Cell culture molding device and method for producing cell culture piece

Abstract

To enhance quality of a produced cell culture piece through improvement in mold releasability.SOLUTION: A cell culture molding device comprises: a pair of resin nets 3A and 3B arranged substantially in parallel with a space therebetween; net frames 11 supporting respectively outer peripheral edges of the pair of resin nets 3A and 3B so as to attach the resin nets 3 under tension; net presser members 12 fixing the resin nets 3 to the net frame 11 in the tensioned state; a gap plate 13 disposed between the pair of resin nets 3A and 3B; a cylindrical outer frame 21 accommodating the net frame 11 and the net presser member 12 with the net faces 3a of the pair of resin nets 3A and 3B orthogonal to a frame axis O; and movement mechanisms 40 configured to move the pair of resin nets 3A and 3B in a direction to approach to or separate from each other within an interior space 21A surrounded 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 manufacturing a cell culture piece. [Background technology]

[0002] In recent years, food demand has increased due to the global population growth, raising concerns about a shortage of meat, a nutrient source of protein. Meanwhile, increasing livestock production poses many problems, including land, feed, waste disposal, and labor. The artificial production of meat through cell culture has been proposed as a way to overcome these problems in one fell swoop (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 the multiple cell modules so that the hole shapes do not overlap when viewed from above, and then proliferating and culturing the skeletal myoblasts in this state and 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 to produce edible meat by forming a three-dimensional scaffold made of 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 techniques, when a metal mesh member is used, the sheet-shaped cell culture sheet formed by filling a mold with cells such as spheroids is difficult to remove from the metal mesh member. As a result, some parts of the cell culture sheet adhere to the metal mesh member, making it difficult to remove the cell culture sheet in a clean state, and there is a problem in that sufficient quality cannot be ensured. Furthermore, in the prior art, the mesh mold is made of metal, which is likely to be deteriorated by the culture medium, making it difficult to use for a long period of time, and there is room for improvement in this respect.

[0008] Therefore, one of the objects of the present invention is to provide a cell culture molding device and a method for manufacturing cell culture pieces that can improve the quality of the cell culture pieces produced by improving the releasability of the mold. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention proposes the following means. The cell culture molding device according to a first aspect of the present invention is a cell culture molding device that creates sheet-shaped or rod-shaped cell culture pieces by injecting spheroids of cultured cells, and comprises a pair of resin meshes arranged at a distance from each other, a mesh frame that supports the outer edges of each of the pair of resin meshes and attaches the resin meshes, a mesh holding member that fixes the resin mesh to the mesh frame, a gap plate that is arranged between the pair of resin meshes, a cylindrical outer frame that houses the mesh frame and the mesh holding member with the mesh surfaces of the pair of resin meshes perpendicular to the frame axis, and a moving mechanism that moves the pair of resin meshes in directions toward and away from each other within the internal space surrounded by the outer frame.

[0010] Another aspect of the present invention relates to a method for manufacturing cell culture pieces, which involves injecting cultured cell spheroids to create sheet-shaped or rod-shaped cell culture pieces, and includes the steps of: arranging a pair of resin meshes at a distance from each other; supporting the outer edges of each of the pair of resin meshes to attach the resin meshes to a mesh frame; fixing the resin meshes to the mesh frame with a mesh holding member; placing a gap plate between the pair of resin meshes; and moving the pair of resin meshes in directions toward and away from each other within the internal space surrounded by the outer frame. [Effects of the Invention]

[0011] According to the cell culture molding device and the method for manufacturing a cell culture piece of the present invention, the quality of the cell culture piece to be produced can be improved by improving the releasability of the mold. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a cross-sectional view of a cell culture molding device according to an embodiment of the present invention, viewed from an axial direction. [Figure 2] 5 is a cross-sectional view taken along line AA in FIG. 4, showing a state of a fixing position that restricts movement of the resin net. FIG. [Figure 3] FIG. 3 is a diagram showing a state of an unlocked position in FIG. 2 that allows the resin net to move. [Figure 4] BB arrow view shown in FIG. 1. [Figure 5] FIG. [Figure 6] 3 is an enlarged cross-sectional view showing the configuration of the net pressing member and the net moving member in FIG. 2. [Figure 7] FIG. 3 is an enlarged cross-sectional view showing the configuration of the outer frame pressing plate of FIG. 2. [Figure 8] FIG. 3 is an enlarged cross-sectional view showing the configuration of the movement mechanism of FIG. 2. [Figure 9] FIG. 10 is a plan view of a gap plate according to a first modified example. [Figure 10] FIG. 10 is a plan view of a gap plate according to a second modified example. [Figure 11] FIG. 10 is a plan view of a gap plate according to a third modified example. [Figure 12] FIG. 12 is an enlarged plan view of region C shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[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 in actual dimensions and shapes.

[0014] An overview of a cell culture molding apparatus 1 according to this embodiment is shown in Figure 1. The cell culture molding apparatus 1 of this embodiment is intended as a food manufacturing apparatus for producing cultured foods from artificially cultured cells. First, desired cells collected from a non-human animal are cultured to form spheroids F (spheroidized cell masses) as shown in Figure 4. Then, using the cell culture molding apparatus 1, multiple spheroids F are arranged in a sheet-like configuration and adhered to each other to obtain a cell culture sheet (cell culture piece). A desired number of cell culture sheets created by the cell culture molding apparatus 1 are then stacked to produce a thick cell-cultured food. The cell culture molding apparatus 1 for obtaining a cell culture sheet will be specifically described below.

