Cell culture molding device and method for producing cell culture sheet

Abstract

To improve the quality of a fabricated cell culture sheet capable of removing the cell culture sheet cleanly from a metal mold.SOLUTION: Provided is a cell culture molding device, in which one outer frame presser plates 23 is secured to an outer frame 21 and the other outer frame presser plate switches the needle grids 3A and 3B between a secured position P1 and a movable position, the cell culture molding device comprising: a pair of needle grids 3A and 3B, arranged with a gap between them and incorporated in a detachable manner configured in a planar manner, where rod-shaped metal needles 30 intersect in both vertical and horizontal directions and are detachably assembled; an inner frame 11 holding an outer peripheral edge of the needle grids 3A and 3B; a gap plate 13 disposed between the needle grids 3A and 3B; a cylindrical outer frame 21 that houses the inner frame 11 while keeping lattice planes 3a of the needle grids 3A and 3B perpendicular to a frame axis; a pair of outer frame presser plates 23 that secure the outer frame 21 from both sides in the frame axial direction and have an opening in the center; and a moving mechanism 40 that brings the needle grids 3A and 3B closer together or farther apart along a frame axis direction Z in an internal space 21A enclosed by the outer frame 21.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a cell culture molding device and a method for producing a cell culture sheet. [Background technology]

[0002] In recent years, food demand has increased with the global population growth, raising concerns about a shortage of meat, a source of protein. On the other hand, increasing livestock production poses many problems in terms of land, feed, waste disposal, and labor. As a way to overcome these problems in one leap, the production of artificial meat through cell culture has been proposed (see, for example, Patent Documents 1 and 2).

[0003] Patent document 1 reports that by forming a hydrogel containing skeletal myoblasts into an approximately rectangular shape, preparing multiple cell modules with holes of a specific shape in the hydrogel, stacking multiple cell modules so that the hole shapes do not overlap when viewed from above, and proliferating and culturing the skeletal myoblasts in this state, and then inducing their differentiation into myotubes, three-dimensional muscle tissue that is expected to have a texture similar to that of conventional edible meat has been obtained.

[0004] Patent Document 2 discloses an idea of ​​producing edible meat by forming a three-dimensional scaffold made of a hydrogel and seeding a population of self-regenerating cells thereon.

[0005] Conventionally, there is also known an apparatus that uses a scaffolding material such as gel and fills only cells (spheroids, etc.) into a mold using a metal mesh to form a sheet-like cell culture sheet. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 7033095 [Patent Document 2] Special Publication No. 2020-523015 Summary of the Invention [Problem to be solved by the invention]

[0007] In conventional technology, when using a metallic mesh member to fill with cells such as spheroids, there was a problem that when removing the created sheet-like cell culture sheet, it was difficult to cleanly remove the cells that had become attached to the mesh, making it impossible to ensure sufficient quality.

[0008] Therefore, one of the objects of the present invention is to provide a cell culture molding device and a method for manufacturing a cell culture sheet that can cleanly peel off a cell culture sheet from a metal mold and improve the quality of the cell culture sheet produced. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention proposes the following means. The cell culture molding apparatus according to a first aspect of the present invention is a cell culture molding apparatus that creates a sheet-shaped cell culture sheet by injecting spheroids obtained by culturing cells taken from an animal other than a human being, and is equipped with a pair of needle lattices arranged at a distance from each other and incorporating rod-shaped metal needles crossed vertically and horizontally in a plane, an inner frame that holds the outer peripheral edge of the needle lattices, a gap plate that is arranged between the pair of needle lattices, a cylindrical outer frame that houses the inner frame with the lattice surfaces of the pair of needle lattices perpendicular to the frame axis, a pair of outer frame pressing plates that fix the outer frame from both sides in the frame axial direction and have an opening in the center, and a moving mechanism that moves the pair of needle lattices toward and away from each other along the frame axial direction in the internal space surrounded by the outer frame, one of the pair of outer frame pressing plates is fixed to the outer frame, and the other is switchably arranged between a fixed position that restricts movement of the needle lattice in the frame axial direction and a movable position that allows the needle lattice to move in the frame axial direction.

[0010] A method for producing a cell culture sheet according to a first aspect of the present invention is a method for producing a sheet-like cell culture sheet by injecting spheroids obtained by culturing cells collected from an animal other than a human being, and includes the steps of: holding the outer peripheral edges of a pair of needle lattices, which are assembled by crossing rod-shaped metal needles vertically and horizontally in a planar manner, with an inner frame; arranging the pair of needle lattices at a distance from each other; arranging a gap plate between the pair of needle lattices; fixing a cylindrical outer frame that houses the inner frame from both sides in the frame axial direction with a pair of outer frame pressing plates having an opening in the center, with the lattice surfaces of the pair of needle lattices being orthogonal to the frame axis; moving the pair of needle lattices toward and away from each other along the frame axial direction in an internal space surrounded by the outer frame; and switching one of the pair of outer frame pressing plates to the outer frame and the other to either a fixed position that restricts movement of the needle lattice in the frame axial direction or a movable position where the needle lattice can be moved in the frame axial direction. Effect of the Invention

