Matrix (Corinig), cooled to 4°C, was injected around the silicone plate to a thickness of 2mm. After warming at 37°C for 30 minutes, the central silicone plate was removed with tweezers to create the Matrigel wall.

[0045] Example 3: Fabrication Example 1 of a Three-Dimensional Sheet of Pig Chondrocytes 192 porcine chondrocyte spheroids, prepared using the above method, were injected into 12.5% ​​GelMA frames (4 mm × 8 mm) prepared using the above method. The culture medium used for expansion culture was then mixed with ascorbic acid (50 μg / ml concentration) and cultured for 14 days. Figure 1 shows macro and micrographs immediately after the spheroids were injected into the GelMA frames. The spheroids are in contact with each other, and with the GelMA frames. Figure 2 shows macro and micrographs after 1, 3, 7, and 14 days of culture. After 1 day of culture, the spheroids were beginning to adhere to each other, and with the GelMA frames. After 7 days of culture, the gaps disappeared, and a sheet-like structure was formed. Histological analysis of the cell structure after 14 days of culture revealed numerous cell nuclei and sheet-like matrix production by hematoxylin-eosin staining (H·E staining).

[0046] Example 4: Fabrication Example 2 of a Three-Dimensional Structure of Porcine Chondrocytes Spheroids were cultured for 7 days in a 10% GelMA frame (4mm x 4mm), which is half the area of ​​the frame used in Example 1, using the same number of spheroids and method as in Example 1. Figure 3 shows macrographs, H·E stained images, and cell viability findings. Upon examination of the tissue image after H·E staining, it was confirmed that the spheroids were stacked in 2-3 layers, forming a thick cellular structure. Furthermore, a Live / Dead assay was performed to confirm cell viability, and high cell viability was confirmed. The prepared cellular structure could be removed from the GelMA frame with forceps, confirming that it had matured to a state where it could be handled with forceps (Figure 4).

[0047] Example 5: Fabrication example of an HDF three-dimensional structure 96 HDF spheroids prepared using the above method were injected into a silicone wall and a 10% GelMA frame (4 mm x 4 mm) prepared using the above method, and cultured for 7 days in the culture medium used for expansion culture. Figure 5 shows macro and micrographs of the spheroids immediately after injection, after 3 days of culture, and after 7 days of culture. Cells did not adhere to the silicone wall, which has low cell adhesion, and formed a single cell aggregate after 3 days of culture. On the other hand, it was confirmed that spheroids placed in the GelMA frame adhered to each other and to the GelMA frame, regardless of the adhesion of the bottom surface, and that a cell structure was formed along the frame. Furthermore, when a cell suspension using the same number of cells as the injected spheroids was cultured in the same GelMA frame, it was confirmed that a cell structure similar to that of the spheroids was formed along the frame. Note that the ordinary culture dish in Figure 5 was a cell-adherent culture container (CELLSTAR 35 x 10 mm Petri dish [Greiner Japan Co., Ltd.]).

[0048] Example 6: Fabrication example of an MSC three-dimensional structure 192 MSC spheroids prepared using the above method were injected into a 10% GelMA frame (4 mm x 4 mm) prepared using the above method, and cultured for 7 days in chondrogenic medium (MSCgo™ Chondrogenic XF + Supplement Mix [Sartorius]). Figure 6 shows macroscopic and microscopic images of the spheroids immediately after injection, after 3 days of culture, and after 7 days of culture. It was confirmed that the spheroids adhered to each other and to the GelMA frame, and that cellular structures were formed along the frame.

[0049] Example 7: Examination of frame materials 96 HDF spheroids prepared using the above method were injected into alginate gel frames, RGD peptide-modified alginate gel frames, GelMA frames, and Matrigel walls prepared using the above method. 192 MSC spheroids prepared using the above method were injected into chitosan gel frames, and the cells were cultured for 7 days in the culture medium used for expansion culture. Figure 7 shows macroscopic and microscopic images immediately after spheroid injection, after 3 days of culture, and after 7 days of culture. Gel frames made from alginate, which is widely used as a scaffold due to its low cytotoxicity and high biocompatibility, showed poor cell adhesion, with cells detaching from the frame and forming a single cell mass after 3 days of culture. Even when using gel frames based on alginate gel modified with the cell adhesion-active sequence RGD peptide to compensate for the low cell adhesion of alginate gel, cells similarly detached from the frame and formed a single cell mass after 3 days of culture. On the other hand, in GelMA frames (mainly gelatin), Matrigel walls (mainly laminin), and chitosan gel frames (mainly chitin), which are primarily composed of extracellular matrix, it was confirmed that a three-dimensional structure conforming to the shape of the wall surface was formed after 7 days of culture. From the above, it can be concluded that when fabricating HDF three-dimensional structures, the frame and wall surface materials must be formed not only of RGD sequences but also of extracellular matrix with cell adhesion properties (collagen, laminin, etc.).

[0050] Example 8: Fabrication example of a two-dimensional MSC sheet using a bio-3D printer. While several technologies have been developed to form MSCs into sheets, no technology has been developed to freely form sheets into any arbitrary shape. Therefore, we fabricated a two-dimensional MSC sheet with the shape of the frame by using GelMA as a bio-ink to create a frame using a bio-3D printer, and then seeding and culturing an MSC suspension inside the frame (Figure 8). As an example, we fabricated a cylindrical frame with a diameter of 10 mm and cultured MSCs inside the frame for 9 days, confirming that a circular cell sheet conforming to the shape of the frame was formed. Since this cell sheet is adhered to the frame, it can be manipulated without touching the cells by grasping the frame with tweezers or similar tools. [Industrial applicability]

[0051] This invention is useful in fields such as regenerative medicine and therapeutic materials for wound healing, cell research in microenvironments closer to those of living organisms, and transplantation regenerative medicine for various tissues.

Claims

1. A step of seeding multiple cells inside a containment space formed by a cell-non-adherent bottom surface and a wall surface made of a cell-adherent material, A process of culturing multiple cells to form a structure, A method for producing a cell structure, including, The cells to be seeded are a cell suspension or spheroids. The cell adhesion material comprises at least one component selected from gelatin, laminin, and chitin. Manufacturing method.

2. The manufacturing method according to claim 1, wherein the number of spheroids sown is within the product (number of spheroids) of the value obtained by dividing the wall area by the square of the spheroid diameter and the wall height by the spheroid diameter.

3. The method for producing the product according to claim 1, wherein the cells to be seeded are spheroids.

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