Co-culture devices and their use

Abstract

This invention provides a co-culture device and a method for producing co-cultured tissue that can reproduce the junctional state between motor neurons and skeletal muscle tissue in vivo, and that allows the neuro-skeletal muscle co-cultured tissue obtained by co-culture to be easily used in other experiments. [Solution] The co-culture device (1) for co-culturing motor neurons and skeletal muscle tissue comprises a pair of anchor members (2a) each supporting skeletal muscle tissue 10a, a pair of well members (2b) attached to the upper part of the pair of anchor members (2a) and in which motor neurons (10b) are seeded, and a base member (3) that supports the pair of anchor members (2a) from below. Each well member (2b) has multiple through holes (2b1a) formed on the inner bottom surface that penetrate to the bottom surface, and each anchor member (2a) has a pillar portion (2a2) that protrudes upward and supports skeletal muscle tissue (10a), and the pair of anchor members (2a) can be detachably attached to the base member (3).

Description

Technical Field

[0006] , , , , , , , ,

[0005] , , , , , ,

[0007]

[0001] The present invention relates to a co-culture device and its use.

Background Art

[0002] Conventionally, a technique for co-culturing motor neurons and skeletal muscle tissue has been known. The nerve-skeletal muscle co-culture tissue cultured by this co-culture technique has been tried for application in various fields such as disease models for amyotrophic lateral sclerosis (ALS), myasthenia gravis, etc., cultured meat, and drive sources (actuators) for robots.

[0003] For example, Non-Patent Document 1 discloses a technique for directly attaching neurospheres to skeletal muscle tissue and differentiating them into neurons for co-culture. However, the co-culture technique described in Non-Patent Document 1 has a problem that it cannot reproduce the in vivo state where only the axons of nerve cells are joined to skeletal muscle tissue.

[0004] On the other hand, Patent Document 1 discloses a co-culture device including a first unit for forming skeletal muscle tissue, a second unit for culturing motor neurons, and a third unit for communicating the first unit and the second unit.

[0005] In the co-culture device described in Patent Document 1, only the axons of the motor neurons cultured in the second unit are joined to the skeletal muscle tissue cultured in the first unit through the third unit, so that the joining state of motor neurons and skeletal muscle tissue in vivo can be reproduced.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

[0008] However, in the co-culture device described in Patent Document 1, the pair of pillars supporting the skeletal muscle tissue in the first unit are fixed to the substrate 21, making it difficult to use the resulting neuro-skeletal muscle co-cultured tissue for other experiments such as disease elucidation, robot driving, and measurement of muscle contraction force.

[0009] This invention has been made in view of these circumstances, and aims to provide a co-culture device and a method for producing co-cultured tissue that can reproduce the junctional state between motor neurons and skeletal muscle tissue in vivo, and that allows the neuro-skeletal muscle co-cultured tissue obtained by co-culture to be easily used in other experiments. [Means for solving the problem]

[0010] To solve the above problems, one aspect of the present invention includes the following aspects. [1] A co-culture device for co-culturing motor neurons and skeletal muscle tissue, comprising: a pair of anchor members for supporting skeletal muscle tissue; a pair of well members attached to the upper part of the pair of anchor members for seeding motor neurons; and a base member supporting the pair of anchor members from below, wherein a plurality of through holes extending to the bottom surface are formed on the inner bottom surface of each well member; each anchor member has a pillar portion that protrudes upward to support the skeletal muscle tissue; and the pair of anchor members are detachably attached to the base member.

[0011] [2] The base member is roughly plate-shaped, and a pair of recesses are formed on the upper surface of the base member. The co-culture device according to [1], wherein the pair of anchor members can be fitted into the pair of recesses for attachment.

[0012] [3] The co-culture device according to [2], wherein the base member has a shape without a recess between the pair of recesses.

[0013] [4] The co-culture device according to any one of [1] to [3], wherein each well member comprises a membrane portion that constitutes the bottom and a side wall portion that forms a closed annular shape when viewed from above and constitutes the side wall of the well member, and the membrane portion has a plurality of through holes formed therein.

[0014] [5] The co-culture device according to any one of [1] to [4], wherein each well member and anchor member is substantially rectangular in top view, and notches are formed at the four corners of the side wall portion of each well member, and each anchor member has a frame portion at each of the four corners that fits into the notches to position the well member.

[0015] [6] The co-culture device according to any one of [1] to [5], wherein the pillar portion has a columnar portion that extends vertically in a columnar shape and an enlarged diameter portion that expands horizontally from the upper part of the columnar portion.

[0016] [7] The co-culture device according to [4], wherein the thickness of the membrane portion in the vertical direction is 1 to 20 μm and the diameter of the through-hole is 1 to 10 μm.

[0017] [8] A co-culture device according to any one of [1] to [7], wherein each anchor member comprises a plurality of the pillar portions, and each pillar portion is coated with fibronectin.

[0018] [9] A method for producing a co-cultured tissue of motor neurons and skeletal muscle tissue, comprising the steps of: seeding motor neurons in a pair of well members of a co-culture device described in any one of [1] to [8]; forming skeletal muscle tissue connecting the pillar portion of one of the pair of anchor members to the pillar portion of the other anchor member; attaching the well members on which the motor neurons have been seeded to each anchor portion on which the skeletal muscle tissue has been formed, thereby placing the well members on the skeletal muscle tissue; and culturing the motor neurons with the well members placed on the skeletal muscle tissue, thereby joining the axons of the motor neurons to the skeletal muscle tissue through the through-holes.

[0019]

[10] The method for producing co-cultured tissue according to [9], comprising the steps of forming the skeletal muscle tissue, connecting the pillar portion of one of the pair of anchor members to the pillar portion of the other anchor member with a hydrogel containing at least one of skeletal muscle myoblasts and skeletal muscle cells, thrombin, fibrinogen, and a basement membrane matrix, and heating the hydrogel with the pillar portions connected to each other.

[0020]

[11] A method for producing a co-cultured tissue according to [9] or

[10] , wherein the motor neurons seeded in the well member are motor neuron spheroids.

[0021]

[12] A method for producing co-cultured tissue according to any one of [9] to

[11] , further comprising the step of culturing the motor neurons seeded in the well members for 12 hours or more in advance in a state where culture medium is present below the through-holes of the well members, before attaching the well members in which the motor neurons are seeded to each anchor member.

[0022] A co-culture kit comprising the co-culture device described in

[13] [1], and a pair of neuron culture platforms on which the pair of well members of the co-culture device are placed. Each neuron culture platform includes a base portion on which the well member is placed, a leg portion that extends downward from the edge of the base portion and supports the base portion, and a peripheral wall portion that extends upward from the edge of the base portion and surrounds the well member. A through hole penetrating in the vertical direction is formed in the base portion.

[0023]

[14] The co-culture kit according to

[13] , wherein when the well member is placed on each of the base portions of the pair of neuron culture platforms, the vertical distance between the lower surface of the well member and the lower end portion of the leg portion is 0.5 mm or more. [Advantages of the Invention]

[0024] According to the present invention, it is possible to reproduce the junction state between motor neurons and skeletal muscle tissue in vivo, and to provide a co-culture device and a method for manufacturing a co-culture tissue that can easily provide the nerve-skeletal muscle co-culture tissue obtained by co-culture for other experiments. [Brief Description of the Drawings]