[0011] According to the cell culture molding device and the method for manufacturing a cell culture sheet of the present invention, the cell culture sheet can be neatly peeled off from the metal mold, and the quality of the produced cell culture sheet can be improved. [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 is a cross-sectional view of the cell culture molding device according to the embodiment of the present invention, viewed from the axial direction. [Diagram 2] 5 is a cross-sectional view taken along line AA in FIG. 4, showing a state in which the needle lattice is fixed. FIG. [Diagram 3] FIG. 3 is a diagram showing movable positions of the needle lattice in FIG. 2; [Figure 4] BB line arrow view shown in FIG. [Diagram 5] FIG. [Figure 6] FIG. 6 is an exploded perspective view of the inner frame structure shown in FIG. 5 . [Figure 7]FIG. 4 is an enlarged perspective view showing the attachment state of the needle grid. [Figure 8] FIG. 3 is a partially enlarged view of the inner frame in FIG. 2. [Figure 9] FIG. [Figure 10] FIG. 3 is an enlarged cross-sectional view showing the configuration of the outer frame pressing plate of FIG. 2. [Figure 11] FIG. 3 is an enlarged cross-sectional view showing the configuration of the movement mechanism in FIG. 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The jigs and devices shown in the drawings are schematic diagrams for explaining an example of an embodiment, and may differ from the actual dimensions and shapes.

[0014] An outline of the cell culture molding apparatus 1 according to this embodiment is shown in FIG. 1. The cell culture molding apparatus 1 according to this embodiment is intended as a food manufacturing apparatus for manufacturing cultured foods using artificially cultured cells. First, desired cells collected from animals other than humans are cultured to form spheroids F (spheroidized cell masses) as shown in FIG. 4, and then the cell culture molding apparatus 1 is used to obtain a cell culture sheet in which a plurality of spheroids F are arranged in a sheet shape and bonded to each other. Then, a desired number of cell culture sheets made by the cell culture molding apparatus 1 are stacked to obtain a thick cell cultured food. The cell culture molding apparatus 1 for obtaining a cell culture sheet will be specifically described below.

[0015] <Spheroid> A spheroid F is a mass of cells, and is generally roughly spherical. Its size (diameter) varies depending on the type of cell, but is generally about 0.1 to 0.5 mm. The type of cells that form the spheroid F is not particularly limited, and may be selected from any cells derived from cows, pigs, or chickens that have been conventionally used for food. From the viewpoint of mass production of homogeneous spheroid F, immortalized cells that are not limited in the number of times of subculture are preferred. The cells used in this embodiment and the spheroid F, which is an aggregate of the cells, are obtained by known methods.

[0016] ≪Cell culture molding device≫ As shown in FIGS. 1 to 4, the cell culture molding apparatus 1 includes an inner frame structure 10 for fixing a pair of needle lattices 3A, 3B, and an outer frame structure 20 for holding the inner frame structure 10 from the outside.

[0017] In the following description, the pair of needle lattices 3A, 3B are arranged substantially parallel to each other with a gap therebetween, and the direction along the lattice surface 3a of the pair of needle lattices 3A, 3B is defined as the horizontal direction. The pair of needle lattices 3A, 3B are square in plan view and exposed from an opening 23a formed in the outer frame structure 20. In plan view, the direction along one side of the pair of needle lattices 3A, 3B is defined as the X-axis, the direction perpendicular to the X-axis is defined as the Y-axis, and the direction perpendicular to the lattice surface 3a of the pair of needle lattices 3A, 3B is defined as the Z-axis. The Z-axis is a frame axis direction along a frame axis O of the outer frame 21 described later.

[0018] The inner frame structure 10 comprises the pair of needle lattices 3A, 3B described above, an inner frame 11 that holds the outer peripheral edges 3b of each of the pair of needle lattices 3A, 3B in a flat state, and a gap plate 13 arranged between the pair of needle lattices 3A, 3B.

[0019] The outer frame structure 20 includes a cylindrical outer frame 21 that houses the pair of inner frames 11 with the lattice surfaces 3a of the pair of needle lattices 3A, 3B perpendicular to the frame axis O, and a pair of outer frame pressing plates 23 that fix the outer frame 21 from both sides in the frame axial direction Z and have an opening 23a in the center. The outer frame structure 20 includes a movement mechanism 40 that moves the pair of needle lattices 3A, 3B toward and away from each other in the frame axial direction Z in an internal space 21A surrounded by the outer frame 21.

[0020] 5 to 7, the needle lattices 3A and 3B are each configured by intersecting a plurality of rod-shaped metal needles 30 in the vertical and horizontal directions (X-axis and Y-axis directions) in a planar manner relative to the inner frame 11 and detachably incorporating them. The needle lattices 3A and 3B are detachable from one surface side of the inner frame 11. The metal needles 30 are made of a metal that has affinity for living organisms, such as tungsten, and are formed from a thin rod material with an outer diameter of, for example, about 2 mm.

[0021] The metal needles 30 arranged vertically and horizontally all have the same length and are set to a dimension longer than the length of each side of the opening 11a of the inner frame 11 described later. That is, each metal needle 30 is attached so as to bridge the opening edge of the inner frame 11 (the inner frame opening edge 14b described later). The needle lattices 3A and 3B each form a lattice surface 3a by overlapping a column group 30y arranged in the vertical direction (here, the Y direction) and a row group 30x arranged in the horizontal direction (here, the X direction) in the frame axis direction Z. Note that the order in which the column group 30y and the row group 30x overlap in the frame axis direction Z is not limited.

