Cell culture molding apparatus, cell culture molding system, and method of producing cell culture sheet

Abstract

To make it possible that a metal or resin mold is filled with only spheroids without using a scaffold material such as gel, and a cell culture sheet is cleanly peeled from the mold, with the result that the quality of the produced cell culture sheet is improved.SOLUTION: Provided is a cell culture molding apparatus that includes: a cylindrical molding portion 20 having a cylindrical accommodating portion side 20A which accommodates spheroids F; and a pair of weight bodies 32 that are disposed on both sides of the accommodating portion side 20A in a z-axis direction and are capable of reciprocating in the z-axis direction inside the accommodating portion side 20A; wherein the cylindrical molding portion 20 includes a cylindrical shaft 21 and an outer cylinder 22 which is provided on a radially outer side of the cylindrical shaft 21 and forms the accommodating portion side 20A between the cylindrical shaft 21 and the outer cylinder 22, the outer cylinder 22 has a culture solution passing hole 20A which communicates with the accommodating portion side 20A and allows a culture solution to pass therethrough from the outside of the outer cylinder 22 to the inside of the accommodating portion side to replenish the culture solution, and a compressive force is applied in the z-axis direction by the pair of weight bodies 32 which reciprocate in the accommodating portion side 20A.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a cell culture molding device, a cell culture molding system, 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, a cell culture molding system, and a method for manufacturing a cell culture sheet that can fill a metal or resin mold with only spheroids without using a scaffold material such as gel, and then cleanly peel off the cell culture sheet from the mold, thereby improving the quality of the cell culture sheet to be created. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention proposes the following means. A cell culture molding apparatus according to a first aspect of the present invention is a cell culture molding apparatus that creates a cylindrical cell culture sheet by injecting cultured cell spheroids, and includes a cylindrical forming part having a cylindrical storage part that stores the spheroids, and a cone that is arranged on at least one side of the storage part in the cylindrical axis direction and can move back and forth in the cylindrical axis direction inside the storage part, and the cylindrical forming part includes a cylindrical axis and an outer tube that is provided radially outside the cylindrical axis and forms the storage part between itself and the cylindrical axis, and the outer tube has a culture medium passing hole that communicates with the storage part and passes culture medium from the outside of the outer tube to the inside of the storage part to replenish it, and the cone that moves back and forth in the storage part applies a compressive force in the cylindrical axis direction.

[0010] A cell culture molding system according to a second aspect of the present invention is a cell culture molding system equipped with the above-mentioned cell culture molding device, and includes the cell culture molding device, a culture medium tank that houses the cell culture molding device and stores culture medium, and a reciprocating movement mechanism that moves the weight back and forth relative to the cell culture molding device inside the culture medium tank.

[0011] A manufacturing method for a cell culture sheet according to a third aspect of the present invention is a manufacturing method for a cell culture sheet that includes the above-mentioned cell culture molding device and creates a sheet-shaped cell culture sheet by injecting spheroids in which cells are cultured, and includes the steps of: injecting the spheroids into the storage portion of the cylindrical formation part; moving the cones arranged on both sides of the storage portion in the cylindrical axis direction back and forth in the cylindrical axis direction, and applying a compressive force to the spheroids in the storage portion in the cylindrical axis direction by the cones; and during culture with the cones moving back and forth, passing the culture medium through the culture medium passing hole of the outer tube to replenish the inside of the storage portion. Effect of the Invention

[0012] According to the cell culture molding device, cell culture molding system, and cell culture sheet manufacturing method of the present invention, without using a scaffolding material such as gel, only spheroids can be filled into a metal or resin mold, and the cell culture sheet can be cleanly peeled off from the mold, improving the quality of the cell culture sheet produced. [Brief description of the drawings]

[0013] [Figure 1] 1 is a perspective view showing a cell culture molding system according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a perspective view showing the configuration of a culture molding device. [Diagram 3] FIG. 2 is a longitudinal cross-sectional view of the culture molding device as viewed from the front and rear directions. [Figure 4] FIG. [Diagram 5] An enlarged view of area A in Figure 4. [Figure 6] FIG. [Figure 7] FIG. [Figure 8] FIG. 2 is a longitudinal cross-sectional view showing a state in which spheroids are injected into a container in the culture molding device. [Figure 9] 1A and 1B are diagrams illustrating a state in which a weight moves from top to bottom, where (a) is a diagram of the weight moving, and (b) is a diagram of the weight moving downward. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 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.