[0025] [Figure 1] FIG. 1 is a schematic perspective view showing a state of culturing a nerve (motor neuron)-skeletal muscle co-culture tissue using the co-culture device according to a preferred embodiment of the present invention. [Figure 2] FIG. 2 is a schematic perspective view showing a state where the culture part is removed from the culture platform of the co-culture device shown in FIG. 1. [Figure 3] FIG. 3 is a schematic top view of each anchor member. [Figure 4] FIG. 4 is a schematic perspective view of each anchor member. [Figure 5] FIG. 5 is a schematic longitudinal sectional view of the well member along the line W-W shown in FIG. 1. [Figure 6] FIG. 6 is a schematic perspective view showing the side wall portion of each well member. [Figure 7]This is a schematic longitudinal cross-sectional view of the well member and the vicinity of the neuron culture stand, showing the preliminary culture process using a neuron culture stand. [Figure 8] Figure 8 is a schematic perspective view of the neuron culture stand used in the preliminary culture process. [Figure 9] Figure 9 is a schematic longitudinal cross-sectional view of the vicinity of the well component, showing how the well component is placed directly in a petri dish for cultivation without using a neuron culture stand. [Figure 10] Figure 10 is a schematic front view showing the flow of the skeletal muscle tissue formation step and the well placement step in a method for producing co-cultured tissue according to a preferred embodiment of the present invention. [Figure 11] Figure 11 is a schematic, partially enlarged perspective view of the vicinity of the neuromuscular junction formation process in the neuro-skeletal muscle co-cultured tissue. [Figure 12] Figure 12 is a schematic perspective view of the culture stage of a co-culture device according to another preferred embodiment of the present invention. [Figure 13] Figure 13 is a schematic perspective view showing the mold for the side wall portion of the well member. [Figure 14] Figure 14 is a schematic perspective view of the well member fabricated in Experimental Example 3. [Figure 15] Figure 15 is a reference image showing the anchor member fabricated in Experimental Example 2 with the well member fabricated in Experimental Example 3 attached. [Figure 16] Figure 16 shows an image of a neuron culture stand with well components fitted into it. [Figure 17] Figure 17 shows images of the co-cultured tissue and co-culture device prepared in Experimental Example 6. [Figure 18] Figure 18 shows the co-cultured tissue prepared in Experimental Example 6 after being removed from the co-culture device using tweezers. [Figure 19] Figure 19 is a microscopic image showing the through-holes formed in the membrane fabricated in Experimental Example 3. [Figure 20] Figure 20 is a fluorescence microscope image showing motor neuron spheroids seeded on the membrane in Experimental Example 5. [Figure 21] Figure 21 is a fluorescence microscope image showing the axons of motor neuron spheroids passing through through-holes formed in the membrane fabricated in Experimental Example 3. [Figure 22] Figure 22 is a graph showing the change in contraction distance of skeletal muscle tissue when electrical stimulation is applied. [Modes for carrying out the invention]

[0026] The following describes a preferred embodiment of the present invention, including a method for producing a co-culture device or co-cultured tissue, with reference to the drawings as needed. Note that in all the following drawings, the dimensions and proportions of the components have been appropriately altered for clarity.

[0027] [Co-culture device] Figure 1 is a schematic perspective view showing the cultivation of nerve (motor neuron)-skeletal muscle co-cultured tissue 10 in a co-culture device 1 according to a preferred embodiment of the present invention. Figure 2 is a schematic perspective view showing the co-culture device 1 shown in Figure 1 with the culture section 2 removed from the culture stage 3.

[0028] As shown in Figures 1 and 2, the co-culture device 1 comprises a culture section 2 for culturing neuro-skeletal muscle co-cultured tissue 10 and a culture stand 3 that supports the culture section 2 from below. The culture stand 3 is an example of a stand member of the present invention.

[0029] The culture stand 3 is roughly plate-shaped. A pair of recesses 3a are formed on the upper surface of the culture stand 3. As shown in Figure 2, each recess 3a reaches only one end in the short direction of the culture stand 3, which is rectangular in top view. Therefore, the bottom surface 3a1 of the recess 3a has an open end 3a2 and a closed end 3a3 consisting of a wall located on the other side in the short direction.

[0030] In the following explanation, for convenience, the side of the closing end 3a3 relative to the opening end 3a2 will be referred to as "front" (in the +Y direction in the diagram), and the opposite side will be referred to as "rear" (in the -Y direction in the diagram). Also, the left side when facing forward will be referred to as "left" (in the +X direction in the diagram), and the opposite side will be referred to as "right" (in the -X direction in the diagram). Furthermore, the vertically upward side will be referred to as "up" (in the +Z direction in the diagram), and the vertically downward side will be referred to as "down" (in the -Z direction in the diagram).

[0031] The culture stage 3 maintains the relative positions of a pair of anchor members, which will be explained later. In this embodiment, the culture stage 3 has a shape without a recess between the pair of left and right recesses 3a. Therefore, the upper surface 3b1 of the portion 3b (hereinafter also referred to as the "recessed portion") located between the pair of left and right recesses 3a on the upper surface of the culture stage 3 is located above the bottom surface 3a1 of the recesses 3a. The recessed portion 3b is positioned below the skeletal muscle tissue 10a produced by the co-culture device 1. Therefore, when forming the skeletal muscle tissue 10a, the recessed portion 3b located between the recesses 3a supports the skeletal muscle tissue 10a from below, preventing downward bending of the skeletal muscle tissue 10a, and enabling the production of a firm skeletal muscle tissue 10a that is substantially straight when viewed from the front or rear.

[0032] The neuro-skeletal muscle co-cultured tissue 10 is placed together with the co-culture device 1 in a container such as a petri dish containing the culture medium and cultured. For this reason, it is preferable that the culture stand 3 is made of a biocompatible material. Examples of biocompatible materials include, but are not limited to, cyclic olefin copolymers (COC), polyglycolic acid, and polydimethylsiloxane (PDMS).

[0033] The culture section 2 includes a pair of left and right anchor members 2a for supporting one and the other portion of the skeletal muscle tissue 10a in the longitudinal direction (left-right direction), and a pair of left and right well members 2b attached to the upper part of the pair of left and right anchor members 2a, into which motor neurons 10b are seeded. In other words, the co-culture device 1 includes a pair of left and right anchor members 2a, a pair of left and right well members 2b, and a culture stage 3. In this embodiment, each anchor member 2a and each well member 2b are configured with 4 rotational symmetry around a virtual axis of rotation that passes through the horizontal center and extends vertically. Therefore, each anchor member 2a and each well member 2b maintain the same shape even when rotated 90 degrees in a top view. The pair of anchor members will be described below.

[0034] (Anchor member) Figure 3 is a schematic top view of each anchor member 2a. Figure 4 is a schematic perspective view of each anchor member 2a.

[0035] As shown in Figure 3, each anchor member 2a has a roughly rectangular shape when viewed from above. As shown in Figures 3 and 4, each anchor member 2a has a roughly plate-shaped bottom portion 2a1, a pillar portion 2a2 that protrudes upward from the approximate center of the top surface of the bottom portion 2a1, and columnar frame portions 2a3 that extend upward from the four corners of the bottom portion 2a1.

[0036] The bottom portion 2a1 is configured to have approximately the same dimensions in the left-right, front-back, and up-down directions as the recesses 3a shown in Figure 2. The left and right anchor members 2a are fitted into the left and right recesses 3a and are removed from the culture stand 3 by sliding them backward or lifting them upward. In other words, the pair of left and right anchor members 2a can be detachably attached to the culture stand 3.

[0037] As described above, the pair of left and right anchor members 2a are configured to be detachably attached to the culture stage 3, allowing the neuro-skeletal muscle co-cultured tissue 10 produced by the co-culture device 1 to be removed from the culture stage 3 along with the culture section 2 (see Figure 2). This makes it easy to use the tissue for other experiments such as elucidating diseases like amyotrophic lateral sclerosis (ALS) and myasthenia gravis, screening for drugs effective against these diseases, driving robots, and measuring muscle contraction force as a driving source.

[0038] As described above, each anchor member 2a is configured to be rotationally symmetrical four times when viewed from above. Therefore, as long as one side of the bottom 2a1 is parallel to the rear surface 3c of the culture stand 3 when viewed from above, it can be attached to the culture stand 3 in any orientation rotated by 90 degrees, making it highly convenient.

[0039] The pillar portion 2a2 of the left anchor member 2a of the pair of left and right anchor members 2a supports the left side of the skeletal muscle tissue 10a, and the pillar portion 2a2 of the right anchor member 2a supports the right side of the skeletal muscle tissue 10a. In other words, the skeletal muscle tissue 10a, which extends long from side to side, is suspended by the pillar portions 2a2 of the pair of left and right anchor members 2a.