[0022] The mesh size (the size of the gaps in the mesh) of the needle lattices 3A and 3B is set to 1 / 2 or less of the average outer diameter of the spheroids F. Since the needle lattices 3A and 3B come into contact with the spheroids F, a metal having affinity for living organisms, such as tungsten, is used. The spheroids F are injected into the gap S between the pair of needle lattices 3A and 3B through an injection port 26 (see FIG. 4) formed in the outer frame 21, which will be described later. The gap S between the needle lattices 3A and 3B is set to be less than twice the average outer diameter of the spheroids F.

[0023] As shown in Figs. 5 and 6, the inner frame 11 has a frame shape. The inner frame 11 has a predetermined thickness, and the thickness direction coincides with the frame axis direction Z. The needle lattices 3A and 3B are arranged on one side of the inner frame 11 in the thickness direction. That is, one side of the inner frame 11 (a surface 16b of a fixed plate 16 described later) and the lattice surfaces 3a of the needle lattices 3A and 3B are approximately flush with each other. In the inner frame 11, the side on which the needle lattices 3A and 3B are arranged is referred to as the lattice surface side. The inner frame 11 includes a frame main body 12, a needle holding frame 14, a needle pressing member 15, and a fixed plate 16.

[0024] The frame main body 12 has a square opening (not shown) in a plan view seen from the frame axis direction Z. The frame main body 12 has four corners with legs 121 extending toward the opposite side to the lattice surface side in the frame axis direction Z. As shown in FIG. 2, an outer frame pressing plate 23, which will be described later, can abut against a bottom surface 121a of the legs 121. That is, the outer frame pressing plate 23 restricts the movement of the pair of inner frames 11 in the frame axis direction Z within a predetermined range. As shown in FIG. 6, the frame main body 12 has a plurality of bolt holes 122 penetrating in the thickness direction. Fixing bolts 17 for fixing the fixing plate 16 to the frame main body 12 are inserted into these bolt holes 122.

[0025] As shown in Figs. 5 to 7, the needle holding frame 14 protrudes from the outer peripheral edge of the opening of the frame main body 12 toward the lattice surface side in the frame axis direction Z. That is, the needle holding frame 14 has an opening 14a that is coaxial with and has the same shape as the opening of the frame main body 12. A plurality of engagement grooves 141 that engage the ends 30a of the metal needles 30 are formed in the inner frame opening edge 14b that forms the protruding end face of the needle holding frame 14. The engagement grooves 141 include a plurality of first engagement grooves 141A that engage the vertical row group 30y of the metal needles 30 and a plurality of second engagement grooves 141B that engage the horizontal row group 30x. A plurality of first engagement grooves 141A are formed on two opposing sides (first opening edge 14c) of the inner frame opening edge 14b surrounded by four sides, and a plurality of second engagement grooves 141B are formed on the other opposing two sides (second opening edge 14d). The first opening edge 14c and the second opening edge 14d are offset in a step in the frame axis direction Z. That is, the metal needle 30 engaged with the first engagement groove 141A and the metal needle 30 engaged with the second engagement groove 141B are arranged offset in the frame axis direction Z and are perpendicular to each other when viewed from the frame axis direction Z.

[0026] The needle pressing member 15 presses the inner frame opening edge 14b with the metal needle 30 engaged in the engagement groove 141. A needle pressing member 15 is provided for each of the opening edges 14c, 14d of the needle holding frame 14. The needle pressing member 15 has a pressing surface 151 that presses the metal needle 30 engaged in the engagement groove 141, and a fixing surface 152 that is connected to the pressing surface 151 via a connecting surface 153 and is fixed to the surface 12b on the lattice surface side of the frame main body 12.

[0027] The pressing surface 151 is provided so as to cover the needle lattices 3A, 3B engaged with the engagement groove 141, and is therefore formed in a thin plate shape so as not to affect the gap S between the needle lattices 3A, 3B. The fixing surface 152 is formed with a bolt hole 152a through which the fixing bolt 17 passes. The needle pressing member 15 is fixed to the frame main body 12 by the fixing bolt 17 with the fixing surface 152 sandwiched between the surface 12b of the frame main body 12 and the fixing plate 16 (see FIG. 2).

[0028] The fixed plates 16 are plate members having an L shape in a plan view, and are provided in a pair. The pair of fixed plates 16A, 16B are fixed to the surface 12b of the frame body 12 by fixing bolts 17. When the pair of fixed plates 16A, 16B are attached to the frame body 12, the surface 12b of the frame body 12 is covered, and the needle lattices 3A, 3B held by the needle holding frame 14 are arranged in an exposed state in the opening 16a formed by the pair of fixed plates 16A, 16B (see FIG. 1). The surface 16b of the fixed plate 16 and the needle lattices 3A, 3B are approximately flush with each other.