[0015] An overview of the cell culture molding apparatus 1 according to this embodiment is shown in FIG. 1. The cell culture molding apparatus 1 of this embodiment is intended as a food manufacturing apparatus for manufacturing cultured foods using artificially cultured cells. First, desired cells 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 multiple spheroids F are formed into a cylindrical shape. The cell culture sheet produced by the cell culture molding apparatus 1 is then appropriately processed, for example, by stacking a desired number of sheets or changing the cylindrical shape into a flat shape, to produce a cell culture food. The cell culture molding apparatus 1 for obtaining a cell culture sheet will be specifically described below.

[0016] <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.

[0017] <Cell culture molding system> 1, the cell culture molding apparatus 1 of this embodiment is included in a cell culture molding system 10. That is, the cell culture molding system 10 includes the above-mentioned cell culture molding apparatus 1, a culture solution tank 11 that houses the cell culture molding apparatus 1 and stores a culture solution E, and a reciprocating mechanism 12 that reciprocates a weight (described later) relative to the cell culture molding apparatus 1 inside the culture solution tank 11.

[0018] The culture solution tank 11 is provided with a size and shape that ensures an area in which the cell culture molding apparatus 1 can rotate around a predetermined rotation axis J and an area in which a part of the reciprocating mechanism 12 can be accommodated, and that allows sufficient storage of the culture solution E. In this embodiment, the culture solution tank 11 has a cubic shape. The culture solution E is stored in an amount that allows at least the storage section 20A (see FIG. 3) of the cell culture molding apparatus 1 rotating inside the culture solution tank 11 to be constantly immersed in the culture solution E.

[0019] The composition of the culture medium E is not particularly limited, and a known composition that maintains the vital activity of the spheroid F may be used. The culture medium E may contain a known additive that induces the differentiation of each cell that constitutes the spheroid F. The temperature of the culture medium E is preferably within a temperature 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.

[0020] ≪Reciprocating mechanism≫ The reciprocating mechanism 12 has a function of reciprocating the weight 32 (see Figs. 2 and 3) by rotating the cell culture molding apparatus 1 in the culture solution E around a central axis (rotation axis J) extending in the horizontal direction. The reciprocating mechanism 12 includes a holding plate 13 having a U-shape in a top view and holding the center of the cell culture molding apparatus 1, a first gear 14 that is coaxial with the rotation axis J and fixed by a bolt or the like to a plate surface of the holding plate 13 perpendicular to the rotation axis J, a second gear 15 that meshes with the first gear 14, a drive motor 16 with a rotation shaft 16a fixed to the center of the second gear 15, and a pair of support legs 17A and 17B that rotatably support the rotation shaft 16a inside and outside the culture solution tank 11.

[0021] The pair of holding plates 13 are plate-shaped and fixed in a state in which the cylindrical forming portion 20 is sandwiched between them on both sides of the rotation axis J. The cylindrical forming portion 20 is rotatable around the rotation axis J together with the pair of holding plates 13.

[0022] The rotating shaft 16a penetrates the upper side wall of the culture medium tank 11, and the drive motor 16 is disposed outside the culture medium tank 11. The second gear 15, the first gear 14, and the holding plate 13 provided at the tip of the rotating shaft 16a are disposed inside the culture medium tank 11. The first support leg 17A is disposed outside the culture medium tank 11, and supports the drive motor 16 together with the rotating shaft 16a. The second support leg 17B is disposed inside the culture medium tank 11, and rotatably supports the tip of the rotating shaft 16a and the rotating shaft 14a of the first gear 14.

[0023] By the rotational drive of the drive motor 16, the rotational power is transmitted from the rotating shaft 16a to the second gear 15 and the first gear 14, and the cell culture molding device 1 rotates around the rotation axis J in the culture solution E via the holding plate 13 together with the first gear 14. That is, the cylindrical forming part 20 is provided so as to be upside down by the reciprocating mechanism 12.

[0024] ≪Cell culture molding device≫ As shown in Figures 2 and 3, the cell culture molding apparatus 1 comprises a cylindrical formation part 20 having a cylindrical storage section 20A for storing spheroids F, a pair of movable parts 30 (30A, 30B) arranged on both sides of the storage section 20A in the direction of the cylindrical axis O and having a weight 32 that can move back and forth in the direction of the cylindrical axis O in the storage section 20A, and an outer shell body 40 that integrally incorporates the cylindrical formation part 20 and the movable part 30.