[0040] Each pillar portion 2a2 has a columnar portion 2a2a that extends vertically in a columnar shape, and an enlarged diameter portion 2a2b that widens horizontally (in a direction perpendicular to the vertical direction) from the upper part of the columnar portion 2a2a. The presence of the enlarged diameter portion 2a2b in the pillar portion 2a2 effectively prevents the skeletal muscle tissue 10a from detaching from the pillar portion 2a2. The shape of the enlarged diameter portion is not particularly limited; for example, it may be rectangular prism-shaped (cuboid-shaped) or cylindrical, but the enlarged diameter portion 2a2b in this embodiment is rectangular prism-shaped (cuboid-shaped).

[0041] In this embodiment, each left and right anchor member 2a has nine pillar portions 2a2, but the number of pillar portions 2a2 may be less than eight, one, or ten or more. It is preferable that each anchor member 2a has multiple pillar portions 2a2. By having multiple pillar portions 2a2 in each anchor member 2a, the fixing force of the skeletal muscle tissue 10a can be increased, and the tensile force exerted on the skeletal muscle tissue 10a by each pillar portion 2a2 when the skeletal muscle tissue 10a contracts can be weakened, thereby suppressing fracture of the skeletal muscle tissue 10a.

[0042] The pair of left and right anchor members 2a in this embodiment are made of resin formed by a 3D printer, and their surfaces are coated with parylene (polyparaxylene). The material and forming method of the anchor members are not particularly limited.

[0043] The anchor member is preferably made of a biocompatible material. Examples of biocompatible materials include the materials mentioned above.

[0044] If the material of the anchor member is not biocompatible, it is preferable to coat the surface of the anchor member with parylene. This prevents the resin from adversely affecting cells.

[0045] Regardless of whether the anchor member material is biocompatible or not, it is preferable that each pillar portion be coated with a cell matrix component such as fibronectin, laminin, and / or collagen. This improves the adhesion of skeletal muscle tissue 10a to each pillar portion and suppresses fracture of the skeletal muscle tissue 10a. In this embodiment, each pillar portion 2a2 is further coated with fibronectin over a parylene coating.

[0046] The frame portion 2a3 holds the well member 2b above the bottom portion 2a1 of the anchor member 2a. More specifically, the frame portion 2a3 fits into the notch 2b2c of the well member 2b, which will be described later, to position the well member 2b. In other words, the frame portion 2a3 fits into the side wall portion 2b2 of the well member 2b to position the well member 2b. In this embodiment, the frame portion 2a3 has a rectangular prism shape in a top view, matching the shape of the L-shaped notch 2b2c. The left and right pair of anchor members 2a have been described in detail above, and now the left and right pair of well members 2b will be described in detail below.

[0047] (Well component) Figure 5 is a schematic longitudinal cross-sectional view of the well member 2b along the WW line shown in Figure 1. Figure 6 is a schematic perspective view showing the side wall portion 2b2 of each well member 2b. The well member 2b has a neuron housing space 2b3 on the inside for housing motor neurons 10b, and has a plurality of through holes 2b1a in its inner bottom surface 2b1b that penetrate to the lower surface 2b1c of the well member 2b.

[0048] As shown in Figure 5, each well member 2b comprises a membrane portion 2b1 that forms its bottom and a side wall portion 2b2 that forms a closed annular shape in top view and constitutes the side wall of the well member 2b. Each well member 2b is substantially rectangular in top view and is detachably attached to the upper part of a pair of anchor members 2a (see Figures 1 and 2). The membrane portion 2b1 and the side wall portion 2b2 may be integrally molded or separate, but in this embodiment they are formed as separate parts.

[0049] The membrane portion 2b1 is a sheet-like member with multiple through holes 2b1a formed therein, and in this embodiment, it is fixed to the lower surface 2b2a of the side wall portion 2b2 to form a neuron housing space 2b3. However, the membrane portion 2b1 may be located radially inward of the annular side wall portion 2b2.

[0050] Motor neurons 10b are seeded on the upper surface (inner bottom surface of the well member 2b) 2b1b of the membrane portion 2b1 in the neuron housing space 2b3. Therefore, in order to prevent adverse effects on the motor neurons 10b, it is preferable that the membrane portion 2b1 be made of a biocompatible material.

[0051] The material of the membrane portion 2b1 is not particularly limited as long as it has through holes 2b1a or as long as through holes can be formed, but may be, for example, medical-grade silicone rubber, medical-grade polyester, medical-grade polyethylene, medical-grade polycarbonate, cyclic olefin copolymer, polyglycolic acid, polydimethylsiloxane, etc. A commercially available track-etched membrane can preferably be used for the membrane portion 2b1.

[0052] The thickness of the membrane portion 2b1 in the vertical direction is not particularly limited, but may be, for example, 1 to 50 μm, 1 to 20 μm, 1 to 15 μm, or 5 to 15 μm.

[0053] To improve the adhesion of motor neurons 10b, the membrane portion 2b1 is preferably coated with fibronectin, laminin, collagen, and / or polyethyleneimine. The membrane portion 2b1 may also have a coating of laminin on top of the polyethyleneimine coating. These coatings can promote cell adhesion and the elongation of axons 10b1 (see Figure 7, which will be described in detail later).

[0054] The through-holes 2b1a that penetrate the membrane portion 2b1 may penetrate approximately vertically or obliquely. However, considering that the axons 10b1 of the motor neurons 10b extend through the through-holes 2b1a to the lower part of the membrane portion 2b1, as will be described later, it is preferable that they penetrate approximately vertically. This is thought to shorten the culture time until the terminals of the axons 10b1 attach to the skeletal muscle tissue 10a.

[0055] The diameter of the through-hole 2b1a may be, for example, 1 to 10 μm, 1 to 8 μm, or 3 to 8 μm, but is not limited to these ranges. By setting the diameter of the through-hole within these ranges, only the axon 10b1 of the motor neuron 10b can be extended to and joined to the skeletal muscle tissue 10a, making it possible to reproduce the connection state between motor neurons and skeletal muscle tissue in vivo.

[0056] The number of through-holes 2b1a in the membrane portion 2b1 is 2 or more, and may be, for example, 10 or more, 100 or more, 500 or more, or 1000 or more. Furthermore, the number of through-holes 2b1a may be 300 or less, 800 or less, 1500 or less, or 3000 or less. These lower and upper limits for the number of through-holes 2b1a can be combined in any way.

[0057] On the other hand, as shown in Figure 6, the side wall portion 2b2 of this embodiment has L-shaped notches 2b2c formed at each of its four corners when viewed from above. Therefore, as shown in Figures 1 and 2, the well member 2b can be attached to the anchor member 2a by fitting the frame portions 2a3 at the four corners of the anchor member 2a with the four notches 2b2c of the well member 2b (side wall portion 2b2), and pushing it down to a position just above the pillar portion 2a2. The vertical position of the well member 2b can be adjusted by the amount the well member 2b is pushed down.

[0058] Here, it is preferable that the side wall portion 2b2 is made of a biocompatible and highly flexible material. The high flexibility of the side wall portion 2b2 allows the well member 2b and the frame portion 2a3 of the well member 2b and / or anchor member 2a to be fitted together accurately, preventing horizontal and vertical displacement of the well member 2b during the culture of the neuro-skeletal muscle co-cultured tissue 10.

[0059] Examples of biocompatible and highly flexible materials include, but are not limited to, polydimethylsiloxane and medical-grade silicone.

[0060] The inner diameter of the hollow side wall portion 2b2 is not particularly limited, but may be, for example, 2 to 10 mm, or 3 to 8 mm, and preferably about 5 mm.

[0061] The height of the neuron housing space 2b3, that is, the vertical length from the upper surface 2b1b of the membrane portion 2b1 to the upper surface 2b2b of the side wall portion 2b2, is preferably in the range of 1 to 5 mm, but is not limited to this range, in order to prevent the motor neuron 10b from being too far from the air and to supply an appropriate culture medium.