[0015] <Spheroid> Spheroids F are aggregates of cells and are usually roughly spherical. Their 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 spheroids F is not particularly limited and can be selected from, for example, any cells derived from cows, pigs, or chickens that have traditionally been used for food. From the perspective of mass production of homogeneous spheroids F, immortalized cells that can be passaged without any restrictions on the number of times 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 device 1 includes a mesh structure 10 for fixing a pair of resin meshes 3 (3A, 3B) and an outer frame structure 20 for holding the mesh structure 10 from the outside.

[0017] In the following description, the pair of resin nets 3A, 3B are arranged approximately parallel to each other with a gap between them, and the direction along the mesh surfaces 3a of the pair of resin nets 3A, 3B is defined as the horizontal direction. The pair of resin nets 3A, 3B are square in plan view and exposed to the outside of the outer frame structure 20. In plan view, the direction along one side of the pair of resin nets 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 mesh surfaces 3a of the pair of resin nets 3A, 3B is defined as the Z-axis. The Z-axis is a direction along the frame axis O of the outer frame 21, which will be described later.

[0018] The mesh structure 10 comprises the pair of resin meshes 3 (3A, 3B) described above, a mesh frame 11 that supports the outer peripheral edges 3b of each of the pair of resin meshes 3A, 3B and attaches the resin meshes 3A, 3B in a substantially planar shape, a mesh pressing member 12 that fixes the resin meshes 3A, 3B to the mesh frame 11 while applying tension to them, and a gap plate 13 that is arranged between the pair of resin meshes 3A, 3B.

[0019] The outer frame structure 20 comprises a cylindrical outer frame 21 that houses the mesh frame 11 and the mesh pressing member 12 with the mesh surfaces 3a of the pair of resin meshes 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 axis direction (Z axis direction) and have an opening 23a in the center. The mesh structure 10 and the outer frame structure 20 are provided with a movement mechanism 40 that moves the pair of resin meshes 3A, 3B in the direction of approaching and separating them (Z-axis direction) in the internal space surrounded by the outer frame 21.

[0020] The mesh size (the size of the gaps in the mesh) of the resin nets 3A and 3B is set to be equal to or less than half the average outer diameter of the spheroids F. The spheroids F are injected into the gap S between the pair of resin nets 3A and 3B through an injection port 26 formed in the outer frame 21, which will be described later. The gap S between the resin nets 3A and 3B is set to be less than twice the average outer diameter of the spheroids F.

[0021] The resin meshes 3A and 3B are made of polyolefin or fluorine-based resin that has releasability to cells. Specific materials for the resin meshes 3A and 3B include, for example, polytetrafluoroethylene (PTFE), tetrafluoroethylene-ethylene copolymer (ETFE), polyvinylidene fluoride (PVDF), etc. If the resin meshes 3A and 3B are made of a material with elasticity (e.g., properties such as flexibility) such as PTFE, the tension when they are attached to the mesh frame 11 can be adjusted.

[0022] The mesh frame 11 has a frame shape with a square opening 11a in plan view. A resin mesh 3 is attached to one surface of the mesh frame 11 under tension. Resin meshes 3A and 3B are attached to each of the pair of mesh frames 11. That is, the pair of resin meshes 3A and 3B fixed to each mesh frame 11 are arranged with their mesh surfaces 3a facing each other and are movable toward and away from each other in the frame axial direction by a movement mechanism 40, and are arranged while maintaining a predetermined gap S. Spheroids F are injected into the gap S between the pair of resin meshes 3A and 3B. The gap size of the resin meshes 3A and 3B is changed by the movement mechanism 40 so that the gap S is wide when the spheroids F are injected and narrowed after injection.

[0023] A gap plate 13 shown in Fig. 5 is provided in the gap S between the pair of resin nets 3A and 3B. 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). The gap plate 13 has a plate body 131 and an injection port plug 132. As shown in Fig. 5, the plate body 131 has a recess 131a that is approximately U-shaped in plan view. The injection port plug 132 can be fitted into the recess 131a with a gap therebetween. When fitted into the recess 131a as shown in Fig. 5, the gap plate 13 has a linear tip 132a that forms a square space in plan view within the gap plate 13. The shape of the gap plate 13 can be changed as appropriate to suit the size and shape of the cell culture sheet to be produced (see a first modified example shown in FIG. 9 and a second modified example shown in FIG. 10, which will be described later).

[0024] As shown in Figure 2, a pair of resin meshes 3A and 3B are arranged facing each other in the recess 131a of the gap plate 13, ensuring a gap S. The thickness of the gap plate 13 is set to at least half the average outer diameter of the spheroids F. Since the gap plate 13 comes into contact with the spheroids F, a metal with affinity for living organisms, such as tungsten, is used for the gap plate 13. Furthermore, because the gap plate 13 is made of metal, it can be easily sterilized and has excellent durability.

[0025] As shown in Figures 1 to 3, the net pressing member 12 has a frame shape with a square opening that is approximately the same shape as the net frame 11 in a plan view, and is divided into each side that forms the square. That is, the net pressing member 12 has a pair of horizontal net pressing members 12A extending in the X-axis direction and a pair of vertical net pressing members 12B extending in the Y-axis direction. The net pressing member 12 is fixed from the outside in the frame axis direction by pressing bolts 14. As shown in Figure 6, a net moving member 15 is provided between the net frame 11 and the net pressing member 12, which is capable of moving horizontally in the X-axis and Y-axis directions along the resin net 3 together with the net pressing member 12.

[0026] 1 and 6, the net moving member 15 has a frame shape in plan view so as to form a square opening that is approximately the same shape as the net pressing member 12, and is divided into each side that forms the square, just like the net pressing member 12. In other words, the net moving member 15 has a pair of horizontal moving bars 15A extending in the X-axis direction and a pair of vertical moving bars 15B extending in the Y-axis direction. In this way, the mesh frame 11, mesh moving member 15, and mesh pressing member 12 are arranged coaxially with the frame axis O, and overlap in the frame axis direction (Z-axis direction).