[0025] In the following description, the direction of the cylinder axis O of the cylindrical forming part 20 is referred to as the z-axis direction, the direction perpendicular to the z-axis direction is referred to as the radial direction, and the direction going around the z-axis as viewed from the z-axis direction is referred to as the circumferential direction. The cylindrical forming part 20 performs cell culture with the z-axis direction (cylinder axis O) facing up and down. In addition, in the following description, the direction in which the outer shell 40 is sandwiched between the supports 40A and 40B described below is referred to as the x-axis direction (or width direction x), and the direction perpendicular to the x-axis direction when viewed from the z-axis direction is referred to as the y-axis direction (or front-to-back direction y).

[0026] <External shell> 2 and 3, the outer shell 40 has a pair of supports 40A, 40B each having a rectangular cross section extending in one direction. The supports 40A, 40B are arranged parallel to each other with a gap therebetween, sandwiching the cylindrical forming portion 20 from the left and right. The length of the supports 40A, 40B in the z-axis direction is set to be longer than the length of the cylindrical forming portion 20 in the z-axis direction.

[0027] 3, the opposing surfaces 40a of the pair of supports 40A, 40B are each formed with a locking portion 41 for locking the cylindrically formed portion 20. The locking portion 41 protrudes in a direction perpendicular to the opposing surface 40a. The locking portion 41 has a first locking portion 41A for locking one end (first end surface 20a) of the cylindrically formed portion 20 from the z-axis direction, and a second locking portion 41B for locking a side surface (first side surface 20c) of the cylindrically formed portion 20 in the front-rear direction y from the width direction x.

[0028] Both end faces (first end face 40b and second end face 40c) of the supports 40A and 40B in the z-axis direction are provided with female threaded holes 42 for fixing the movable part 30. The pair of supports 40A and 40B arranged as the cell culture molding device 1 have female threaded holes 42 provided at intervals in the width direction x, respectively. The tip of a guide shaft 31 of the movable part 30, which will be described later, is screwed into the female threaded hole 42.

[0029] A screw hole 43 is provided in the axial center of the supports 40A and 40B on the surface (outer surface 40d) opposite to the opposing surface 40a. The holding plate 13 of the reciprocating mechanism 12 shown in FIG.

[0030] A connecting plate 44 that holds the supports 40A, 40B at a fixed distance in the width direction X1 is fixed to the front side surface 40e and the rear side surface 40f of the supports 40A, 40B with screw members 44A. An opening 44a is formed in the center of the connecting plate 44. Through the opening 44a of the connecting plate 44, the culture solution E can easily flow to the outer circumferential surface of the cylindrical formation portion 20 disposed between the supports 40A, 40B.

[0031] <Cylindrical forming section> 3 and 4, the cylindrical forming part 20 includes a cylindrical shaft 21, an outer tube 22 that is provided radially outward of the cylindrical shaft 21 and forms a storage part 20A between the cylindrical shaft 21, and a protrusion 23 that protrudes radially outward from both ends in the z-axis direction of the outer tube 22. As described above, the cylindrical forming part 20 is provided to be rotatable about a direction perpendicular to the cylindrical axis O by the reciprocating mechanism 12 (see FIG. 1).

[0032] The cylindrical shaft 21 and the outer cylinder 22 in the cylindrical forming portion 20 are made of a metal having affinity for the living body, such as tungsten, or a resin, and are members that can be easily sterilized and have excellent durability.

[0033] The cylindrical shaft 21 is hollow or solid, has a circular cross section, and has the same diameter over the entire length. The cylindrical shaft 21 is arranged coaxially with the outer tube 22. When the cylindrical shaft 21 is inserted into the outer tube 22, the fitting protrusions 21a at both ends in the z-axis direction are fixed to the fixing plates 33 (described later) of the movable part 30. That is, the cylindrical shaft 21 is longer than the outer tube 22, and both ends of the cylindrical shaft 21 in the z-axis direction protrude outward from the outer tube 22 by the same length. In this embodiment, the cylindrical shaft 21 is divided into two in the z-axis direction, but it is sufficient that the cylindrical shaft 21 is provided coaxially in the z-axis direction. It may be a single piece, or may be divided into three or more parts in the z-axis direction.

[0034] The outer diameter φ3 of the cylindrical shaft 21 is smaller than the inner diameter φ1 of the outer tube 22 (see FIG. 7). A cylindrical storage section 20A is formed having a gap S(φ1-φ3) / 2 shown in FIG. 7 in the radial direction between the cylindrical shaft 21 and the outer tube 22. The gap S corresponds to the diameter dimension of the cell mass (spheroid F) formed in the storage section 20A, i.e., the thickness of the cylindrical cell culture sheet.