[0062] [Method for producing co-cultured tissue] A method for producing co-cultured tissue of motor neurons and skeletal muscle tissue according to another preferred embodiment of the present invention includes the steps of: seeding motor neurons in a pair of well members of a co-culture device (hereinafter also referred to as the "motor neuron seeding step"); forming skeletal muscle tissue that connects the pillar portion of one of a pair of anchor members to the pillar portion of the other anchor member (hereinafter also referred to as the "skeletal muscle tissue formation step"); attaching well members on which motor neurons have been seeded to each anchor portion where skeletal muscle tissue has been formed, and placing the well members on the skeletal muscle tissue (hereinafter also referred to as the "well placement step"); and culturing motor neurons with the well members placed on the skeletal muscle tissue to connect the axons of the motor neurons to the skeletal muscle tissue through the through-holes of the well members (hereinafter also referred to as the "neuromuscular junction formation step").

[0063] In the manufacturing method of the present invention, the above-described co-culture device 1 can be suitably used, but the device used to produce co-cultured tissue of motor neurons and skeletal muscle tissue (neuro-skeletal muscle co-cultured tissue) is not limited to the above-described co-culture device 1. In this embodiment, for the sake of explanation, a method for producing neuro-skeletal muscle co-cultured tissue using co-culture device 1 will be described in detail. During the culture of skeletal muscle tissue 10a, motor neurons 10b, and their co-cultured tissues using the co-culture device 1, the culture is performed with some components of the co-culture device 1 or the entire co-culture device 1 contained in a container such as a petri dish containing the culture medium.

[0064] The motor neuron seeding process and the skeletal muscle tissue formation process may be performed in either order, or both may be performed simultaneously. After the motor neuron seeding and skeletal muscle tissue formation processes are completed, the well placement process is performed, followed by the neuromuscular junction formation process. Each process is described in detail below.

[0065] (Motor neuron seeding process) In the motor neuron seeding process, motor neurons 10b are seeded on the inner bottom surface of a pair of well members 2b. In the co-culture device 1 described above, the inner bottom surface of the pair of well members 2b is the upper surface 2b1b of the membrane portion 2b1.

[0066] The motor neurons 10b seeded in the well member 2b are preferably motor neuron spheroids. Motor neuron spheroids have high adhesion to the membrane portion 2b1, which helps to prevent them from detaching from the membrane portion 2b1 during culture.

[0067] The motor neuron spheroids may be motor neuron spheroids differentiated from iPS cells. If motor neuron spheroids are not used, motor nerve cells or the like may be used. In the case of motor nerve cells, the number of cells seeded on the inner bottom surface of the pair of well members 2b is not particularly limited; for example, it may be 1, 10 or more, or 50 or more.

[0068] The motor neurons 10b seeded in the well member 2b can be mammalian cells, and the type of animal is not particularly limited. The motor neurons 10b may be, for example, human motor neurons 10b, or motor neurons 10b of a mammal other than a human. Examples of mammals other than humans include mice, rats, rabbits, guinea pigs, hamsters, hedgehogs, dogs, cats, monkeys, horses, cows, pigs, sheep, etc.

[0069] After seeding the motor neurons 10b, the motor neurons 10b, along with the well members 2b, are immersed in a culture medium and cultured. The type of culture medium is not particularly limited; for example, a culture medium commonly used for culturing motor neurons can be used. For example, if the height of the neuron housing space 2b3 is 1 to 5 mm and the inner diameter of the hollow side wall portion 2b2 is 5 mm, the amount of culture medium is preferably about 20 to 100 μL.

[0070] Alternatively, before attaching the pair of well members 2b on which motor neurons 10b are seeded to each anchor member 2a, a step may be performed in which the motor neurons 10b seeded in the well members 2b are cultured for 12 hours or more in advance with culture medium present below the through-holes 2b1a of the well members 2b (hereinafter also referred to as the "preliminary culture step"). The preliminary culture step will be explained below.

[0071] (Preliminary culture process) Figure 7 is a schematic longitudinal cross-sectional view of the well member 2b and the vicinity of the neuron culture platform 4, showing the preliminary culture process using the neuron culture platform 4. Figure 8 is a schematic perspective view of the neuron culture platform 4 used in the preliminary culture process. Figure 9 is a schematic longitudinal cross-sectional view of the vicinity of the well member 2b, showing the process of culturing by directly placing the well member 2b in a petri dish without using the neuron culture platform 4.

[0072] As shown in Figures 7 and 8, the neuron culture stand 4 comprises a base portion 4a on which the well members 2b are placed, four legs 4b extending downward from the edge of the base portion 4a and supporting the base portion 4a, and a peripheral wall portion 4c extending upward from the edge of the base portion 4a and surrounding the well members 2b.

[0073] The base portion 4a has a circular opening (through hole) 4a1 when viewed from above. When the well member 2b is placed on the base portion 4a, the edge of the lower surface 2b1c of the membrane portion 2b1 abuts against the base portion 4a, while the radial center of the lower surface 2b1c of the membrane portion 2b1 faces the opening 4a1 and therefore does not abut against the base portion 4a. Therefore, when the well member 2b is placed on the base 4a, as shown in Figure 7, a culture medium storage space 4d is formed, defined by the lower surface of the well member 2b (the lower surface of the membrane portion 2b1) 2b1c, the bottom surface 5 of a container such as a petri dish, and the inner surface of the neuron culture stand 4. Culture medium is stored in this culture medium storage space 4d.

[0074] Thus, by culturing motor neurons 10b in the well member 2b with culture medium present in the culture medium reservoir space 4d formed below the through-hole 2b1a formed in the well member 2b, the axons 10b1 of the motor neurons 10b are more easily extended downward through the through-hole 2b1a. Therefore, in the subsequent neuromuscular junction formation process, more axons 10b1 can be joined to the skeletal muscle tissue 10a, and the number of culture days until the axons 10b1 join can be shortened. Furthermore, since the axon 10b1 extends along the surrounding objects, after extending through the through-hole 2b1a to its lower side, it extends in a substantially horizontal direction along the lower surface 2b1c of the well member 2b (the lower surface of the membrane portion 2b1 in the co-culture device 1).

[0075] In contrast, when motor neurons 10b are cultured by directly placing the well member 2b in a container such as a petri dish without using the neuron culture stand 4, as shown in Figure 9, the through-hole 2b1a is closed by the bottom surface 5 of the container such as a petri dish, making it impossible to promote the extension of the axon 10b1.

[0076] The height of the culture medium storage space 4d, that is, the vertical length L (see Figure 7) between the lower surface 2b1c of the membrane portion 2b1 and the lower end of the neuron culture stand 4 (the lower end of the leg portion 4b), may be 0.5 mm or more, preferably 0.7 mm or more, and more preferably 1 mm or more.

[0077] The culture time for motor neurons 10b in the preliminary culture step is not particularly limited as long as it is 12 hours or more, but may be, for example, 12 to 45 hours, 15 to 35 hours, or 20 to 30 hours. The culture time for motor neurons 10b in the preliminary culture step can be appropriately adjusted considering the degree of axon 10b1 elongation, the length of the through-hole 2b1a, i.e., the thickness of the membrane portion 2b1, etc.

[0078] After the motor neuron seeding step and during the pre-culture step, it is preferable to store in the well member 2b an amount of culture medium sufficient to immerse the motor neurons 10b, as shown in Figure 7.

[0079] (Skeletal muscle tissue formation process) The method for forming skeletal muscle tissue 10a that connects the pillar portion 2a2 of one anchor member 2a to the pillar portion 2a2 of the other anchor member 2a in the skeletal muscle tissue formation process is not particularly limited, but the skeletal muscle tissue formation process in this embodiment includes the steps of connecting the pillar portion 2a2 of one of the pair of anchor members 2a to the pillar portion 2a2 of the other anchor member 2a with a hydrogel containing at least one of skeletal muscle myoblasts and skeletal muscle cells, and the steps of heating the hydrogel connecting the pillar portions 2a2.

[0080] Figure 10 is a schematic front view showing the flow of the skeletal muscle tissue formation step and the well placement step in a method for producing co-cultured tissue according to a preferred embodiment of the present invention.

[0081] In the process of joining the pillar sections 2a2 together, first, hydrogel H is applied to each pillar section 2a2 of the pair of anchor members 2a. Next, the pillar section 2a2 of one anchor member 2a and the pillar section 2a2 of the other anchor member 2a are joined together with hydrogel H.

