Cultivation method and culture vessel

Abstract

In a culture vessel having a plurality of wells, culturing can be performed under optimum culture conditions according to the types of cells to be cultured in each well. This culture method comprises: filling, with a nerve cell medium, a first well 1 of a culture vessel, and a second well 2 communicating with the first well 1 through a flow path 3; seeding a nerve cell N in the first well 1; culturing the axon a of the nerve cell N until the axon a of the nerve cell N reaches the second well 2 through the flow path 3 and blocks the flow path 3; then removing the medium in the second well 2 and filling the second well 2 with a medium for a co-cultured cell that is co-cultured with the nerve cell N; and seeding a cell C to be co-cultured in the second well 2, and culturing the cell C together with the axon a of the nerve cell N.

Description

[Technical field]

[0001] The present invention relates to a cell culture method and a culture vessel, and in particular to a method for culturing nerve cells, a method for co-culturing nerve cells and cells other than nerve cells, and a culture vessel for carrying out these cultures. [Background technology]

[0002] In vivo, axons extending from nerve cells are connected to cells other than nerve cells, such as skeletal muscle and skin cells, and therefore co-culture of nerve cells with other cells is expected to be an effective screening tool in the field of drug discovery. For example, Patent Document 1 describes a method in which a cell mass of nerve cells is cultured in a specified culture vessel, and the axons of the nerve cells are bundled and extended, and then connected to skeletal muscle at the end of the axons.

[0003] On the other hand, in the drug discovery stage, high-throughput drug screening is required. As a culture vessel used for high-throughput screening, a multi-well plate with 96 or more wells is known. For example, Patent Document 2 describes that wells of a multi-well plate are connected to each other, and nerve cells and non-neuronal cells are co-cultured to create a neuronal network. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2017 / 187696 [Patent Document 2] Special Publication No. 2018-536424 Summary of the Invention [Problem to be solved by the invention]

[0005] Here, co-culture is a method of seeding different cells in the culture space of a culture vessel and culturing them, but since the suitable medium differs depending on the type of cell, it is difficult to culture different cells in the same vessel. One method is to develop a medium in which both types of cells can be cultured, but developing such a medium is not necessarily easy.

[0006] In the co-culture of nerve cells and cells other than nerve cells, the objective is to use different media suitable for the culture of the well in which the cell body of the nerve cells is cultured and the well in which the axon extending from the cell body and the cells such as skeletal muscle cells connected thereto are cultured. Even if co-culture is not performed, it is also an objective to use different media suitable for the culture of the well in which the cell body of the nerve cells is cultured and the well in which the tip of the axon extending from the cell body is cultured. However, neither Cited Documents 1 nor 2 describes a specific means for making the media different between the communicating wells.

[0007] The present invention has been made in consideration of the above-mentioned background and technical problems, and aims to provide a culture method and a culture vessel having multiple wells, which enable culture to be performed under optimal culture conditions depending on the type of cell to be cultured in each well. [Means for solving the problem]

[0008] As a result of intensive research into this technical background, the inventors discovered that by controlling the number of neurons and the thickness (cross-sectional area) of the flow channel and blocking the channel with the axons themselves extending through the channel, they could isolate each well that was previously connected by a flow channel, and thus completed the present invention. Furthermore, they found that by controlling the thickness (cross-sectional area) and length of the flow channels connecting the wells in the culture vessel within a specific range, the liquids, such as culture medium, filled in each well can be retained between the wells connected by the flow channels without mixing with each other, even when no cells are present in the flow channels.

[0009] The present invention has been made based on the above findings and aims to advantageously solve the above problems. A first aspect of the present invention is a culture method for co-culturing a plurality of types of cells including nerve cells in a culture vessel having a plurality of wells, which comprises filling a first well of the culture vessel and a second well communicating with the first well via a flow path with a medium for nerve cells, seeding nerve cells in the first well, culturing the axons of the nerve cells until the axons of the nerve cells reach the second well through the flow path and block the flow path, removing the medium in the second well and filling the second well with a medium for co-cultured cells to be co-cultured with the nerve cells, seeding the second well with cells to be co-cultured and culturing them together with the axons of the nerve cells. By culturing the axons of the neurons until the flow path between the wells is blocked, the connection between the first well and the second well via the flow path can be severed and the second well can be filled with a culture medium different from that used for the first well for culture. Therefore, in a culture vessel having multiple wells, the cell body of a neuronal cell cultured in a first well, and the tip of an axon extending from the cell body cultured in the first well toward the second well and another cell connected to the axon, such as a skeletal muscle cell cultured in the second well, can be cultured under separate suitable medium conditions.