[0027] As shown in Figures 2, 3, and 6, the net moving member 15 is clamped between the net frame 11 and the net pressing member 12 by the pressure bolt 14 and fixed in a state where movement in the Z-axis direction is restricted. The net moving member 15 has a bolt hole 15a through which the pressure bolt 14 is inserted. The bolt hole 15a has an inner diameter larger than the bolt diameter of the pressure bolt 14. In other words, the net moving member 15 is horizontally movable in the X-axis direction and the Y-axis direction within the gap between the bolt hole 15a and the pressure bolt 14. Also, as shown in Figure 2, the net moving member 15 is fixed integrally to the net pressing member 12 by a fixing bolt 16. The fixing bolt 16 is shorter than the pressure bolt 14 and has a length that does not reach the net frame 11. In this way, movement of the net moving member 15 in the X-axis direction, Y-axis direction, and Z-axis direction relative to the net pressing member 12 is restricted.

[0028] 6, the outer peripheral edges 3b of the resin nets 3A and 3B are clamped and fixed between the net moving member 15 and the net pressing member 12. That is, the resin nets 3A and 3B are clamped between the net moving member 15 and the net pressing member 12 in a state where they are wrapped around the outside of the net frame 11 and the net moving member 15.

[0029] As shown in FIGS. 1 and 2, the net moving member 15 can move horizontally in the X-axis and Y-axis directions together with the net pressing member 12. Specifically, in the net moving member 15, by moving a pair of horizontal movement bars 15A in the Y-axis direction toward the frame axis O, a tensile force in the Y-axis direction is applied to the resin nets 3A and 3B sandwiched between the net moving member 15 and the net pressing member 12. Furthermore, by moving the vertical movement bar 15B in the X-axis direction toward the frame axis O, a tensile force in the X-axis direction is applied to the resin nets 3A and 3B. The horizontal movement bars 15A and the vertical movement bars 15B can be moved, for example, by a person pushing them with their fingers or by pushing them with a push screw (not shown) provided on the outer frame 21, which will be described later. As a result, the resin nets 3A and 3B are attached without any slack to the mesh frame 11. Furthermore, tension is applied to the resin nets 3A and 3B, so that the gap S between the pair of resin nets 3A and 3B can be set with high precision.

[0030] As shown in FIGS. 1 and 2, the outer frame 21 is formed into a square cylindrical shape when viewed in the frame axis direction. An outer frame presser plate 23, having a square opening 23a in a plan view, is fixed to both ends of the outer frame 21 in the frame axis direction by fixing bolts 24 (24A, 24B). The opening 23a of the outer frame presser plate 23 is coaxial with the opening 11a of the mesh frame 11 and is set to be larger than the opening 11a. The height of the outer frame 21 in the frame axis direction in the internal space 21A matches the length of the mesh structure 10 in the frame axis direction when the gap S between the pair of resin nets 3A and 3B described above is maximum. In other words, in this case, the length of the mesh structure 10 when the gap S is minimum is shorter than the height of the internal space 21A of the outer frame 21, so the mesh structure 10 can move in the frame axis direction (Z-axis direction) in the internal space 21A.

[0031] 2 and 3, of the pair of outer frame retaining plates 23A, 23B, one first outer frame retaining 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 retaining plate 23B is provided so as to be switchable between a fixed position P1 that restricts movement of the resin meshes 3A, 3B in the frame axis direction (Z-axis direction) and a fixed release position P2 that allows movement of the resin meshes 3A, 3B in the frame axis direction (Z-axis direction).

[0032] 2 is a state in which a clearance C is formed between the second outer frame presser plate 23B and the outer frame 21 to ensure a wide gap S between the resin webs 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 resin webs 3A and 3B, and at this time, the positions of the resin webs 3A and 3B are fixed together with the pair of mesh frames 11. In other words, fixed position P1 is the injection position when the resin webs 3A and 3B are positioned with the predetermined gap S maintained and spheroids F are injected into the gap S between the pair of resin webs 3A and 3B.

[0033] The release position P2 shown in FIG. 3 is a state in which the gap adjustment screw 42 of the movement mechanism 40 is loosened in the direction retracting from between the resin meshes 3A and 3B, thereby releasing the restriction for ensuring the gap S between the resin meshes 3A and 3B and abutting the second outer frame retaining plate 23B against the outer frame 21 without any gap between them. At this time, the pair of mesh frames 11, 11, and the resin meshes 3A and 3B are released from their fixed positions. At the release position P2, the resin meshes 3A and 3B can move closer to or away from each other. That is, the release position P2 is a load-applying position where, after injecting spheroids F, the frame axis direction (Z-axis direction) of the cell culture molding device 1 is turned up or down, narrowing the gap between the resin meshes 3A and 3B due to the weight of the upper mesh frame 11 and outer frame 21. This allows a certain load to be applied to the spheroids F, promoting the spheroids F to adhere to each other and become one.

[0034] As shown in FIG. 7, the second outer frame retaining plate 23B is fixed to the other second fixed end 21b of the outer frame 21 by a second fixing bolt 24B via a spacer 25. The second outer frame retaining plate 23B has an insertion hole 23b through which a spacer 25, which has a diameter larger than that of the second fixing bolt 24B, passes. The axial length of the spacer 25 is longer than the axial length of the insertion hole 23b. A clearance C is generated between the second fixed end 21b of the outer frame 21, which is fixed by the second fixing bolt 24B via the spacer 25, and the fixed surface 23c of the second outer frame retaining plate 23B. As shown in FIG. 2, when the gap S between the pair of resin nets 3A and 3B is minimized, the length of the mesh structure 10 along the frame axis direction becomes smaller than the distance between the pair of outer frame retaining plates 23A and 23B (the height of the internal space 21A of the outer frame 21 along the frame axis direction), and the mesh structure 10 becomes movable in the frame axis direction (Z-axis direction).