[0029] As shown in FIG. 2, in the cell culture molding apparatus 1, the needle lattices 3A and 3B fixed to a pair of inner frames 11 are arranged with their lattice surfaces 3a facing each other. The pair of inner frames 11 are provided so that they can approach and move away from each other in the frame axis direction Z by a moving mechanism 40. At this time, the opposing needle lattices 3A and 3B are arranged while maintaining a predetermined gap S. A spheroid F is injected into the gap S between the pair of needle lattices 3A and 3B. The gap size is changed by the moving mechanism 40 so that the gap S is wide when the spheroid F is injected and narrowed after the injection.

[0030] A gap plate 13 is provided in the gap S between the pair of needle lattices 3A and 3B. As shown in FIG. 8, the minimum dimension of the gap S is the thickness dimension of the gap plate 13. Therefore, the gap S does not become smaller than a certain gap (here, the thickness of the gap plate 13). As shown in FIG. 9, the gap plate 13 forms an opening recess 13a that is U-shaped in plan view. In the opening recess 13a, a pair of needle lattices 3A and 3B are arranged opposite each other to ensure the gap S. The thickness of the gap plate 13 is set to be 1 / 2 or more of the average outer diameter of the spheroids F. Since the gap plate 13 comes into contact with the spheroids F, the same material as the needle lattices 3A and 3B is used, for example, a metal that has affinity with living bodies, such as tungsten, which is the same material as the needle lattices 3A and 3B. In addition, since the needle lattices 3A and 3B are made of metal, sterilization is easy and the durability is excellent.

[0031] 1 and 2, the outer frame 21 is formed into a square cylindrical shape when viewed from the frame axis direction Z. At both ends of the outer frame 21 in the frame axis direction Z, outer frame pressing plates 23 having square openings 23a in a plan view are fixed by fixing bolts 24 (24A, 24B).

[0032] The opening 23a of the outer frame pressing plate 23 is provided coaxially with the opening 11a of the inner frame 11, and is set to have a larger opening cross section than the opening 11a of the inner frame 11. The height in the frame axial direction Z in the internal space 21A of the outer frame 21 matches the length in the frame axial direction Z when the gap S between the pair of needle lattices 3A, 3B described above in the inner frame structure 10 is maximum. In other words, in this case, the length of the inner frame structure 10 when the gap S is minimum is smaller than the height of the internal space 21A of the outer frame 21, so that the inner frame structure 10 can move in the frame axial direction Z in the internal space 21A.

[0033] 2 and 3, of the pair of outer frame pressing plates 23, one first outer frame pressing plate 23A is fixed by a first fixing bolt 24A to one first fixed end 21a of the outer frame 21. The other second outer frame pressing plate 23B is provided switchably between a fixed position P1 (see FIG. 2) that restricts movement of the needle grids 3A, 3B in the frame axial direction Z and a movable position P2 (see FIG. 3) that allows movement of the needle grids 3A, 3B in the frame axial direction Z.

[0034] 2 is a state in which a clearance C is provided between the second outer frame pressing plate 23B and the outer frame 21 so as to ensure a wide gap S between the needle lattices 3A and 3B by tightening a gap adjustment screw 42 (described later) of the moving mechanism 40 in the forward direction toward the gap between the needle lattices 3A and 3B, and in which the positions of the needle lattices 3A and 3B are fixed together with the pair of inner frames 11. In other words, the fixed position P1 is an injection position when the needle lattices 3A and 3B are positioned with a predetermined gap S maintained and a spheroid F is injected into the gap S between the pair of needle lattices 3A and 3B.

[0035] The movable position P2 shown in Figure 3 is a state in which the restriction for ensuring the gap S between the needle grids 3A, 3B is released by loosening the gap adjustment screw 42 of the moving mechanism 40 in the direction of retreating from between the needle grids 3A, 3B, and further the second outer frame pressing plate 23B is abutted against the outer frame 21 without any gap.At this time, the fixed positions of the needle grids 3A, 3B together with the pair of inner frames 11, 11 are released, and they become freely movable.

[0036] At the movable position P2, the needle lattices 3A and 3B can approach and separate from each other. That is, the movable position P2 is a load application position where, by orienting the frame axis direction Z of the cell culture molding apparatus 1 in the vertical direction after injection of the spheroid F, the distance between the needle lattices 3A and 3B is narrowed by the weight of the upper inner frame 11 and outer frame 21, so that a certain load can be applied to the spheroid F, and the spheroids F are promoted to stick to each other and become integrated.

[0037] As shown in FIG. 10, the second outer frame pressing plate 23B is fixed to the other second fixed end 21b of the outer frame 21 by the second fixing bolt 24B via the spacer 25. The second outer frame pressing plate 23B has an insertion hole 23b through which a spacer 25 having a diameter larger than that of the second fixing bolt 24B is inserted. The axial length of the spacer 25 is greater than the hole axial length of the insertion hole 23b. A clearance C is generated between the second fixed end 21b of the outer frame 21 fixed by the second fixing bolt 24B via the spacer 25 and the fixed surface 23c of the second outer frame pressing plate 23B. At this time, as shown in FIG. 2, when the gap S between the pair of needle lattices 3A and 3B is minimized, the length of the inner frame structure 10 along the frame axis direction Z becomes smaller than the separation between the pair of outer frame pressing plates 23A and 23B (the height along the frame axis direction Z of the internal space 21A of the outer frame 21) and the inner frame structure 10 becomes movable in the frame axis direction Z.