[0035] As shown in Fig. 3, the movable cylinders 35 of the weights 32 of the pair of movable parts 30 (30A, 30B) are provided in the storage section 20A between the cylindrical shaft 21 and the outer cylinder 22 so as to be capable of reciprocating in the z-axis direction (see Fig. 6). The first movable cylinder 35A of the first movable part 30A is inserted into the storage section 20A from one side in the z-axis direction (the upper side in Fig. 3), and the second movable cylinder 35B of the second movable part 30B is inserted from the other side (the lower side in Fig. 3). The storage section 20A is a region in which cell clusters (spheroids F) are filled and arranged (see Fig. 7).

[0036] As shown in FIG. 4, the outer cylinder 22 is a generally cylindrical shape with an inner circumferential shape having a circular cross section and an outer circumferential shape having a hexagonal cross section. The outer circumferential shape is not limited to a hexagonal cross section, and may be a circle or another rectangular cross section. The upper end 22a and the lower end 22b of the outer cylinder 22 have protrusions 23 that protrude in both directions away from the cylinder axis O in the x-axis direction. As shown in FIG. 3, the protrusions 23 are engaged with the engagement portions 41 of the supports 40A and 40B of the outer shell 40, so that the outer cylinder 22 is fixed in a state of being sandwiched between the pair of supports 40A and 40B.

[0037] As shown in FIG. 4 and FIG. 5, the outer cylinder 22 has a number of culture medium passing holes 22c that communicate with the storage section 20A and pass the culture medium E from the outside of the outer cylinder 22 to the inside of the storage section 20A to replenish it. The number of culture medium passing holes 22c are provided almost uniformly on almost the entire surface of the outer cylinder 22. Specifically, the number of culture medium passing holes 22c are arranged vertically and horizontally. The interval P between the culture medium passing holes 22c adjacent vertically and horizontally is set to be the same distance both vertically and horizontally. This allows the culture medium E to pass through the outer cylinder 22 uniformly and be supplied to the storage section 20A inside. Furthermore, by making the interval P between the culture medium passing holes 22c equal to or less than the diameter dimension of the spheroid F, all cell clusters (spheroids F) arranged in the storage section 20A can be constantly in contact with the culture medium E.

[0038] Furthermore, the hole diameter D (see FIG. 7) of the culture medium passing hole 22c is smaller than the radial width dimension (gap S) of the accommodating section 20A, and is preferably set to ½ or less of the radial width dimension (gap S) of the accommodating section 20A. When the diameter of the cell cluster is d and the hole diameter D of the culture medium passing holes 22c is d / 5 to d / 3, it is preferable that the distance between the culture medium passing holes 22c is equal to or less than P. By setting the hole diameter D to d / 5 or more, it is possible to prevent air bubbles from getting into the hole diameter D. Furthermore, by setting the hole diameter D to d / 3 or less, it is possible to prevent the moving cell cluster (spheroid F) from jumping out.

[0039] ≪Movable part≫ 2, 3 and 6, the movable part 30 (30A, 30B) includes a pair of guide shafts 31 fixed to the outer shell 40 and extending in the z-axis direction, a weight 32 that moves while being guided by the pair of guide shafts 31, and a fixed plate 33 that fixes the guide shafts 31 and the cylindrical shaft 21 of the cylindrical forming part 20. The movable parts 30 are disposed on both sides of the cylindrical forming part 20 in the z-axis direction, one of which is a first movable part 30A and the other is a second movable part 30B. In the cell culture molding apparatus 1, the cylinder axis O is inverted up and down by rotation by the reciprocating mechanism 12 (see FIG. 1), so that the cone 32 reciprocates up and down (in the z-axis direction).

[0040] As shown in FIG. 3, a pair of guide shafts 31 are provided corresponding to each of the pair of supports 40A, 40B. The guide shaft 31 is hollow, cylindrical, and has a circular cross section, and has the same diameter over its entire length. A shaft portion 333a of a screw member 333 is inserted into the guide shaft 31. The screw member 333 has a male screw 333b at the tip of the shaft portion 333a which is screwed into a female screw hole 42 of the outer shell body 40, and is fastened together with the fixing plate 33. As a result, the guide shaft 31 is attached in a state parallel to the cylinder axis O, that is, extending in the z-axis direction. The pair of guide shafts 31 are arranged at positions symmetrical on both sides in the x-axis direction across the cylindrical axis 21.

[0041] The fixed plate 33 fixes the pair of guide shafts 31 and the cylindrical shaft 21 in parallel along the z-axis direction. The fixed plate 33 has a pair of screw holes 331 spaced apart from each other, and a fitting hole 332 penetrating the plate in the plate thickness direction at the center of the pair of screw holes 331. The center distance between the pair of screw holes 331 matches the distance between the central axes of the pair of guide shafts 31. The fitting protrusion 21a of the cylindrical shaft 21 is fitted into the fitting hole 332 from the inside in the z-axis direction. The cylindrical shaft 21 may be either fixed or unfixed with respect to the fitting hole 332.