[0010] A second aspect of the present invention is a culture method for culturing neurons in a culture vessel having a plurality of wells, comprising filling a first well of the culture vessel and a second well communicating with the first well via a flow path with culture medium for neurons, seeding neurons into the first well, culturing the axons of the neurons until they reach the second well through the flow path and block the flow path, and then removing the culture medium from the second well, filling it with culture medium for axon culture, and culturing the axons. By culturing the axons of the neurons until the flow path between the wells is blocked, the connection between the first well and the second well via the flow path can be severed and the second well can be filled with a culture medium different from that used for the first well for culture. Thus, in a culture vessel having multiple wells, the cell body of a neuron can be cultured in a first well under suitable medium conditions, while the tip of the axon extending from the cell body of the neuron can be cultured in a second well under different medium conditions to promote axon growth.

[0011] In the first and second aspects, it is preferable that the first well has a first recess in the bottom capable of accommodating a cell cluster, and that the cell cluster of nerve cells is seeded in the first recess. By doing this, the cell clusters are less likely to fall apart, and the axons extending from the cell bodies of each nerve cell are more likely to grow together in one direction.

[0012] In the first and second aspects, the nerve cells may be central nerve cells or peripheral nerve cells. Specifically, the central nerve cells include cells derived from glutamatergic nerves, dopaminergic nerves, and GABAergic nerves. The peripheral nerve cells include cells derived from motor nerves and sensory nerves. The cells may be directly collected from animals or may be established cell lines. The cells may also be cells obtained by differentiation and culture of various stem cells.

[0013] A third aspect of the present invention is a culture vessel having multiple wells for culturing neurons or for co-culturing multiple types of cells including neurons, the culture vessel comprising a first well having a first recess at the bottom capable of accommodating a cell cluster, and a second well communicating with the first well via a flow path, the maximum width of the flow path in a direction perpendicular to the longitudinal direction being 20 to 100 μm, and the depth of the flow path being 20 to 100 μm. By controlling the flow path connecting the wells within the above range, the flow path can be blocked by axons of neurons cultured in an appropriate number according to the type of neurons. After that, the second well is filled with a medium different from that in the first well, cells other than neurons (cells for co-culture) are seeded, cultured, and joined to the tips of the axons, and cultured in a medium suitable for co-culture. Even if co-culture is not performed, different media suitable for each culture can be used for the well in which the cell bodies of neurons are cultured and the well in which the tips of the axons extending from the cell bodies are cultured.

[0014] In the third aspect, the nerve cells are peripheral nerve cells, and the number of peripheral nerve cells seeded in the first well is 1×10 4 ~4×10 4 cells, and the maximum cross-sectional area of ​​the flow channel is 4×10 -6 ~1×10 -4 cm 2 The peripheral nerve cells are preferably motor nerve cells. When the cells to be seeded are peripheral nerve cells, the flow channel can be blocked with higher precision by controlling the number of cells and the cross-sectional area of ​​the flow channel within the above ranges.

[0015] In the third aspect, the nerve cells are central nerve cells, and the number of central nerve cells seeded in the first well is 4×10 4 ~8×10 4 cells, and the maximum cross-sectional area of ​​the flow channel is 4×10 -6 ~1×10 -4 cm 2 The central nerve cells are preferably glutamatergic nerve cells. When the cells to be seeded are central nerve cells, the flow channel can be blocked with higher precision by controlling the number of cells and the cross-sectional area of ​​the flow channel within the above ranges.

[0016] In the third aspect, the second well may have a second recess in the bottom portion thereof capable of accommodating a cell cluster. By doing so, even when cell clusters for co-culture are seeded in the second well, the cell clusters are less likely to crumble, and culture can be carried out in a cell cluster state with high cell density.

[0017] A fourth aspect of the present invention is a culture vessel having a plurality of wells for culturing neurons or for co-culturing a plurality of types of cells including neurons, the culture vessel comprising a first well having a first recess in the bottom capable of accommodating a cell cluster, and a second well communicating with the first well via a flow path, one end of the flow path being connected to the bottom of the first well and the other end of the flow path being connected to the bottom of the second well, the maximum width of the flow path in a direction perpendicular to the longitudinal direction being 20 to 120 μm, the depth of the flow path being 20 to 120 μm, and the longitudinal length of the flow path being 2 to 6 mm. In this way, by controlling the width and length of the flow channel within a specific range, it is possible to prevent the liquid in the first well from mixing with the liquid in the second well through the flow channel even when the flow channel is not filled with cells, and therefore it is possible to fill the second well with a medium different from that of the first well for culture. Therefore, in a culture vessel having multiple wells, the cell body of a neuronal cell cultured in a first well, and the tip of an axon extending from the cell body cultured in the first well toward the second well and another cell connected to the axon, such as a skeletal muscle cell cultured in the second well, can be cultured under separate suitable medium conditions.