[0082] The animal species of skeletal muscle myoblasts and / or skeletal muscle cells contained in hydrogel H are not particularly limited. For example, they may be human skeletal muscle myoblasts and / or skeletal muscle cells, or skeletal muscle myoblasts and / or skeletal muscle cells of non-human mammals as exemplified in the motor neuron seeding step. The skeletal muscle myoblasts and / or skeletal muscle cells and the motor neurons 10b may be of the same animal species or different animal species, but it is preferable that they be of the same animal species.

[0083] Hydrogel H is preferably a gel obtained by mixing skeletal muscle myoblasts and / or skeletal muscle cells, in addition to thrombin, fibrinogen, and basement membrane matrix. Hydrogel H may also separately contain other components such as gelatin, agar, agarose, hyaluronic acid, glycosaminoglycans, and proteoglycans.

[0084] According to the above hydrogel H, the hydrogel H can be held in a connected state by a first coagulation action using thrombin and fibrinogen and a second coagulation action using the basement membrane matrix.

[0085] In the first coagulation process, thrombin breaks down fibrinogen into fibrin through an enzymatic reaction, thereby promoting coagulation. It is preferable to add thrombin to the hydrogel H, which contains fibrinogen and basement membrane matrix, just before attaching the hydrogel H to the pillar portions 2a2. This causes the hydrogel H to begin coagulating in about 1-2 minutes, making it easier to maintain the shape of the hydrogel H that connects the pillar portions 2a2.

[0086] In the second coagulation process, coagulation proceeds by heating (warming) the basement membrane matrix contained in the hydrogel H. The heating method in the step of heating the hydrogel H is not particularly limited, but for example, the culture stand 3 with the pair of anchor members 2a attached is placed in a 37°C incubator and incubated for 30 minutes or more. In the step of heating the hydrogel, it is preferable to heat it to 35°C or higher.

[0087] As the hydrogel H solidifies due to the first and second coagulation processes, the upper part of the pillar portion 2a2 of each anchor member 2a becomes embedded in the hydrogel H. Therefore, as described above, the presence of an enlarged diameter portion 2a2b in the pillar portion 2a2 allows the hydrogel H to be firmly fixed to the pillar portion 2a2.

[0088] The basement membrane matrix is ​​an extracellular matrix found in animal tissues between epithelial and stromal cell layers, and around muscle cells, adipocytes, and nerve tissue. The basement membrane matrix can be any basement membrane matrix present in animal tissue; the type of animal is not particularly limited.

[0089] Examples of basement membrane matrix materials include non-fibrous collagen such as type IV collagen, laminin, nidogen, heparan sulfate proteoglycan, or mixtures of two or more of these, but the most preferred is Matrigel® basement membrane matrix (Corning).

[0090] When connecting pillar sections 2a2 with hydrogel H, attaching a pair of left and right anchor members 2a to the culture stand 3 allows the relative positions of the left and right anchor members 2a to be maintained, thereby enabling the formation of robust skeletal muscle tissue.

[0091] Furthermore, as described above, the formation of inter-recess protrusions 3b between the left and right recesses 3a of the culture stand 3, which protrude upward when viewed from the recesses, effectively prevents the hydrogel H connecting the pillar portions 2a2 of the left and right anchor members 2a from bending downward (in other words, the central part of the hydrogel H in the left-right direction from sagging downward), thereby enabling the formation of a firm skeletal muscle tissue 10a that is roughly linear when viewed from the front or back.

[0092] It is preferable to culture the skeletal muscle tissue 10a formed on the pair of left and right anchor members 2a together with the culture stage 3 and the pair of anchor members 2a in a container such as a petri dish containing culture medium. Therefore, it is convenient to perform the skeletal muscle tissue formation process in a container such as a petri dish, as this eliminates the need to move the skeletal muscle tissue 10a into the container afterward. It is also preferable to add culture medium to the container after the skeletal muscle tissue 10a has been formed, up to a height that immerses the skeletal muscle tissue 10a.

[0093] Skeletal muscle myoblasts proliferate and fuse within the solidified hydrogel H to form muscle fibers. Similarly, skeletal muscle cells fuse within the solidified hydrogel H to form muscle fibers. As a result, artificial skeletal muscle tissue 10a is formed, connecting the pillar portions 2a2. Hydrogel H acts as a scaffold for forming the three-dimensional skeletal muscle tissue 10a. During the skeletal muscle tissue formation process, the concentration of skeletal muscle myoblasts and / or skeletal muscle cells in hydrogel H can be appropriately adjusted so that the formed skeletal muscle tissue 10a exhibits the function of contracting in response to electrical signals, etc.

[0094] (Well placement process) In the well placement step, well members 2b on which motor neurons 10b have been seeded are attached to a pair of anchor members 2a that support the skeletal muscle tissue 10a formed in the skeletal muscle tissue formation step, and the lower surface of the well member 2b (in the case of co-culture device 1, the lower surface 2b1c of the membrane portion 2b1) is brought into contact with the upper surface of the skeletal muscle tissue 10a (see Figure 10). It is preferable to add culture medium to a container such as a petri dish beforehand, either after or prior to the well placement step, to a height that immerses the motor neurons 10b in the well member 2b.

[0095] (Neuromuscular junction formation process) In the neuromuscular junction formation process, the motor neurons 10b and skeletal muscle tissue 10a are cultured with well members 2b on which motor neurons 10b have been seeded, which were placed on skeletal muscle tissue 10a in the well placement process.

[0096] Figure 11 is a schematic magnified partial perspective view of the vicinity of the neuromuscular junction formation process of the neuro-skeletal muscle co-cultured tissue 10. For ease of explanation, Figure 11 shows the membrane portion 2b1, the left portion of the skeletal muscle tissue 10a, and the motor neuron 10b with the side wall portion 2b2 of the well member 2b removed.

[0097] By culturing skeletal muscle tissue 10a and motor neurons 10b with the well member 2b placed on the skeletal muscle tissue 10a, the axons 10b1 of the motor neurons 10b can be extended and joined to the upper surface of the skeletal muscle tissue 10a, as shown in Figure 11, thereby forming a neuromuscular junction 10c. For example, if the motor neuron seeding step and pre-culture step are performed using a membrane portion 2b1 with a thickness of approximately 10 μm, and then the well placement step and neuromuscular junction formation step are performed, it is expected that the axons 10b1 will join to the skeletal muscle tissue 10a within one day or about one day after the well member 2b is placed on the skeletal muscle tissue 10a.

[0098] In the well placement process, as described above, the side wall portion 2b2 of the well member 2b is fitted with the frame portion 2a3 of the anchor member 2a (see Figure 1, etc.), thereby maintaining the relative position between the motor neuron 10b and the skeletal muscle tissue 10a. As a result, it is possible to form a robust neuromuscular junction 10c in the neuromuscular junction formation process (see Figure 11). Furthermore, it is expected that this culture will cause acetylcholine receptors on the upper surface of the skeletal muscle tissue 10a to aggregate at the contact point with the terminal of the axon 10b1 of the motor neuron 10b.

[0099] By carrying out the motor neuron seeding step, skeletal muscle tissue formation step, well placement step, and neuromuscular junction formation step described in detail above (and preferably a pre-culture step as well), a neuro-skeletal muscle co-cultured tissue 10 can be obtained.

[0100] The resulting neuromuscular co-cultured tissue 10 can be used, for example, as artificial meat having a neuromuscular junction 10c, as a drive system for robots, as well as as a model for measuring muscle contraction force and as an in vitro evaluation model for neuromuscular diseases. Therefore, the manufacturing method of this embodiment can also be considered as a method for manufacturing an in vitro evaluation model for neuromuscular diseases. However, the uses of the neuromuscular co-cultured tissue 10 are not limited to these.

[0101] When manufacturing in vitro evaluation models for neuromuscular diseases, it is possible to accurately reproduce the disease state by using cells that have the gene mutation causing the disease, for example, motor neurons 10b and / or the aforementioned skeletal muscle myoblasts or skeletal muscle cells. For example, when creating an in vitro evaluation model for Parkinson's disease, it is conceivable to use cells with the VPS35 gene mutation. Similarly, when creating an in vitro evaluation model for spinocerebellar degeneration, it is conceivable to use cells with the CACNA1G gene mutation.