[0042] 3 and 6, the weight body 32 has a weight body 34 and a movable cylinder 35. The weight body 34 is a member having a predetermined thickness and weight. The weight body 34 has a pair of guide holes 34a penetrating in the thickness direction (z-axis direction) and through which the pair of guide shafts 31 are inserted. The weight body 34 is capable of reciprocating movement in the z-axis direction along the guide shafts 31.

[0043] A movable cylinder 35 is fixed to the center between the pair of guide holes 34a of the weight body 34. The movable cylinder 35 is provided so as to protrude from the surface 34b of the weight body 34 facing the cylindrical forming part 20 side, and is movably fitted to the outside of the cylindrical shaft 21. One end (base end 35a) of the movable cylinder 35 is fixed to the weight body 34, and the other end (pressing end 35b) is provided in a state of being inserted into the storage part 20A of the cylindrical forming part 20. The movable cylinder 35 can reciprocate in the z-axis direction together with the weight body 34. The first movable cylinder 35A of the first movable part 30A is inserted into the storage part 20A from one side in the z-axis direction (upper side in FIG. 3), and the second movable cylinder 35B of the second movable part 30B is inserted from the other side (lower side in FIG. 3).

[0044] When the weight body 34 of the movable part 30 is located above the cylindrically formed part 20, the weight body 32 moves downward due to its own weight (see FIGS. 9(a) and 9(b)). That is, the pressing end 35b of the movable cylinder 35 moves in the direction in which the movable cylinder 35 advances in the storage part 20A. On the other hand, when the weight body 34 is located below the cylindrically formed part 20, the weight body 32 moves downward due to its own weight. That is, the pressing end 35b of the movable cylinder 35 moves in the direction in which the movable cylinder 35 retreats from the storage part 20A.

[0045] As shown in FIG. 7, the relationship between the inner diameter φ1 of the outer cylinder 22, the outer diameter φ2 of the movable cylinder 35 of the weight 32, and the hole diameter D of the culture medium passing hole 22c satisfies the range of formula (1).

[0046]

number

[0047] Further, the relationship between the outer diameter φ3 of the cylindrical shaft 21, the inner diameter φ4 of the movable cylinder 35 of the weight 32, and the hole diameter D of the culture medium passing hole 22c satisfies the range of formula (2).

[0048]

number

[0049] In this manner, the cell culture molding apparatus 1 is configured such that the spheroid F is placed in the storage section 20A, and a compressive force is applied to the spheroid F by the weight 32 of the movable section 30 moving in the z-axis direction.

[0050] <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 Fig. 8, the cylindrical forming part 20, the first movable part 30A, and the outer shell 40 are assembled. That is, the first movable part 30A is attached to only one side of the cylindrical forming part 20 in the z-axis direction, and the first movable part 30A is arranged so that it is on the lower side. Then, a funnel 50 for introducing the spheroids F into the storage part 20A is arranged on the other side of the cylindrical forming part 20 in the z-axis direction. The funnel 50 has a mortar-shaped injection port 50a that communicates with the outer cylinder 22 of the cylindrical forming part 20.

[0051] Spheroids F are placed into the injection port 50a of this funnel 50, and the spheroids F are injected into the storage section 20A formed between the outer cylinder 22 and the cylindrical shaft 21. When the spheroids F are injected, the injection liquid flows down within the storage section 20A according to the direction of gravity, and the spheroids F gradually pile up in the storage section 20A, until the storage section 20A is filled with the spheroids F to the point where it is completely filled. In the storage section 20A, the hole diameter D (see FIG. 7) of the culture medium passing hole 22c of the outer tube 22 is smaller than the gap S of the storage section 20A. Therefore, the spheroids F having an average outer diameter equivalent to the gap S are prevented from leaking out of the culture medium passing hole 22c.

[0052] As shown in Fig. 3, after the injection of the spheroids F into the storage section 20A is completed, the funnel 50 is removed, and the second movable section 30B is attached to the other side in the z-axis direction of the cylindrical forming section 20. Note that Fig. 3 shows a state in which the second movable section 30B is facing downward.