[0035] As shown in Fig. 4, a tapered injection port 26 for injecting spheroids F is provided on one first side surface 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 resin networks 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 with affinity for living organisms, such as tungsten, is used.

[0036] 8, the movement mechanism 40 is a mechanism that can widen the gap between the meshes when injecting spheroids, etc., 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 resin meshes 3A and 3B apart.

[0037] 2 and 4, the female through-threads 41 into which the gap adjustment screws 42 are threaded are arranged 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 resin meshes 3A and 3B in the Z-axis direction. On each of the second side surface 21d and the third side surface 21e, a pair of female through-threads 41 is provided spaced apart 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 resin meshes 3A and 3B.

[0038] As shown in FIGS. 2 and 8 , the gap adjustment screw 42 has a sharp tip 42a that can enter the gap S between the pair of resin nets 3A and 3B. A hexagonal socket set screw, for example, can be used as the gap adjustment screw 42. The movement mechanism 40 adjusts the size of the gap S by moving the gap adjustment screw 42 back and forth to adjust the insertion dimension of the tip 42a into the gap S. That is, the maximum dimension of the gap S corresponds to the outer diameter of the gap adjustment screw 42. Specifically, the gap S can be widened by tightening the gap adjustment screw 42 to advance it toward the gap S during injection of the spheroids F. Furthermore, the gap S can be narrowed by loosening the gap adjustment screw 42 and retracting it after injection of the spheroids F. Note that narrowing the gap S allows the mesh structure 10 to move in the frame axis direction (Z-axis direction) within the internal space 21A of the outer frame 21.

[0039] As shown in Figure 4, each side surface 21c, 21d, 21e, 21f (see Figure 1) of the outer frame 21 is formed with a female threaded portion 28 into which a push-in screw 27 is threaded to press at least one of the net moving member 15 and the net holding member 12, thereby moving the net moving member 15 and the net holding member 12 horizontally in the X-axis direction or Y-axis direction.

[0040] <<Cell culture sheet manufacturing method>> Next, the procedure for producing a cell culture sheet using the above-described cell culture molding apparatus 1 will be specifically described. 1 to 4, the cell culture molding device 1 is first assembled with the mesh structure 10 housed inside the outer frame structure 20. Then, in the assembled cell culture molding device 1, the gap adjustment screw 42 of the movement mechanism 40 is tightened into the through-hole female screw 41 of the outer frame 21, thereby restricting the gap S between the pair of resin meshes 3A and 3B to a state where it is widened to its maximum.

[0041] Next, with the resin nets 3A and 3B oriented vertically with the injection port 26 facing up (see FIG. 4), the cell culture molding device 1 is introduced into a culture tank (not shown) with the injection port stopper 261 of the injection port 26 open, and a mixture of culture fluid and spheroids F is injected through the injection port 26 into the gap S between the pair of resin nets 3A and 3B. At this time, the outer periphery of the gap S is sealed by the outer frame 21, preventing the injected spheroids F 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, eventually filling the gap S completely with spheroids F.

[0042] After the spheroids F are injected, the gap adjustment screw 42 is loosened and moved back in a direction away from the gap S, thereby releasing the constraint on 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 network structure 10 to move in the frame axis direction (Z-axis direction) within the internal space 21A of the outer frame 21.

[0043] Next, the cell culture molding apparatus 1 is turned sideways in the culture tank so that the weight of the mesh structure 10 is applied to the spheroids F filled in the gap S between the pair of resin meshes 3A and 3B. Specifically, the cell culture molding apparatus 1 is rotated and positioned so that the outer frame presser plate 23, which is movable within 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 applies a certain load (stress) to the spheroids F, promoting them to adhere to each other and become one. If this state is maintained for the number of days (for example, about one day) required for the spheroids F to adhere to each other, the desired cell culture sheet is formed in the cell culture molding apparatus 1.

[0044] The composition of the culture medium is not particularly limited, and a known composition that maintains the vital activity of spheroid F may be used. The culture medium may contain known additives that induce differentiation of each cell that constitutes spheroid F. The temperature of the culture medium is preferably within a temperature range that maintains the vital activity of spheroid F. The pH, CO2 concentration, oxygen concentration, temperature, etc. of the culture medium may be controlled using common culture techniques.

[0045] Next, the outer frame presser plate 23 is removed, and the cell culture sheet is taken out from the gap S in the outer frame 21. Specifically, the pair of mesh structures 10 are taken out from the outer frame 21, and the pair of resin meshes 3A, 3B are removed, thereby obtaining a cell culture sheet of the desired thickness formed in the gap S.

[0046] The number of types of spheroids contained in the cell culture sheet formed by the cell culture molding apparatus 1 may be one or two or more. The number of types of cells constituting the individual spheroids contained in the cell culture sheet may be one or two or more. In the cell culture sheet, the individual spheroids F are adjacent to and adhere to each other along the surface of the sheet. The preferred adhesion form is spontaneous adhesion between the cells present on the spheroid surface, for example, binding to each other via proteins such as fibronectin present on the cell surface.

[0047] <Actions and Effects of the Embodiment> The cell culture molding apparatus 1 of this embodiment creates a sheet-like cell culture sheet by injecting cultured cell spheroids F. The cell culture molding apparatus 1 includes a pair of resin nets 3A, 3B arranged approximately parallel to each other with a gap between them, a mesh frame 11 that supports the outer edges of each of the pair of resin nets 3A, 3B and attaches the resin nets 3, a mesh pressing member 12 that fixes the resin net 3 to the mesh frame 11 while applying tension, a gap plate 13 that is arranged between the pair of resin nets 3A, 3B, a cylindrical outer frame 21 that houses the mesh frame 11 and the mesh pressing member 12 with the mesh surfaces 3a of the pair of resin nets 3A, 3B perpendicular to the frame axis O, and a movement mechanism 40 that moves the pair of resin nets 3A, 3B toward and away from each other within an internal space 21A surrounded by the outer frame 21.