[0102] When the neuro-skeletal muscle co-cultured tissue 10 is used, for example, as a drive system for a robot, sensors and / or electrodes may be connected to the neuro-skeletal muscle co-cultured tissue 10. Even in such cases, the neuro-skeletal muscle co-cultured tissue 10 can be easily used for experiments by removing the pair of anchor members 2a from the culture stand 3. When using the neuro-skeletal muscle co-cultured tissue 10 for various experiments, it is also possible to use the tissue 10 immersed in a buffer or physiological saline instead of the culture medium for a short period of time.

[0103] [Co-culture kit] In one embodiment, the present invention provides a co-culture kit comprising a co-culture device for co-culturing motor neurons and skeletal muscle tissue, and a pair of neuron culture platforms on which a pair of well members of the co-culture device are placed.

[0104] The co-culture device is the same as co-culture device 1 detailed in the above-described embodiment of the [co-culture device], but instead of the culture stage 3 shown in Figure 2, it may be equipped with a culture stage 30 (see Figure 12), which will be explained later.

[0105] Each neuron culture stand is similar to the neuron culture stand 4 detailed in the above-described embodiment of the [method for manufacturing co-cultured tissue], and comprises a base portion 4a on which the well member 2b is placed, four legs 4b extending downward from the edge of the base portion 4a and supporting the base portion 4a, and a peripheral wall portion 4c extending upward from the edge of the base portion 4a and surrounding the well member 2b. A circular opening (a through hole penetrating the base portion 4a vertically) 4a1 is formed approximately in the radial center of the base portion 4a when viewed from above.

[0106] When the well members 2b are placed on each of the platform portions 4a of the pair of neuron culture stands 4, the opening (through hole) 4a1 is located below at least some of the numerous through holes 2b1a formed in the well member 2b. At this time, the vertical length L (see Figure 7) between the lower surface of the membrane portion 2b1 (lower surface of the well member 2b) 2b1c and the lower end of the neuron culture stand 4 (lower end of the leg portion 4b) is not particularly limited, but may be 0.2 mm or more, 0.5 mm or more, preferably 0.7 mm or more, and more preferably 1 mm or more (lower limit of length L). In particular, a vertical length L of 0.7 mm or more ensures sufficient height for the culture medium storage space 4d. The upper limit of length L may be 3 mm or less, 2 mm or less, or 1.5 mm or less, but is not limited to these. For example, length L may be 0.5 to 3 mm, 0.7 to 2 mm, or 1 to 2 mm. The lower and upper limits of length L mentioned above can be combined in any way.

[0107] According to the co-culture kit of this embodiment, which also includes a neuron culture platform 4, a preliminary culture step can be performed using the neuron culture platform 4 prior to the well placement step, thereby promoting the elongation of the axons 10b1 of the motor neurons 10b.

[0108] Although preferred embodiments have been described in detail above, the present invention is not limited to these embodiments, and various modifications are possible within the scope of the invention as described in the claims.

[0109] For example, as shown in Figure 12 (a schematic perspective view of the culture stage 30 of a co-culture device according to another preferred embodiment of the present invention), the co-culture device may include a culture stage 30 that does not have the inter-recessed protrusions 3b, instead of the culture stage 3 shown in Figure 2. The co-culture device according to this embodiment is configured in the same way as the co-culture device 1 shown in Figure 1, except for the points described in detail below.

[0110] The culture stand 30, like the culture stand 3, has a pair of left and right recesses 3a on its upper surface, but does not have a protrusion 3b between the recesses located below the skeletal muscle tissue 10a being prepared. In the culture stand 3 shown in Figure 2, while the hydrogel H can be supported by the protrusion 3b between the recesses when preparing the skeletal muscle tissue 10a, the protrusion 3b between the recesses may obstruct the supply of culture medium to the skeletal muscle tissue 10a. However, with the culture stand 30, which does not have the protrusion 3b between the recesses, it is possible to distribute the culture medium throughout the skeletal muscle tissue 10a.

[0111] As shown in Figure 12, the culture stage 30 is equipped with a displacement prevention piece 31 that protrudes upward at a position behind the anterior-posterior position where the skeletal muscle tissue 10a is produced, preventing lateral displacement of the pair of left and right anchor members 2a. Furthermore, it is equipped with a displacement prevention part 32 that protrudes backward (towards the displacement prevention piece 31) at a position in front of the anterior-posterior position where the skeletal muscle tissue 10a is produced. Therefore, it is possible to prevent displacement of the pair of left and right anchor members 2a while preventing obstruction of the supply of culture medium to the skeletal muscle tissue 10a. [Examples]

[0112] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0113] [Experimental Example 1] In this experiment, culture stages were prepared using PDMS (Dow Corning). In detail, first, a mold was created to produce the culture stand. The mold was designed using 3D CAD software (AutoCAD, registered trademark) and then fabricated using a 3D printer (Shape1+HD, Rayshape). After that, the mold was cleaned in an ultrasonic cleaner (MCS-3, AS ONE) for about 30 minutes and dried in a dryer (Shapecure, Rayshape) for 40 minutes. Furthermore, it was dried further by vacuuming in a desiccator (VL type, AS ONE).

[0114] Next, to make it easier to remove the polysiloxane from the mold, parylene (Dichloro-p-cyclophane, SCS) was coated to a thickness of 2 μm using a vacuum deposition apparatus. Then, a mixture of PDMS liquid and a hardener (Dow Corning) in a 10:1 ratio was poured into the mold, and the mold was vacuumed in a desiccator for 20-30 minutes to remove air bubbles. An OHP sheet was then placed over the mold containing the PDMS liquid to prevent air bubbles from forming. This was then heated on a hot plate heated to 75°C for 90 minutes to cure the PDMS. The PDMS culture platform was removed while ethanol was dripped between the PDMS and the mold.

[0115] Subsequently, the culture trays were sterilized with ozone gas using an ozone gas device (Coolclave plus Ozone and UV Personal Sterilizer, Funakoshi) and UV light using a UV light device (Sterilizer, Fifty Visionary).

[0116] Next, the culture medium (PDMS) was coated. Specifically, to prevent skeletal muscle tissue from adhering to the PDMS, a mixture of PBS (Dulbeccio phosphate buffer, Cell Chemistry Laboratory) and BSA (Bovine Serum Albumin, Sigma-Aldrich) at a concentration of 1% was added to the PDMS until it was submerged, and the medium was placed in an incubator maintained at 37°C and a carbon dioxide concentration of 5% for 40 minutes.

[0117] Finally, culture stages were prepared by washing the PDMS three times with PBS to remove the BSA (see Figure 2 for the shape of culture stage 3, which has protrusions between recesses, and Figure 12 for the shape of culture stage 30, which does not have protrusions between recesses). Each culture stage was degassed in a desiccator for about 15 minutes before being used to prepare skeletal muscle tissue, which will be explained later.

[0118] [Experimental Example 2] (Fabrication of anchor members) An anchor member for attaching skeletal muscle tissue was fabricated. The design of the anchor member and the parylene deposition process were the same as in the fabrication of the culture stand in Experimental Example 1. As with the culture stand, ozone gas sterilization and UV sterilization were performed. Please refer to Figures 3 and 4 for the shape of the anchor member.

[0119] Next, the anchor members were coated with fibronectin to allow skeletal muscle tissue to grip them. A mixture of fibronectin (fibronectin derived from bovine plasma, Sigma-Aldrich) and PBS in a 1:10 ratio was poured onto the pillars and placed in an incubator overnight. Finally, the anchor members were washed three times with PBS to remove the fibronectin solution before use.

[0120] [Experimental Example 3] (Fabrication of well components and seeding of motor neurons) First, a mold for the side wall portion of the well member was prepared in the same manner as the culture stand. Figure 13 is a schematic perspective view showing the mold set for the side wall portion of the well member. A mixture of PDMS liquid and hardener in a 10:1 ratio was poured into mold b. After vacuuming in a desiccator for 20-30 minutes to remove air, another mold a was placed in place, and the PDMS was cured by heating on a hot plate heated to 75°C for 90 minutes. After that, the wells (side walls) were removed with tweezers while ethanol was added dropwise.