[0053] Next, as shown in Fig. 1, the cell culture molding apparatus 1 with the spheroids F injected into the storage section 20A is attached to the reciprocating mechanism 12 and immersed in the culture solution E in the culture solution tank 11. This allows the cell culture molding apparatus 1 to rotate around the rotation axis J in the culture solution E by the rotation drive of the drive motor 16 of the reciprocating mechanism 12, and to be inverted up and down in the z-axis direction. With the storage section 20A immersed in the culture solution E, the culture solution E passes through the culture solution passing hole 22c of the outer cylinder 22 (see Fig. 4) to replenish the interior of the storage section 20A.

[0054] Next, as shown in Figures 9(a) and (b), the weights 32 arranged on both sides in the z-axis direction inside the storage section 20A are moved back and forth in the z-axis direction, and the weights 32 apply a compressive force in the z-axis direction to the spheroid F inside the storage section 20A. Specifically, as shown in FIG. 1, the cell culture molding device 1 is rotated around the rotation axis J by the rotation drive of the drive motor 16 of the reciprocating movement mechanism 12 so that, for example, the second movable part 30B located on the lower side as shown in FIG. 3 is located on the upper side as shown in FIG. 9(a), and is turned upside down. As a result, as shown in FIGS. 9(a) and (b), the weight of the weight body 32 of the cylindrical forming part 20 is guided by the guide shaft 31 and moves downward, and the upper second movable cylinder 35B and the lower first movable cylinder 35A, which are integrally provided with the weight body 34, move downward along the cylindrical axis 21. At this time, the arranged cell clusters (spheroids F) also move downward together with the upper and lower movable cylinders 35A and 35B in the storage part 20A. Furthermore, at this time, the weight body 32 having the weight body 34 moves, so that a desired compressive force is applied to the spheroids F in the storage part 20A.

[0055] Furthermore, when the cell culture molding apparatus 1 is turned upside down, the first movable part 30A is located above the cylindrical forming part 20, and the second movable part 30B is located below, as described above. Since the weights 32 of the movable parts 30A and 30B are located above the cylindrical shaft 21, they are guided by the guide shaft 31 by the weights of the weight bodies 34 and move downward. At this time, the cell clusters (spheroids F) arranged in the storage part 20A also move downward together with the upper and lower movable cylinders 35A and 35B.

[0056] By continuing in this state for a number of days (eg, 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.

[0057] Next, the cell culture molding device 1 is removed from the reciprocating mechanism 12, and the cylindrical forming part 20, the movable parts 30A and 30B, and the outer shell 40 are disassembled to remove the cylindrical forming part 20. Then, the cylindrical shaft 21 is removed from the outer tube 22 to remove the cylindrical cell culture sheet formed in the storage part 20A, thereby obtaining the manufactured cell culture sheet.

[0058] 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 addition, in the cylindrical cell culture sheet, each spheroid F can be flatly stretched in the surface direction of the sheet, and multiple sheets can be stacked and adhered to each other to produce the sheet. In this case, 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 bond to each other via a protein such as fibronectin present on the cell surface.

[0059] <Actions and Effects of the Embodiment> The cell culture molding apparatus 1 of this embodiment creates a cylindrical cell culture sheet by injecting spheroids F obtained by culturing cells. The cell culture molding apparatus 1 includes a cylindrical forming part 20 having a cylindrical storage part 20A for storing spheroids F, and cones 32 arranged on both sides of the storage part 20A in the z-axis direction and capable of reciprocating in the z-axis direction inside the storage part 20A. The cylindrical forming part 20 includes a cylindrical shaft 21 and an outer cylinder 22 provided on the radial outside of the cylindrical shaft 21 and forming the storage part 20A between the cylindrical shaft 21. The outer cylinder 22 has a culture medium passing hole 22c that communicates with the storage part 20A and passes the culture medium E from the outside of the outer cylinder 22 to the inside of the storage part 20A to replenish it. A compressive force is applied in the z-axis direction by a pair of cones 32 that reciprocate in the storage part 20A.

[0060] In this embodiment, after the spheroid F is placed in the storage section 20A of the cylindrical formation section 20, the weight of the weight 32 of the movable section 30 moves back and forth in the z-axis direction. At this time, the weight of the weight 34 moves the weight of the weight 32 in the z-axis direction, and the arranged cell mass (spheroid F) also moves in the z-axis direction together with the weight 32 in the storage section 20A. Therefore, a desired compressive force is applied to the spheroid F in the storage section 20A by the weight of the weight 32. By repeating the reciprocating movement of the weight 32 in the z-axis direction in the storage section 20A in this manner, a certain load (stress) can be applied to the spheroid F, and the spheroids F are promoted to stick to each other and become integrated. During the culture of the spheroid F, the storage position of the spheroid F in the storage section 20A also moves back and forth in the z-axis direction together with the weight 32, so that the spheroid F can be cultured without contacting the cylindrical shaft 21 and the outer tube 22 for a long time. Therefore, in this embodiment, without using a scaffolding material such as gel, adhesion of spheroids F to the cylindrical axis 21 and outer tube 22 can be suppressed, and the created cell culture sheet can be cleanly peeled off from the cylindrical formation portion 20, thereby improving the quality of the created cell culture sheet.