[0048] Therefore, in this embodiment, when injecting the spheroids F, the gap S between the pair of resin nets 3A, 3B can be widened by moving the pair of resin nets 3A, 3B away from each other using the movement mechanism 40, and after injecting the spheroids F, the gap S can be narrowed by moving the pair of resin nets 3A, 3B toward each other using the movement mechanism 40. Then, by narrowing the gap S, the mesh structure 10 can move in the frame axis direction (Z-axis direction) within the internal space 21A of the outer frame 21, and the resin nets 3A, 3B can move in the frame axis direction.

[0049] More specifically, as shown in FIG. 2, by tightening the gap adjustment screw 42 of the moving mechanism 40 in the direction of moving forward toward the resin meshes 3A and 3B, a wide gap S between the resin meshes 3A and 3B can be secured, and spheroids F can be injected into the gap S between the pair of resin meshes 3A and 3B. After the injection of the spheroids F, as shown in FIG. 3, by loosening the gap adjustment screw 42 of the moving mechanism 40 in the direction of moving backward from the resin meshes 3A and 3B, the restriction for securing the gap S between the resin meshes 3A and 3B is released, allowing the resin meshes 3A and 3B to move toward and away from each other. Therefore, by positioning the cell culture molding device 1 so that the movable outer frame 21 in the culture tank is on top after the injection of the spheroids F, a certain load can be applied to the spheroids F, promoting their adhesion and integration. In this way, this embodiment ensures that necessary components are received from the culture medium, improving the quality of the cell culture sheet produced.

[0050] In this embodiment, the resin meshes 3A and 3B have good releasability to cells, so the sheet-like cell culture sheet can be easily removed from the resin meshes 3A and 3B. Furthermore, in this embodiment, deterioration of the resin meshes 3A and 3B due to the culture medium can be suppressed, improving the durability of the mesh as a mold and enabling long-term use. In addition, in this embodiment, a gap plate 13 is provided between the pair of resin nets 3A and 3B, so that the gap S between the pair of resin nets 3A and 3B does not become smaller than a certain gap equal to the thickness of the gap plate 13, thereby preventing the resin nets 3A and 3B from coming into contact with each other.

[0051] The cell culture molding apparatus 1 of this embodiment is provided with a pair of outer frame retaining plates 23 that secure the outer frame 21 from both sides in the frame axial direction and have an opening in the center. One of the pair of outer frame retaining plates 23 is fixed to the outer frame 21, and the other is switchable between a fixed position P1 that restricts movement of the resin meshes 3A and 3B in the frame axial direction and a released position P2 that allows movement of the resin meshes 3A and 3B in the frame axial direction. For this reason, in this embodiment, when injecting spheroids F, the pair of resin nets 3A, 3B can be moved away from each other by the movement mechanism 40 to widen the gap S between the pair of resin nets 3A, 3B to a fixed position P1 shown in Fig. 2, and after injecting spheroids F, the pair of resin nets 3A, 3B can be moved toward each other by the movement mechanism 40 to narrow the gap S to an unlocked position P2 shown in Fig. 3. Then, by narrowing the gap S, the mesh structure 10 can move in the frame axial direction (Z-axis direction) within the internal space 21A of the outer frame 21, and the other of the outer frame presser plates 23 can be positioned at an unlocked position P2 where the resin nets 3A, 3B can be moved in the frame axial direction.

[0052] More specifically, as shown in FIG. 2, the gap adjustment screw 42 of the moving mechanism 40 is tightened in the direction of moving forward toward the resin webs 3A and 3B, thereby widening the gap S between the resin webs 3A and 3B and providing a clearance C between the second outer frame retaining plate 23B and the outer frame 21. At this time, the positions of the resin webs 3A and 3B are fixed together with the pair of mesh frames 11. This positions the resin webs 3A and 3B at fixed position P1, maintaining the predetermined gap S, and spheroids F can be injected into the gap S between the pair of resin webs 3A and 3B. After the injection of spheroids F, as shown in FIG. 3, the gap adjustment screw 42 of the moving mechanism 40 is loosened in the direction of moving backward from the resin webs 3A and 3B, thereby releasing the restriction for ensuring the gap S between the resin webs 3A and 3B and bringing the second outer frame retaining plate 23B into contact with the outer frame 21 without any gaps. At this time, the pair of mesh frames 11, 11 are released from the fixed positions of the resin nets 3A, 3B to a fixed release position P2, and the resin nets 3A, 3B are allowed to approach and separate from each other.

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

[0054] Furthermore, in the cell culture molding device 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.

[0055] Furthermore, in the cell culture molding device 1 of this embodiment, the gap S between the resin networks 3A and 3B into which the spheroids F are injected is less than twice the average outer diameter of the spheroids F. This prevents the spheroids F from overlapping, allowing a high-quality cell culture sheet to be formed.

[0056] Furthermore, in the cell culture molding apparatus 1 of this embodiment, the movement mechanism 40 includes female through-threads 41 (through-holes) 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 female through-threads 41 from the outside of the outer frame 21 and pushes the gap between the pair of resin meshes 3A and 3B apart. Therefore, in the movement mechanism 40, the gap S can be widened by tightening the gap adjustment screw 42 to advance 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 mesh structure 10 to move in the frame axis direction (Z-axis direction) within the internal space 21A of the outer frame 21.