[0038] 4, a tapered injection port 26 for injecting the spheroids F is provided on one of the first side surfaces 21c of the outer frame 21. An injection port plug 261 is provided on the injection port 26. The injection port 26 communicates with the gap S between the pair of needle lattices 3A, 3B from the outside of the first side surface 21c of the outer frame 21. Since the injection port 26 comes into contact with the spheroids F, a metal having affinity for living organisms, such as tungsten, is used.

[0039] 11, the movement mechanism 40 is a mechanism that can widen the gap between the meshes when injecting spheroids or the like, and narrow the gap after injection. The movement mechanism 40 has a female through-thread 41 (through-hole) formed in the outer frame 21 that penetrates the outer frame 21 in the thickness direction, and a gap adjustment screw 42 (pushing member) that is inserted into the female through-thread 41 from the outside of the outer frame 21 and pushes the gap between the pair of needle lattices 3A and 3B shown in FIG. 2 apart.

[0040] 2 and 4, the female through-threads 41 into which the gap adjustment screws 42 are screwed are disposed on the second side surface 21d and the third side surface 21e, which are opposed to each other and perpendicular to the first side surface 21c among the side surfaces of the outer frame 21, at positions corresponding to the gap S between the pair of needle grids 3A, 3B in the frame axis direction Z. On each of the second side surface 21d and the third side surface 21e, a pair of female through-threads 41 are provided at intervals in the Y-axis direction. In this embodiment, a total of four gap adjustment screws 42 adjust the size of the gap S between the pair of needle grids 3A, 3B.

[0041] As shown in FIG. 2 and FIG. 11, the gap adjustment screw 42 has a sharp tip 42a that can enter the gap S between the pair of needle lattices 3A and 3B. For example, a hexagonal socket set screw can be used as the gap adjustment screw 42. In the movement mechanism 40, the gap adjustment screw 42 is moved forward and backward to adjust the insertion dimension of the tip 42a into the gap S, thereby adjusting the size of the gap S. That is, the maximum dimension of the gap S coincides with the outer diameter dimension of the gap adjustment screw 42. Specifically, the gap S can be widened by tightening the gap adjustment screw 42 to advance toward the gap S when injecting the spheroids F, and the gap S can be narrowed by loosening the gap adjustment screw 42 to move backward after injecting the spheroids F. Note that narrowing the gap S allows the inner frame structure 10 to move in the frame axis direction Z within the internal space 21A of the outer frame 21.

[0042] <Production method of cell culture sheet> Next, a procedure for producing a cell culture sheet using the above-mentioned cell culture molding apparatus 1 will be specifically described. First, as shown in Figs. 1 to 4, the cell culture molding device 1 is assembled in a state where the inner frame structure 10 is accommodated inside the outer frame structure 20. As shown in Figs. 5 to 7, the inner frame structure 10 is assembled by fitting the metal needles 30 into the multiple engagement grooves 141 of the needle holding frame 14 in the frame body part 12 of the inner frame 11. Then, the pair of fixing plates 16A, 16B are fixed with fixing bolts 17 in a state where the needle pressing member 15 is interposed at a predetermined position relative to the frame body part 12. As a result, the needle grids 3A, 3B are pressed by the pressing surface 151 of the needle pressing member 15, and assembled without falling or shifting. Then, the outer frame structure 20 is assembled to the inner frame structure 10, and the assembly of the cell culture molding device 1 is completed.

[0043] Then, as shown in Figure 11, in the cell culture molding device 1 where assembly is completed, the gap adjustment screw 42 of the moving mechanism 40 is tightened into the through female screw 41 of the outer frame 21, thereby restricting the gap S between the pair of needle lattices 3A, 3B to its maximum widened state.

[0044] Next, the cell culture molding device 1 is introduced into a culture tank (not shown) with the injection port 26 facing up and the needle grids 3A and 3B facing vertically (see FIG. 4), and the injection port plug 261 of the injection port 26 is opened. A mixture of the culture fluid and the spheroids F is injected from the injection port 26 into the gap S between the pair of needle grids 3A and 3B. At this time, the outer periphery of the gap S is sealed by the outer frame 21, so that the injected spheroids F are prevented from spilling out of the gap S. When the spheroids F are injected, the injection fluid flows down in the direction of gravity, and the spheroids F gradually pile up in the gap S, and finally the gap S is filled with the spheroids F to the extent that it completely fills it.

[0045] After the spheroids F are injected, the gap adjustment screw 42 is loosened and moved backward in a direction away from the gap S, thereby releasing the constraint of the gap S. Then, the injection port plug 261 is closed to prevent the spheroids F from leaking from the injection port 26. This narrows the gap S, allowing the inner frame structure 10 to move in the frame axis direction Z within the internal space 21A of the outer frame 21.