[0061] Furthermore, in this embodiment, since the reciprocating mechanism 12 that reciprocates the weight 32 is provided, the weight 32 can be reciprocated efficiently.

[0062] Furthermore, in this embodiment, the cylindrical forming part 20 is provided to be rotatable around a rotation axis J perpendicular to the z-axis direction. By turning the cylindrical forming part 20, which has the z-axis direction facing the up-down direction, up and down, the weight 32 moves back and forth in the up-down direction. Therefore, by rotating the cylindrical forming part 20 around the rotation axis J and repeatedly turning it upside down, the weight 32 can be easily moved back and forth in the z-axis direction. That is, by providing a simple rotation mechanism, a structure for moving the weight 32 back and forth can be realized. In addition, in this case, the surrounding culture solution E can be stirred by the rotating cylindrical forming part 20, and the uniformity of the culture solution E can be maintained.

[0063] In addition, in the cell culture molding device 1 of this embodiment, the hole diameter D of the culture medium passing hole 22c is smaller than the radial width dimension (gap S) of the storage section 20A. Therefore, in this embodiment, it is possible to prevent the spheroids F in the storage section 20A from flowing out from the culture medium passing hole 22c, and to suppress air bubbles mixed in the culture medium E from flowing into the storage section 20A from the culture medium passing hole 22c.

[0064] In addition, in the cell culture molding device 1 of this embodiment, the hole diameter D of the culture medium passing hole 22c is equal to or less than 1 / 2 of the radial width dimension (gap S) of the storage section 20A. This makes it possible to more reliably prevent the spheroids F in the storage section 20A from flowing out from the culture medium passing hole 22c, and further suppresses air bubbles mixed in the culture medium E from flowing into the storage section 20A through the culture medium passing hole 22c.

[0065] In the cell culture molding apparatus 1 of this embodiment, the relationship between the inner diameter φ1 of the outer cylinder 22, the outer diameter φ2 of the weight 32, and the hole diameter D of the culture medium passing hole 22c satisfies the range of the above formula (1). Therefore, the spheroids F in the storage section 20A can be prevented from jumping out of the outer cylinder 22.

[0066] In the cell culture molding apparatus 1 of this embodiment, the relationship between the outer diameter φ3 of the cylindrical shaft 21, the inner diameter φ4 of the weight 32, and the hole diameter D of the culture medium passing hole 22c satisfies the range of the above formula (2). Therefore, the spheroids F in the storage section 20A can be prevented from jumping outward from the outer cylinder 22.

[0067] Furthermore, in the cell culture molding apparatus 1 of this embodiment, at least the cylindrical shaft 21 and outer tube 22 in the cylindrical formation section 20 are made of metal or resin that has affinity for the living body, so that the cylindrical shaft 21 and outer tube 22 that come into contact with the cell mass (spheroid F) can be prevented from causing harm to the living body.

[0068] Moreover, the cell culture molding system 10 of this embodiment includes the above-mentioned cell culture molding device 1. The cell culture molding system 10 includes the cell culture molding device 1, a culture fluid tank 11 that houses the cell culture molding device 1 and stores the culture fluid E, and a reciprocating movement mechanism 12 that reciprocates the weight 32 relative to the cell culture molding device 1 inside the culture fluid tank 11. In this embodiment, the weight of the weight body 32 of the cell culture molding apparatus 1 accommodated in the culture solution E stored in the culture solution tank 11 can be reciprocated in the z-axis direction by the reciprocating mechanism 12. As a result, the weight of the weight body 34 moves the weight body 32 in the z-axis direction, and the cell mass (spheroid F) arranged in the storage unit 20A also moves together with the weight body 32 in the z-axis direction, and the weight of the weight body 32 can apply a desired compressive force to the spheroid F in the storage unit 20A.

[0069] 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, a structure is adopted in which the reciprocating mechanism 12 is used to rotate the weight 32 of the cylindrical forming unit 20 around the rotation axis J as a drive device for reciprocating the weight 32, but the present invention is not limited to such a structure in which the weight 32 is reciprocated by a rotary drive. For example, the weight 32 may be reciprocated by a linearly moving telescopic cylinder.