[0057] Furthermore, in the cell culture molding device 1 of this embodiment, the gap adjustment screw 42 has a sharp tip 42a that can enter between the pair of resin nets 3A and 3B. This allows the pair of resin nets 3A and 3B to be reliably spread apart, and also makes it possible to adjust the size of the gap S according to the distance the gap adjustment screw 42 advances into the gap S.

[0058] Furthermore, in the cell culture molding apparatus 1 of this embodiment, a mesh moving member 15 is provided between the mesh frame 11 and the mesh pressing member 12. The mesh moving member 15 is horizontally movable along the mesh surfaces 3a of the resin meshes 3A and 3B together with the mesh pressing member 12. The ends of the resin meshes 3A and 3B are clamped and fixed between the mesh moving member 15 and the mesh pressing member 12. Therefore, by moving the mesh moving member 15 in a direction approaching the frame axis O, a tensile force is applied to the resin meshes 3A and 3B. As a result, tension is applied to the resin meshes 3A and 3B without any slack relative to the mesh frame 11, and the gap S between the pair of resin meshes 3A and 3B can be set with high precision.

[0059] Furthermore, in the cell culture molding apparatus 1 of this embodiment, the outer frame 21 is formed with a female thread portion 28 into which a press screw 27 is threadedly engaged. The press screw 27 presses at least one of the mesh moving member 15 and the mesh pressing member 12 to horizontally move the mesh moving member 15 and the mesh pressing member 12. Therefore, by tightening or loosening the press screw 27 relative to the female thread portion 28, the mesh moving member 15 and the mesh pressing member 12 can be moved horizontally to adjust the tension of the resin mesh 3. In this case, the tension of the resin mesh 3 can be adjusted from outside the outer frame, improving operability.

[0060] Furthermore, in the cell culture molding device 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 over the surrounding area.

[0061] Furthermore, in the cell culture molding device 1 of this embodiment, the mesh size of the resin meshes 3A and 3B is less than half the average outer diameter of the spheroids F, so that the spheroids F can be prevented from jumping outward from the gap S between the pair of resin meshes 3A and 3B.

[0062] Furthermore, in the cell culture molding apparatus 1 of this embodiment, 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 gap plate 13 and the injection port 26 can be suppressed.

[0063] Furthermore, in the cell culture molding apparatus 1 of this embodiment, the resin meshes 3A and 3B are made of a fluorine-based resin that has the ability to peel off cells, so that cells can be reliably peeled off from the resin meshes 3A and 3B, and a higher quality cell culture sheet can be formed.

[0064] Furthermore, in the cell culture molding device 1 of this embodiment, the material of the resin nets 3A and 3B is polytetrafluoroethylene, so that the above-mentioned peeling effect can be more reliably achieved.

[0065] Furthermore, in the cell culture molding apparatus 1 of this embodiment, the resin meshes 3A and 3B have elasticity, and the tension can be adjusted when they are attached to the mesh frame 11. In this way, in this embodiment, since the resin meshes 3A and 3B are elastic and flexible members, when attaching the resin meshes to the mesh frame 11, the degree of attachment can be set by adjusting the tension of the resin meshes 3A and 3B.

[0066] (First Modification) The gap plate 13A according to a first modified example shown in Fig. 9 is for forming a cell culture sheet that is a regular hexagon in plan view. The gap plate 13A has a plate body 133 and an injection port plug 134. That is, the plate body 133 has a recess 133a that is approximately U-shaped in plan view and has a triangular bottom. The injection port plug 134 can be fitted into the recess 133a with a gap therebetween, and has a triangular recessed tip 134a at its tip.

[0067] In the gap plate 13A of the first modification, when the injection port plug 134 is fitted into the recess 133a of the plate body 133, an opening for the culture medium that is regular hexagonal in plan view is formed in the gap plate 13A. In other words, a regular hexagonal cell culture sheet can be produced. In the gap plate 13A used to form a polygonal cell culture sheet other than a square like the first modified example, a load can be applied not only from one direction (vertical direction in FIG. 9) but also from all four directions.

[0068] (Second Modification) The gap plate 13B according to a second modification shown in FIG. 10 is for forming rod-shaped cell culture pieces in a plan view. The gap plate 13B includes a plate body 135 and an inlet plug 136. The plate body 135 has a recess that is approximately U-shaped in a plan view, with multiple elongated slits 135a formed at the bottom. The inlet plug 136 is capable of fitting into the recess, and has a protrusion 136a at its tip that closes the multiple slits 135a. FIG. 10 shows the inlet plug 136 in the process of fitting into the recess of the plate body 135. The width dimension D1 of the slit 135b of the plate body 135 is, for example, 1.2 to 1.5 times the diameter of the spheroid. The width dimension D2 of the protrusion 136b of the inlet plug 136 is the width dimension D1 of the slit 135b minus half the diameter of the spheroid.

[0069] In the gap plate 13B of the second modification, when the injection port plug 136 is fitted into the recess of the plate body 135, an opening for the culture medium, which is slit-shaped in plan view, is formed in the gap plate 13B. That is, by injecting spheroids into this slit 135a, rod-shaped cell culture pieces can be created. Furthermore, in the second modification, there are multiple fine slits 135b into which the spheroids can be inserted, so multiple thin rod-shaped cell culture pieces can be formed simultaneously. In this way, with the gap plate 13B formed in a slit shape according to the second modification, it is possible to create a cell culture piece that is useful for measuring the elasticity of a spheroid, for example.