[0121] Next, the side wall portion and the track-etched membrane film (membrane portion) were bonded together. In detail, the PDMS liquid was heated on a hot plate at 75°C for approximately 10 minutes to promote curing, and then used as an adhesive. The PDMS liquid was applied to the edge of the side wall with a toothpick, and the coated side was placed face down inside a culture insert (24-Well Insert 8.0μm PET clear, cellQART). It was then heated on a hot plate for approximately 80 minutes to bond the side wall and membrane. After that, the excess portion of the membrane (the part that protruded from the bottom surface of the side wall) was cut off to create the well member 2b. Figure 14 is a schematic perspective view of the well member 2b prepared in Experimental Example 3.

[0122] The lengths of the notches formed at the four corners of the side wall portion 2b2 of the obtained well member 2b in the front-rear and left-right directions (see Figure 6) were 0.8 mm each, the outer dimensions of the well member 2b in the front-rear and left-right directions (see Figures 5 and 6) were 6.6 mm, the height was 5 mm, and the inner diameter of the hollow side wall portion 2b2 was 5 mm. The diameter of the numerous through holes formed in the membrane portion 2b1 was approximately 8 μm, and the thickness of the membrane portion 2b1 in the vertical direction was approximately 10 μm.

[0123] Next, the membrane portion 2b1 of the well member 2b was coated. First, a 1X borate buffer was prepared by mixing 20X borate buffer (Pierce® Concentrated Buffer Stocks, Thermo Fisher) with sterilized pure water. Using this buffer, PEI (polyethyleneimine solution, Sigma-Aldrich) was diluted to a concentration of 0.07% and sterilized by passing it through a 0.22 μm filter. 30 μL of this solution was dropped onto both sides of the membrane portion 2b1 (for the bottom side, 30 μL was dropped onto a dish and the well 2b was placed on top), and incubated in an incubator for 1 hour. After that, the solution was aspirated and washed three times with sterilized pure water, and then left to stand in a clean bench for 1 hour.

[0124] Subsequently, laminin (laminin EHS derived from sarcoma basement membrane, Sigma-Aldrich) was dissolved in a basal salt solution (pH 7.4) consisting of 130 mM (mol / L) NaCl (Fujifilm Wako Pure Chemical Corporation), 5.4 mM KCl (Fujifilm Wako Pure Chemical Corporation), 1.8 mM CaCl2 (Kanto Chemical Co., Ltd.), 5.5 mM D-glucose (D(+)-glucosamine hydrochloride, Chemical Co., Ltd.), and 20 mM HEPES (Fujifilm Wako Pure Chemical Corporation). The laminin concentration was adjusted to 0.02 mg / mL, and 30 μL was dropped onto each side of the membrane, as in the PEI procedure, and incubated in an incubator for 1 hour. Next, the solution was aspirated, and motor neuron spheroids were rapidly seeded.

[0125] Figure 15 is a reference image showing the well member prepared in Experimental Example 3 attached to the anchor member prepared in Experimental Example 2. In Figure 15, the pillar portion 2a2 of the anchor member 2a and the well member 2b are in contact, but when actually preparing co-cultured tissue, skeletal muscle tissue 10a is present between the anchor member 2a and the well member 2b, as shown in the lower part of Figure 10.

[0126] [Experimental Example 4] (Preparation of skeletal muscle tissue) Skeletal muscle tissue was constructed using cultured human skeletal myoblast cell lines. Two solutions, A and B, were required for tissue construction. Solution A consisted of a culture medium containing cells and thrombin (thrombin derived from human plasma, Sigma-Aldrich), while Solution B was a mixture of culture medium, fibrinogen (fibrinogen derived from human plasma, Sigma-Aldrich), and Matrigel (Matrigel basement membrane matrix, Corning). Table 1 below shows the compositions of Solution A and Solution B.

[0127] [Table 1]

[0128] Solutions A and B were pipetted carefully to avoid introducing air bubbles, quickly mixed, and first dropped onto the pillar portion 2a2 of the anchor member 2a, then joined together to construct the skeletal muscle tissue. Subsequently, to prevent the cells from drying out and dying, the prepared skeletal muscle tissue, along with the anchor member 2a and well member 2b, was placed in a 15 cm dish containing water-moistened Bencot, and incubated in a 37°C incubator for 40 minutes.

[0129] Next, the skeletal muscle tissue was removed from the incubator and immediately placed in growth medium. The growth medium used was 100 mL of the growth medium (Lonza Corporation) used for culturing human skeletal myoblasts, with 1 mL of 1 mg / mL ACA (6-Aminocaproic acid, Sigma-Aldrich) added. The medium was changed every 1-2 days.

[0130] After constructing skeletal muscle tissue, the tissue was cultured in differentiation medium either until it contracted or 3-5 days after the start of culture. The differentiation medium used was a 100 mL solution of DMEM low glucose (Dalbeck modified Eagle medium (low glucose), Sigma-Aldrich), 2% (v / v) HS (horse serum, Gibco), and 1% (v / v) P / S, to which 1 mL of 200 mg / mL ACA, SB (SB 431542), and IGF (IGF-I, Human, Recombinant, Animal Free) were added. The medium was changed once every 1-2 days.

[0131] [Experimental Example 5] (Creation of motor neuron spheroids) First, the culture medium was prepared. iCell Neural Supplement A (FUJIFILM Cellular Dynamics) and iCell Nervous System Supplement (FUJIFILM Cellular Dynamics) were melted at room temperature. In addition, the supplements dissolved in the basal medium (iCell Neural Basal Medium, FUJIFILM Cellular Dynamics) were dissolved in a clean bench.

[0132] For the first week, 12.5 μL of a solution prepared by mixing DAPT (DAPT>=$98% (HPLC), solid, Sigma-Aldrich) with DMSO (Dimethysiol Sulfoxide, Kanto Chemical) to a concentration of 8.6 mg / mL was dissolved in 50 mL of culture medium and filtered through a 0.22 μm syringe filter.

[0133] In the second week, a culture medium without DAPT and DMSO was used. The medium was stored at 4°C, protected from light by aluminum foil. Before use, it was left at room temperature for at least 30 minutes. Next, the cells were awakened and seeded onto plates. Frozen motor neurons (iCell® motor neurons, Fujifilm) were thawed and seeded at a rate of 100 μL per well in a 96-well V-bottom plate, resulting in 2500 cells per spheroid.

[0134] The culture medium was changed every 2-3 days after seeding, with 70 μL of medium being replaced in each well. On the 7th day after the start of culture, motor neuron spheroids were transferred to well member 2b. To promote spheroid adhesion and axonal extension, the well members were fitted into a neuron culture stand fabricated with a 3D printer (see Figures 7 and 8 for shape), and cultured for 1 day. Figure 16 shows the neuron culture stand 4 with well member 2b fitted inside.

[0135] In Experimental Example 5, the external dimensions of the neuron culture platform 4 used were 9 mm in the front-to-back and left-to-right directions, the dimensions of the platform 4a in the front-to-back and left-to-right directions were 7 mm, the diameter of the opening 4a1 was 6 mm, the vertical length from the top surface of the platform 4a to the top end of the neuron culture platform 4 was 2.5 mm, the vertical length from the bottom surface of the platform 4a to the bottom end of the neuron culture platform 4 was 1 mm, the vertical length of the platform 4a was 0.5 mm, and the vertical length L between the bottom surface 2b1c of the membrane portion 2b1 and the bottom end of the neuron culture platform 4 was 1.5 mm.

[0136] [Experimental Example 6] (Co-culture of tissue) Using tweezers, the well member 2b was fitted into the anchor member 2a, and the lower part of the membrane portion 2b1 shown in Figure 14 was brought into contact (adhered) with the skeletal muscle tissue to initiate co-culture. After one day of co-culture, movement (contraction) of the skeletal muscle tissue was observed, confirming that the axons had joined to the skeletal muscle tissue within one day. After co-culture, half of the culture medium was added to the skeletal muscle tissue differentiation medium and half to the motor nerve medium without DAPT, and half of the culture medium was replaced daily.