[0070] The direction of reciprocating movement of the weight 32 (z-axis direction) is not limited to the up-down direction as in this embodiment. For example, the direction of reciprocating movement of the weight 32 may be oriented sideways (horizontally).

[0071] In addition, in the present embodiment, an example is shown in which weight 32 is disposed on both sides of storage section 20A in the cylindrical axis direction, but this is not limited to both sides of storage section 20A, and a configuration in which the weight is disposed on either side of storage section 20A is also possible. Even if the weight is provided on one side in this manner, the weight can reciprocate in the cylindrical axis direction inside the storage section.

[0072] Furthermore, the shape, size, and other configurations of each of the cylindrical forming portion 20, the movable portion 30, and the outer shell 40 can be changed as appropriate. [Explanation of symbols]

[0073] 1...Cell culture molding device 10. Cell culture molding system 11...Culture solution tank 12...Reciprocating mechanism 16...Drive motor 20...Cylindrical forming section 20A…Housing section 21...Cylindrical shaft 22…Outer cylinder 22c…Culture solution passage hole 30, 30A, 30B...Movable part 31...Guide shaft 32...pyramid 33…Fixing plate 34…Sinker body 35, 35A, 35B...Movable tube 40…Exoshell 40A, 40B...Support E…Culture solution F…Spheroid J…Rotation axis O…Cylinder shaft

Claims

1. A cell culture molding device that creates a cylindrical cell culture sheet by injecting cultured cell spheroids, a cylindrical forming part having a cylindrical container part for accommodating the spheroids; a weight body that is disposed on at least one side of the housing portion in the cylindrical axis direction and is capable of reciprocating in the housing portion in the cylindrical axis direction, The cylindrical forming portion is A cylindrical shaft; an outer cylinder provided radially outward of the cylindrical shaft and defining the accommodation portion between the outer cylinder and the cylindrical shaft; the outer cylinder has a culture medium passing hole that communicates with the storage portion and passes a culture medium from the outside of the outer cylinder to the inside of the storage portion to replenish the culture medium; The cell culture molding device applies a compressive force in the cylindrical axis direction by the weight that reciprocates in the storage section.

2. The cell culture molding device according to claim 1 , wherein the weights are disposed on both sides of the storage portion in the cylindrical axis direction.

3. The cell culture molding apparatus according to claim 1 , further comprising a reciprocating mechanism for reciprocating the weight.

4. The cylindrical forming portion is provided rotatably about a rotation axis perpendicular to the cylinder axis direction, The cell culture molding apparatus according to claim 1 , wherein the cylindrical forming part is inverted up and down such that the cylinder axis direction is oriented in a vertical direction, thereby causing the cone to reciprocate in the vertical direction.

5. The cell culture molding device according to claim 1 , wherein a hole diameter of the culture medium passing hole is smaller than a radial width dimension of the storage portion.

6. The cell culture molding device according to claim 5 , wherein the hole diameter of the culture medium passing hole is equal to or smaller than ½ of a radial width dimension of the storage portion.

7. The cell culture molding device according to claim 1 , wherein a relationship between an inner diameter φ1 of the outer cylinder, an outer diameter φ2 of the cone, and a hole diameter D of the culture medium passing hole satisfies a range of formula (1). [0010]

8. The cell culture molding device according to claim 1 , wherein a relationship between an outer diameter φ3 of the cylindrical shaft, an inner diameter φ4 of the cone, and a hole diameter D of the culture medium passing hole satisfies a range of formula (2). [0025]

9. The cell culture molding device according to claim 1 , wherein at least the cylindrical shaft and the outer cylinder in the cylindrical forming portion are made of a metal or resin having affinity for the living body.

10. A cell culture molding system comprising the cell culture molding apparatus according to any one of claims 1 to 9, The cell culture molding device; A culture solution tank that houses the cell culture molding device and stores a culture solution; A reciprocating mechanism that reciprocates the weight with respect to the cell culture molding device inside the culture medium tank; A cell culture molding system comprising:

11. A method for producing a cell culture sheet, comprising the cell culture molding apparatus according to any one of claims 1 to 9, and injecting the spheroids in which cells are cultured to produce a sheet-shaped cell culture sheet, Injecting the spheroids into the container of the cylindrical forming part; a step of reciprocating the weights arranged on both sides of the container in the axial direction in the axial direction of the cylinder, and applying a compressive force in the axial direction to the spheroids in the container by the weights; a step of passing the culture medium through the culture medium passing hole of the outer cylinder into the inside of the storage portion and replenishing the culture medium during the culture in which the cone is reciprocated; The method for producing a cell culture sheet comprising the steps of:

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