[0046] Next, the cell culture molding apparatus 1 is turned sideways in the culture tank so that the weight of the inner frame structure 10 is applied to the spheroids F filled in the gap S between the pair of needle lattices 3A and 3B. Specifically, the cell culture molding apparatus 1 is rotated and positioned so that the outer frame pressing plate 23, which is movable in the culture tank, is on top. Then, the culture medium is replenished until the spheroids F in the cell culture molding apparatus 1 are immersed in the culture medium. This allows a certain load (stress) to be applied to the spheroids F, promoting them to stick to each other and become integrated. When this state is maintained for a number of days (for example, about one day) required for the spheroids F to adhere to each other, the intended cell culture sheet is formed in the cell culture molding apparatus 1.

[0047] The composition of the culture medium is not particularly limited, and a known composition that maintains the vital activity of the spheroid F is applied. The culture medium may contain a known additive that induces the differentiation of each cell that constitutes the spheroid F. The temperature of the culture medium is preferably in a range that maintains the vital activity of the spheroid F. The pH and CO 2 The concentration, oxygen concentration, temperature, etc. may be controlled by general culture techniques.

[0048] Next, the fixing bolts 24A and 24B are loosened to remove the outer frame pressing plate 23 and the outer frame 21, and the inner frame structure 10 is taken out. Then, the pair of inner frames 11 are separated to take out the cell culture sheet formed in the gap S between the inner frames 11. At this time, the needle lattices 3A and 3B are attached to the front and back surfaces of the taken-out cell culture sheet. Therefore, by removing the multiple metal needles 30 one by one or multiple by multiple from the taken-out cell culture sheet, the cell culture sheet can be neatly peeled off from the needle lattices 3A and 3B. In this way, a cell culture sheet of the desired thickness formed in the gap S is obtained.

[0049] The type of spheroids contained in the cell culture sheet formed by the cell culture molding apparatus 1 may be one type, or two or more types. The type of cells constituting each spheroid contained in the cell culture sheet may be one type, or two or more types. In the cell culture sheet, the individual spheroids F are adjacent to each other along the surface direction of the sheet and are adhered to each other. The adhesion form is preferably a form in which the cells present on the spheroid surface spontaneously adhere to each other, for example, a form in which the cells bind to each other via a protein such as fibronectin present on the cell surface.

[0050] <Actions and Effects of the Embodiment> The cell culture molding apparatus 1 of this embodiment creates a sheet-shaped cell culture sheet by injecting spheroids F obtained by culturing cells collected from animals other than humans. The cell culture molding apparatus 1 includes a pair of needle lattices 3A and 3B arranged at intervals from each other and detachably assembled by crossing rod-shaped metal needles 30 in a planar manner in the vertical and horizontal directions, an inner frame 11 that holds the outer periphery of the needle lattices 3A and 3B, a gap plate 13 that is arranged between the pair of needle lattices 3A and 3B, a cylindrical outer frame 21 that houses the inner frame 11 in a state in which the lattice surfaces 3a of the pair of needle lattices 3A and 3B are perpendicular to the frame axis, a pair of outer frame pressing plates 23 that fix the outer frame 21 from both sides in the frame axis direction and have an opening in the center, and a moving mechanism 40 that moves the pair of needle lattices 3A and 3B close to and away from each other along the frame axis direction Z in the internal space 21A surrounded by the outer frame 21. One of a pair of outer frame pressing plates 23 is fixed to the outer frame 21, and the other is switchably arranged between a fixed position P1 which restricts the movement of the needle lattices 3A, 3B in the frame axial direction Z, and a movable position P2 which allows the needle lattices 3A, 3B to move in the frame axial direction Z.

[0051] In this embodiment, when injecting the spheroid F, the pair of needle lattices 3A, 3B can be moved away from each other by the moving mechanism 40 to a fixed position P1 shown in Fig. 2 where the gap S between the pair of needle lattices 3A, 3B is widened, and after injecting the spheroid F, the pair of needle lattices 3A, 3B can be moved toward each other by the moving mechanism 40 to a movable position P2 shown in Fig. 3 where the gap S can be narrowed. Then, narrowing the gap S makes it possible for the inner frame structure 10 to move in the frame axis direction Z within the internal space 21A of the outer frame 21, and the needle lattices 3A, 3B to a movable position P2 where they can move in the frame axis direction Z.

[0052] More specifically, as shown in FIG. 2, the gap adjustment screw 42 of the moving mechanism 40 is tightened in the forward direction toward the needle lattices 3A and 3B, thereby ensuring a wide gap S between the needle lattices 3A and 3B, and a clearance C is provided between the second outer frame pressing plate 23B and the outer frame 21. At this time, the positions of the needle lattices 3A and 3B are fixed together with the pair of inner frames 11. As a result, the needle lattices 3A and 3B are placed at a fixed position P1 while maintaining a predetermined gap S, and spheroids F are injected into the gap S between the pair of needle lattices 3A and 3B. After the injection of the spheroids F, as shown in FIG. 3, the gap adjustment screw 42 of the moving mechanism 40 is loosened in the backward direction from the needle lattices 3A and 3B, thereby releasing the restriction for ensuring the gap S between the needle lattices 3A and 3B, and further, the second outer frame pressing plate 23B is brought into contact with the outer frame 21 without any gap. At this time, the needle grids 3A, 3B are released from the fixed positions together with the pair of inner frames 11, 11 and reach a movable position P2, allowing the needle grids 3A, 3B to move toward and away from each other.