[0070] (Third Modification) The gap plate 13C according to the third modified example shown in FIG. 11 has the same configuration as the gap plate 13B according to the second modified example described above (see FIG. 10), but the length of the area in the slit 135a into which the spheroids F are introduced (spheroid supply area length L1, described below) is adjustable. The gap plate 13C includes a plate body 135 and an injection port stopper 136. The plate body 135 has a plurality of elongated slits 135a formed in a bottom 135c of a recess 135b that is generally U-shaped in plan view. The slits 135a have an elongated shape extending in the depth direction of the plate body 135 (direction away from the bottom). The inlet plug 136 has an inlet plug body 136b that can fit into the recess 135b, and a protrusion 136a that protrudes from the tip 136c of the inlet plug body 136b, blocks the opening 135d at one end of the longitudinal direction of the multiple slits 135a, and fits into the slit 135a.

[0071] 12, in the gap plate 13C according to the third modification, the depth (slit length L) of the slit 135a in the plate body 135 and the protruding length L2 of the convex portion 136a of the injection port plug 136 are different, thereby adjusting the spheroid supply region length L1 (= L - L2), which is the length of the spheroid F-containing spheroid supply region R (internal region). Specifically, the injection port plug body 136b of the injection port plug 136 is fitted into the concave portion 135b of the plate body 135, and the convex portion 136a is inserted into and fitted into the opening 135d of the spheroid 135a, thereby achieving the desired spheroid supply region length L1. As a result, the spheroid F injected into the spheroid supply region R is compressed by being placed in a region with a spheroid supply region length L1 that is smaller than the dimension of the spheroid F in the compression direction E (here, the length direction of the spheroid 135a) before compression. In this way, in the third modified example, a compressive load can be intentionally applied to the spheroids F injected into the slits 135a, and the spheroids F can be compressed at a desired compression rate. The spheroids F are approximately circular in side view, and when compressed, they become approximately elliptical, with adjacent spheroids F joining together.

[0072] The compression at this time can be applied by the weight of injection port plug 136. The method of applying compression is not limited to the weight of injection port plug 136. Furthermore, the compression time and compression method can be appropriately selected. For example, the spheroids F may be injected into the entire slit 135a and compressed at once by the convex portion 136a, or the injection and compression may be performed in stages, which can increase the uniformity of the compressed spheroids F and prevent them from becoming sparse or dense. Furthermore, in the third modified example, the slit 135a is compressed in one direction, but it is also possible to use a method in which compression is performed from both sides.

[0073] The compression ratio applied to the spheroids F by the gap plate 13C is preferably in the range of, for example, more than 10% and less than 40%, and more preferably 20%. If the compression ratio is greater than this range, the spheroids F will be crushed and overcrowded, which may result in a decrease in quality as a cell mass or increase the possibility that the spheroids F will leak out from the gap between the convex portion 136a and the slit 135a.

[0074] For example, when ten spheroids F with an outer diameter of 0.5 mm are placed into the slit 135a, the total arrangement length of the ten placed spheroids F is 5 mm. In this case, by adjusting the protrusion length L2 of the convex portion 136a to 1 mm and setting the spheroid supplying region length L1 to 4 mm, the spheroids F can be cultured in a state where they are compressed by approximately 20%. Furthermore, by adjusting the protrusion length L2 of the convex portion 136a so that the spheroid supplying region length L1 is 3 mm, the compression rate can be set to approximately 40%. Experiments conducted by actually placing spheroids F confirmed that the elasticity of the cell mass improves depending on the compression rate.

[0075] Next, examples conducted to verify the effects of the cell culture molding device and the method for manufacturing the cell culture piece according to the above-described embodiment will be described below.

[0076] (Example) In the examples, the effects of the resin mesh were confirmed by visually evaluating cell culture sheets (cell culture pieces) obtained in a working case using a resin mesh and a comparative case using a metal mesh (metal mesh) instead of the resin mesh in the cell culture molding device of the above-mentioned embodiment. The cell culture sheets to be evaluated were three sheets each for the working case and the comparative case. The tests using three sheets for the working case using the resin mesh are referred to as tests 1 to 3, and the tests using three sheets for the comparative case using the metal mesh are referred to as tests 4 and 5.

[0077] The evaluation method was to visually evaluate the amount of cell culture sheet adhered to the mesh (resin mesh, metal mesh) in both the experimental and comparative cases (the percentage of adhered area (detached area) relative to the total area). Table 1 shows the evaluation results. The evaluation was based on a two-level rating system: "good" (denoted as "◯" in Table 1) indicates that the peeled area was extremely small, between 0 and 20%, and "poor" (denoted as "×" in Table 1) indicates that the peeled area was small and partial, between 20 and 50%.

[0078] [Table 1]

[0079] As shown in Table 1, in the comparative case, the releasability was evaluated as "×" in all three tests (Tests 4 to 6), which was poor. In the practical case, the releasability was evaluated as "◯" in all three tests (Tests 1 to 3), which was good. From the above results, it was confirmed that the use of a resin mesh results in good peelability. Furthermore, when a metal mesh is used, it becomes difficult to adjust the tension of the mesh, making it difficult to adjust when extra gaps occur in the mesh. On the other hand, when a resin mesh is used, it becomes possible to adjust the tension of the mesh, making it easy to adjust when extra gaps occur in the mesh, and it also has excellent handling properties, such as making it easy to assemble the jig. Therefore, it is preferable to use a resin mesh when creating cell culture sheets.

[0080] The present invention is not limited to the above-described embodiment, and various design modifications are possible without departing from the spirit 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-hole female screw 41 that penetrates 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 resin nets 3A and 3B apart, but this configuration is not limited to this. For example, a pin or other member may be used as the pushing member instead of a screw. The point is that any configuration is sufficient as long as it can adjust the size of the gap S between the pair of resin nets 3A and 3B.

[0081] In addition, in this embodiment, the resin networks 3A and 3B are made of a fluorine-based resin such as polytetrafluoroethylene, which has releasability to cells, but this is not limited to such fluorine-based resins, and other resins may also be used.