[0137] Figure 17 shows the co-cultured tissue 10 prepared in Experimental Example 6 and the co-culture device 1. Figure 18 shows the co-cultured tissue 10 prepared in Experimental Example 6 after being removed from the co-culture device 1 using tweezers. As shown in Figure 18, by grasping and lifting the pair of left and right anchor members 2a with tweezers, it was possible to move only the culture section 2.

[0138] [Experimental Example 7] (Observation of axons passing through through holes) Figure 19 is a microscopic image showing the through-pores 2b1a formed in the membrane portion 2b1 prepared in Experimental Example 3. Figure 20 is a fluorescence microscopic image showing motor neuron spheroids 10b seeded in the membrane portion 2b1 in Experimental Example 5. Figure 21 is a fluorescence microscopic image showing the axon 10b1 of the motor neuron spheroid 10b passing through the through-pores 2b1a formed in the membrane portion 2b1 prepared in Experimental Example 3. Figures 20 and 21 are fluorescence staining images using an anti-β-tubulin antibody, where the green fluorescent areas representing the motor neuron spheroids 10b or their axon 10b1 are shown in light color. The anti-β-tubulin antibody used was Anti-β-Tubulin, Neuronal Class III, Mouse-Mono(Tuj-1) from R&D Systems.

[0139] As shown in Figure 19, it can be seen that many through-holes 2b1a are formed in the membrane portion 2b1 (track-etched membrane film). As shown in Figure 20, in the membrane portion 2b1, countless axons extend radially from the motor neuron spheroid 10b. As shown within the circle in Figure 21, it can be seen that the axon 10b1 of the motor neuron spheroid 10b extends downward through the through-hole 2b1a of the membrane portion 2b1.

[0140] [Experimental Example 8] (Observation of muscle contraction by electrical stimulation) In this experiment, electrodes were inserted into both ends of cultured skeletal muscle tissue (without motor neuron connections) fixed with a pair of anchor members 2a, and electrical stimulation at a frequency of 1 Hz and voltage of 60 Vp-p was applied. The distance traveled by a reference point defined at a specific location in the skeletal muscle tissue was measured as the contraction distance.

[0141] Figure 22 is a graph showing the change in contraction distance of skeletal muscle tissue when electrical stimulation is applied. As shown in Figure 22, it can be observed that skeletal muscle tissue repeatedly contracts and relaxes in response to electrical stimulation at a frequency of 1 Hz. This result suggests that skeletal muscle tissue that contracts in response to stimulation can be created by encapsulating myoblasts in hydrogel, connecting them to a pair of anchor members 2a, and culturing them. [Industrial applicability]

[0142] According to the present invention, since only the axons can be joined to skeletal muscle tissue through a plurality of through-holes formed on the inner bottom surface of a pair of well members, the connection state between motor neurons and skeletal muscle tissue in vivo can be reproduced. Furthermore, since the pair of anchor members supporting the skeletal muscle tissue can be removed from the culture stand, the neuro-skeletal muscle co-cultured tissue obtained by co-culture can be easily used in other experiments. Therefore, the present invention has industrial applications. [Explanation of Symbols]

[0143] 1... Co-culture device, 2... Culture section, 2a... Anchor member, 2a2... Pillar section, 2a2a... Columnar section, 2a2b... Enlarged diameter section, 2a3... Frame section, 2b... Well member, 2b1... Membrane section, 2b1a... Through hole, 2b2... Side wall section, 2b3... Neuron housing space, 3... Culture platform, 3a... Recess, 3a1... Bottom surface of recess, 3a2... Open end, 3a3... Closed end, 3b... Protrusion between recesses, 4... Neuron culture platform 4...Neuron culture platform, 4a...Platform, 4a1...Opening, 4b...Legs, 4c...Peripheral wall, 4d...Culture medium storage space, 5...Bottom of container, 10...Skeletal muscle co-cultured tissue, 10a...Skeletal muscle tissue, 10b...Motor neuron, 10b1...Axon of motor neuron, 10c...Neuromuscular junction, 21...Substrate, 30...Culture platform, 31...Anti-displacement piece, 32...Anti-displacement part, A...Mold, B...Mold, H...Hydrogel

Claims

1. A co-culture device for co-culturing motor neurons and skeletal muscle tissue, A pair of anchor members that support skeletal muscle tissue, A pair of well members are attached to the upper part of the pair of anchor members, in which motor neurons are seeded, The system comprises a base member that supports the pair of anchor members from below, Multiple through holes are formed on the inner bottom surface of each well member, extending all the way to the bottom surface. Each anchor member is provided with a pillar portion that protrudes upward and supports the skeletal muscle tissue. A co-culture device in which the pair of anchor members can be detachably attached to the base member.

2. The base member is roughly plate-shaped, and a pair of recesses are formed on the upper surface of the base member. The co-culture device according to claim 1, wherein the pair of anchor members can be fitted into the pair of recesses for attachment.

3. The co-culture device according to claim 2, wherein the base member has a shape without a recess between the pair of recesses.

4. Each well member consists of a membrane portion that forms the bottom, It has a closed annular shape when viewed from above, and includes a side wall portion that constitutes the side wall of the well member, The co-culture device according to claim 1, wherein a plurality of through holes are formed in the membrane portion.

5. Each well member and anchor member has a roughly rectangular shape when viewed from above. Notches are formed at the four corners of the side wall portion of each well member. The co-culture device according to claim 4, wherein each anchor member has a frame portion at each of its four corners that fits into the notches to position the well member.

6. The co-culture device according to claim 1, wherein the pillar portion has a columnar portion that extends vertically in a columnar shape and an enlarged diameter portion that expands horizontally from the upper part of the columnar portion.

7. The thickness of the membrane portion in the vertical direction is 1 to 20 μm. The co-culture device according to claim 4, wherein the diameter of the through-hole is 1 to 10 μm.

8. The co-culture device according to claim 1, wherein each anchor member comprises a plurality of the pillar portions, and each pillar portion is coated with fibronectin.

9. A method for producing co-cultured tissue of motor neurons and skeletal muscle tissue, The steps include seeding motor neurons in the pair of well members of the co-culture device according to claim 1, A step of forming skeletal muscle tissue that connects the pillar portion of one of the pair of anchor members to the pillar portion of the other anchor member, The process involves attaching well members, on which the motor neurons are seeded, to each anchor portion where the skeletal muscle tissue is formed, and placing the well members on the skeletal muscle tissue. A method for producing co-cultured tissue, comprising the steps of: placing a well member on the skeletal muscle tissue, culturing the motor neurons, and connecting the axons of the motor neurons to the skeletal muscle tissue through the through-holes.

10. The process of forming the skeletal muscle tissue is as follows: A hydrogel comprising at least one of skeletal muscle myoblasts and skeletal muscle cells, thrombin, fibrinogen, and a basement membrane matrix, is used to connect the pillar portion of one of the pair of anchor members to the pillar portion of the other anchor member. A method for producing a co-cultured tissue according to claim 9, comprising the step of heating the hydrogel connecting the pillar portions.

11. The method for producing a co-cultured tissue according to claim 9, wherein the motor neurons seeded in the well member are motor neuron spheroids.

12. Before attaching the well members in which the motor neurons are seeded to each anchor member, A method for producing co-cultured tissue according to claim 9, further comprising the step of culturing the motor neurons seeded in the well member for 12 hours or more in advance with a culture medium present below the through-holes of the well member.

13. The co-culture device is as described in claim 1, and the co-culture device comprises a pair of neuron culture platforms on which the pair of well members of the co-culture device are placed, Each neuron culture stand comprises a base on which the well member is placed, legs extending downward from the edge of the base to support the base, and a peripheral wall extending upward from the edge of the base to surround the well member. The base portion has a through hole that penetrates in the vertical direction. The aforementioned through-hole is a co-culture kit.

14. The co-culture kit according to claim 13, wherein when the well member is placed on each of the pair of neuron culture platforms, the vertical distance between the lower surface of the well member and the lower end of the leg portion is 0.5 mm or more.

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