[0053] Therefore, by positioning the cell culture molding device 1 so that the outer frame pressing plate 23, which is movable in the culture tank after injection of the spheroids F, is on top, a certain load can be applied to the spheroids F, promoting the spheroids F to adhere to each other and become integrated. In this way, in this embodiment, the necessary components can be reliably received from the culture solution, and the quality of the created cell culture sheet can be improved.

[0054] In addition, in this embodiment, a gap plate 13 is provided between a pair of needle lattices 3A, 3B, so that the gap S between the pair of needle lattices 3A, 3B never falls below a certain gap equal to the thickness of the gap plate 13, and contact between the needle lattices 3A, 3B can be prevented.

[0055] Furthermore, in this embodiment, the needle grids 3A and 3B are configured by arranging a plurality of metal needles 30 vertically and horizontally in a grid pattern, so that the metal needles 30 that have bitten into the created cell culture sheet can be removed one by one to reduce damage to the cell culture sheet. In other words, the cell culture sheet can be neatly peeled off from the needle grids 3A and 3B, improving the quality of the created cell culture sheet.

[0056] In addition, in the cell culture molding device 1 of this embodiment, the inner frame 11 has an opening 11a that holds the needle grids 3A and 3B. An engagement groove 141 that engages the end 30a of the metal needle 30 is formed in the inner frame opening edge 14b of the opening 11a. A needle pressing member 15 that presses the inner frame opening edge 14b from the outside in a state in which the metal needle 30 is engaged with the engagement groove 141 is provided. Therefore, in this embodiment, the engagement groove 141 formed in the inner frame opening edge 14b is covered by the needle pressing member 15. Therefore, it is possible to prevent the metal needle 30 from falling off or shifting from the engagement groove 141.

[0057] In addition, in the cell culture molding apparatus 1 of this embodiment, the thickness of the gap plate 13 is more than half the average outer diameter of the spheroids F, so that the variation in diameter of the spheroids F can be made uniform and the spheroids F can be prevented from moving to other locations.

[0058] Furthermore, in the cell culture molding apparatus 1 of this embodiment, the gap S between the needle lattices 3A and 3B into which the spheroids F are injected is less than twice the average outer diameter of the spheroids F. This makes it possible to suppress overlapping of the spheroids F, and to form a high-quality cell culture sheet.

[0059] In addition, in the cell culture molding apparatus 1 of this embodiment, the moving mechanism 40 has a through female screw 41 (through hole) formed in the outer frame 21 and penetrating the outer frame 21 in the thickness direction, and a gap adjustment screw 42 (pushing member) that is inserted into the through female screw 41 from the outside of the outer frame 21 to push the gap between the pair of needle lattices 3A and 3B. Therefore, in the moving mechanism 40, the gap S can be widened by tightening the gap adjustment screw 42 to advance toward the gap S when injecting the spheroids F, and the gap S can be narrowed by loosening the gap adjustment screw 42 to move backward after injecting the spheroids F. Therefore, narrowing the gap S allows the inner frame structure 10 to move in the frame axis direction Z within the internal space 21A of the outer frame 21.

[0060] In addition, in the cell culture molding device 1 of this embodiment, the gap adjustment screw 42 has a sharp tip portion 42a that can enter between the pair of needle lattices 3A and 3B. Therefore, the pair of needle lattices 3A and 3B can be reliably widened, and the size of the gap S can be adjusted according to the advancement distance of the gap adjustment screw 42 into the gap S.

[0061] Furthermore, in the cell culture molding apparatus 1 of this embodiment, the outer frame 21 is provided with a tapered injection port 26 for injecting the spheroids F. This allows the spheroids F to be easily injected without spilling out to the surroundings.

[0062] In addition, in the cell culture molding apparatus 1 of this embodiment, the mesh size of the needle lattices 3A, 3B is less than half the average outer diameter of the spheroid F, so that the spheroid F can be prevented from jumping outward through the gap S between the pair of needle lattices 3A, 3B.

[0063] In addition, in the cell culture molding apparatus 1 of this embodiment, the needle lattices 3A and 3B, the gap plate 13, and the injection port 26, which come into contact with the spheroids F, are made of the same metal that has affinity with living organisms. Therefore, the influence on the needle lattices 3A and 3B, the gap plate 13, and the injection port 26 can be suppressed.

[0064] Furthermore, in the cell culture molding apparatus 1 of this embodiment, the needle grids 3A and 3B are made of tungsten, so that the influence on the spheroids F can be more reliably suppressed.

[0065] The present invention is not limited to the above-described embodiment, and various design modifications are possible without departing from the spirit and scope of the present invention. For example, in the above embodiment, the moving mechanism 40 is formed on the outer frame 21 and has a through female screw 41 penetrating the outer frame 21 in the thickness direction, and a gap adjustment screw 42 (pushing member) that is inserted into the through hole from the outside of the outer frame and pushes the gap between the pair of needle lattices 3A and 3B, but is not limited to this configuration. For example, a member such as a pin may be used as the pushing member instead of a screw. In short, any configuration may be used as long as it is possible to adjust the size of the gap S between the pair of needle lattices 3A and 3B.