[0082] Furthermore, in this embodiment, the resin nets 3A and 3B are made of elastic materials that allow the tension to be adjusted when attached to the mesh frame 11, but there is no limitation to resin nets having such characteristics.

[0083] In addition, in this embodiment, a net moving member 15 is provided between the net frame 11 and the net holding member 12, and is capable of moving horizontally along the net surface 3a of the resin nets 3A and 3B together with the net holding member 12.Furthermore, the ends of the resin nets 3A and 3B are fixed in a clamped state between the net moving member 15 and the net holding member 12.However, this configuration is not limited to applying tension to the resin nets 3A and 3B, and other tension applying means may also be provided.

[0084] It is also possible to omit the push-in screws 27 that press the mesh pressing members of the above-described embodiment and the female threaded portions 28 provided on the outer frame 21 into which the push-in screws 27 are screwed. [Explanation of symbols]

[0085] 1, 1A, 1B, 1C…Cell culture molding device 3, 3A, 3B…Resin net 3a…mesh surface 3b…Outer rim 10...Network structure 11...Net frame 12...Net holding member 12A...Horizontal mesh holding member 13, 13A, 13B, 13C...Gap plates 14...Press bolt 15...Net moving member 16...Fixing bolt 20...Outer frame structure 21...Outer frame 21A…Internal space 23...Outer frame retainer plate 26…Inlet 261...Inlet plug 27...Press screw 28...Female thread 40...Movement mechanism 41... Female thread (through hole) 42...Gap adjustment screw (push-in member) 135...Plate body 135a...Slit 135d...Mouth 136...Inlet plug 136a...Convex part F...spheroid L: Slit length L1: Spheroid supply area length L2…Protrusion length O…Frame axis P1…Fixed position P2…Fixing release position S...gap

Claims

1. A cell culture molding device that creates sheet-shaped or rod-shaped cell culture pieces by injecting spheroids in which cells are cultured, A pair of resin nets arranged at an interval from each other; a mesh frame that supports the outer peripheral edges of each of the pair of resin meshes and attaches the resin meshes; a mesh holding member that fixes the resin mesh to the mesh frame; a gap plate disposed between the pair of resin nets; a cylindrical outer frame that accommodates the mesh frame and the mesh pressing member in a state where the mesh surfaces of the pair of resin meshes are perpendicular to the frame axis; A cell culture molding device comprising: a moving mechanism that moves the pair of resin nets in directions toward and away from each other within an internal space surrounded by the outer frame.

2. a pair of outer frame presser plates each having an opening at the center and fixing the outer frame from both sides in the frame axial direction; The cell culture molding device described in claim 1, wherein 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 resin mesh in the frame axial direction and an unlocked position that allows the resin mesh to move in the frame axial direction.

3. The cell culture molding device according to claim 1 or 2, wherein the thickness of the gap plate is at least half the average outer diameter of the spheroids.

4. The cell culture molding device according to claim 1 or 2, wherein the gaps between the resin meshes into which the spheroids are injected are less than twice the average outer diameter of the spheroids.

5. 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 outside the outer frame to push apart the space between the pair of resin nets.

6. The cell culture molding device according to claim 5 , wherein the pushing member is a gap adjustment screw having a sharp tip portion that can enter between the pair of resin nets.

7. Between the mesh frame and the mesh holding member, a mesh moving member is provided which can move horizontally along the mesh surface of the resin mesh together with the mesh holding member, The cell culture molding device according to claim 1 or 2, wherein an end of the resin net is clamped and fixed between the net moving member and the net pressing member.

8. The cell culture molding device of claim 7, wherein the outer frame has a female threaded portion into which a push-in screw is threaded to press at least one of the mesh moving member and the mesh holding member, thereby moving the mesh moving member and the mesh holding member horizontally.

9. The cell culture molding device according to claim 1 or 2, wherein the outer frame is provided with a tapered injection port for injecting the spheroids.

10. The cell culture molding device according to claim 1 or 2, wherein the opening of the resin mesh is equal to or less than half the average outer diameter of the spheroids.

11. The cell culture molding device according to claim 9 , wherein the gap plate and the injection port, which come into contact with the spheroids, are made of the same metal that has affinity for living organisms.

12. The cell culture molding device according to claim 1 or 2, wherein the resin net is made of a polyolefin resin or a fluorine-based resin that has releasability to the cells.

13. The cell culture molding device according to claim 12, wherein the resin mesh is made of polytetrafluoroethylene.

14. The cell culture molding device according to claim 1 or 2, wherein the resin net has elasticity and the tension when the resin net is attached to the net frame is adjustable.

15. the gap plate comprises a plate body having an elongated slit, and an injection port plug having a protrusion that fits into an opening at one end of the slit in the longitudinal direction, The gap plate is applying a compressive force to the spheroids injected into the internal region of the slit by the protrusion that fits into and closes the slit; The cell culture molding device according to claim 1 or 2, wherein the compressive force is adjustable by adjusting the lengthwise depth of the slits and the protruding length of the convex portions.

16. A method for producing a cell culture strip by injecting a spheroid formed by culturing cells to produce a sheet-shaped or rod-shaped cell culture strip, comprising: disposing a pair of resin nets spaced apart from each other; a step of supporting the outer peripheral edges of each of the pair of resin nets and attaching the resin nets to a net frame; a step of fixing the resin net to the net frame by a net pressing member; disposing a gap plate between the pair of resin nets; A process of moving the pair of resin nets in directions approaching and separating in an internal space surrounded by a cylindrical outer frame that accommodates the net frame and the net pressing member, with the net surfaces of the pair of resin nets orthogonal to the frame axis; A method for producing a cell culture piece, comprising:

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