[0066] In addition, in this embodiment, the multiple metal needles 30 are configured to be detachably engaged with the engagement grooves 141 formed on the inner frame opening edge 14b of the inner frame 11, but the configuration is not limited to engaging with the engagement grooves 141. Furthermore, in this embodiment, the needle pressing member 15 is provided to cover the metal needle 30 engaged with the engagement groove 141, but such a needle pressing member 15 may be omitted. [Explanation of symbols]

[0067] 1...Cell culture molding device 3A, 3B...Needle grid 3a...Lattice plane 3b…Outer rim 10...Inner frame structure 11…Inner frame 12...Frame body 13…Gap plate 14...Needle holder frame 14b…Inner frame opening edge 141...Engagement groove 141A…1st engagement groove 141B…Second engagement groove 15...Needle holder 151…Pressing surface 16, 16A, 16B…fixing plate 17…Fixing bolt 20...Outer frame structure 21…Outer frame 21A…Internal space 23…Outer frame support plate 24A…First fixing bolt 24B…Second fixing bolt 25…Spacer 26…Inlet 261...Injection port plug 30…Metal needle 30a...end 40...Movement mechanism 41... Female thread (through hole) 42...Gap adjustment screw (push-in member) F…Spheroid O…Frame axis P1…Fixed position P2…Moveable position S…Gap

Claims

1. A cell culture molding device for producing a sheet-shaped cell culture sheet by injecting spheroids obtained by culturing cells taken from animals other than humans, A pair of needle lattices arranged at intervals from each other and detachably assembled by crossing rod-shaped metal needles in the vertical and horizontal directions on a plane; an inner frame for holding an outer peripheral edge of the needle lattice; a gap plate disposed between the pair of needle grids; a cylindrical outer frame that accommodates the inner frame in a state in which the lattice surfaces of the pair of needle lattices are perpendicular to a frame axis; A pair of outer frame pressing plates that fix the outer frame from both sides in the frame axial direction and have an opening in the center; a moving mechanism for moving the pair of needle lattices toward and away from each other along a frame axis in an internal space surrounded by the outer frame, A cell culture molding device in which one of the pair of outer frame pressing plates is fixed to the outer frame, and the other is switchable between a fixed position that restricts movement of the needle lattice in the frame axial direction, and a movable position that allows the needle lattice to move in the frame axial direction.

2. the inner frame has an opening for holding the needle grid; An engagement groove is formed on the inner frame opening edge of the opening portion to engage with the end of the metal needle, The cell culture molding device according to claim 1 , further comprising a needle pressing member that presses an opening edge of the inner frame from the outside in a state in which the metal needle is engaged with the engagement groove.

3. The cell culture molding device according to claim 1 , wherein the plurality of metal needles are provided so as to be removable from the cell culture sheet one by one or multiple by multiple.

4. The cell culture molding device according to claim 1 or 2, wherein the thickness of the gap plate is equal to or greater than ½ of an average outer diameter of the spheroids.

5. 3. The cell culture molding device of claim 1, wherein the gap between the needle lattices through which the spheroids are injected is less than twice the average outer diameter of the spheroids.

6. The moving mechanism includes: A through hole formed in the outer frame and penetrating the outer frame in a thickness direction; The cell culture molding device according to claim 1 or 2, further comprising: a pushing member that is inserted into the through-hole from the outside of the outer frame to push apart the space between the pair of needle lattices.

7. The cell culture molding apparatus according to claim 6 , wherein the pushing member is a gap adjustment screw having a sharp tip portion capable of entering between the pair of needle lattices.

8. The cell culture molding device according to claim 1 , wherein the outer frame is provided with a tapered injection port for injecting the spheroids.

9. The cell culture molding apparatus of claim 1 , wherein the needle lattice has an opening that is equal to or smaller than ½ of an average outer diameter of the spheroids.

10. The cell culture molding apparatus of claim 8, wherein the needle grid, the gap plate, and the injection port, which come into contact with the spheroids, are made of the same metal that is biocompatible.

11. The cell culture molding apparatus of claim 10 , wherein the metal needle is tungsten.

12. A method for producing a cell culture sheet by injecting spheroids obtained by culturing cells collected from an animal other than a human, thereby producing a sheet-shaped cell culture sheet, comprising the steps of: a step of holding, by an inner frame, the outer periphery of each of a pair of needle lattices, in which rod-shaped metal needles are assembled by crossing each other in the vertical and horizontal directions on a plane; spaced apart the pair of needle lattices; disposing a gap plate between the pair of needle grids; a step of fixing a cylindrical outer frame that houses the inner frame from both sides in the frame axial direction by a pair of outer frame pressing plates having an opening at the center, in a state in which the lattice surfaces of the pair of needle lattices are perpendicular to the frame axis; moving the pair of needle lattices toward and away from each other along the frame axis in an internal space surrounded by the outer frame; a step of fixing one of the pair of outer frame pressing plates to the outer frame and switching the other of the pair of outer frame pressing plates to either a fixed position that restricts movement of the needle lattice in the frame axial direction or a movable position that allows the needle lattice to move in the frame axial direction; The method for producing a cell culture sheet comprising the steps of:

13. The method for producing a cell culture sheet according to claim 12 , wherein the metal needles are removed one by one or a plurality of needles at a time from the cell culture sheet.

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