[0018] In the above aspect, at least the surface of the portion in contact with nerve cells may have a water contact angle of 90 to 15°. In this way, the nerve cells can be more stably attached and cultured on the culture surface of the culture vessel.

[0019] In the above aspect, at least the surface of the portion that comes into contact with nerve cells may be made of an alicyclic structure-containing polymer. In this way, the alicyclic structure-containing polymer has high transparency, little autofluorescence (especially green), and little toxicity, making it possible to stably culture nerve cells in conditions that facilitate observation and analysis during the culture process.

[0020] A fifth aspect of the present invention is a culture method for co-culturing multiple types of cells including neurons using the culture vessel of the fourth aspect described above, comprising filling a first well of the culture vessel with medium for neurons, filling a second well communicating with the first well via a flow path with medium for co-cultured cells to be co-cultured with the neurons, seeding the neurons in the first well and seeding the cells to be co-cultured in the second well, culturing the axons of the neurons until they reach the second well through the flow path, and then culturing the axons of the neurons together with the cells to be co-cultured in the second well. In this way, by using a culture vessel in which the width and length of the flow channel are controlled within a specific range, it is possible to prevent the liquid in the first well from mixing with the liquid in the second well through the flow channel even when there are no cells in the flow channel. Therefore, from the start of culture, the second well can be filled with a medium different from that of the first well for culture.

[0021] A sixth aspect of the present invention is a culture method for culturing neurons using the culture vessel of the fourth aspect described above, comprising filling a first well of the culture vessel with a medium for neurons, filling a second well communicating with the first well via a flow path with a medium for axon culture, seeding neurons in the first well, culturing the axons of the neurons until they pass through the flow path and reach the second well, and then culturing the axons in the second well. In this way, by using a culture vessel in which the width and length of the flow channel are controlled within a specific range, it is possible to prevent the liquid in the first well from mixing with the liquid in the second well through the flow channel even when there are no cells in the flow channel. Therefore, from the start of culture, the second well can be filled with a medium different from that of the first well for culture. Effect of the Invention

[0022] According to the present invention, in a culture vessel having a plurality of wells, culture can be performed under optimal culture conditions according to the type of cell to be cultured in each well. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a plan view of a well body of a 1×8 strip well for a 96-well plate according to one embodiment of the present invention. [Diagram 2] FIG. 2 is a cross-sectional view of the well body. [Diagram 3] FIG. 3 is a partial plan view of a member that constitutes a part of the well bottom and is joined to the well body of FIG. [Figure 4] FIG. 4 is a cross-sectional view of a member that constitutes a part of the well bottom. [Diagram 5] FIG. 5 is a partial plan view of a 1×8 strip well for a 96 well plate according to another embodiment of the present invention. [Figure 6] FIG. 6 is a partial plan view of a 96-well plate according to yet another embodiment of the present invention. [Figure 7] FIG. 7 is a partial plan view of a well body of a 1×8 strip well for a 96-well plate according to yet another embodiment of the present invention. [Figure 8] FIG. 8 is a plan view of a member constituting a part of the well bottom, which is joined to the well body of FIG. [Figure 9] FIG. 9 is a cross-sectional view of a member that constitutes a part of the well bottom of FIG. [Figure 10] FIG. 10 is a plan view of a 6-well plate according to yet another embodiment of the present invention. [Figure 11] FIG. 11 is a cross-sectional view of the six-well plate of FIG. [Figure 12] FIG. 12 is an inverted photomicrograph (×40 magnification) showing axon bundles extending through the channel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. The culture method and culture vessel of the present invention are characterized in that, when co-culturing neurons with other cells, or when culturing the cell body and the tip of the axon of a neuron in separate wells, it is possible to culture the second well in a culture medium different from that of the first well by blocking the flow path connecting the first well and the second well in the culture vessel with the axon of the cultured neuron, or by controlling the width and length of the flow path connecting the first well and the second well in the culture vessel within a specific range. This makes it possible to culture efficiently by using different media for each well depending on the type of cultured cells, without using any special process or member for blocking the flow path.

[0025] FIG. 1 is a plan view of a well body B of a 1×8 strip well for a 96-well plate according to one embodiment of the present invention. As shown in FIG. 1, the well body B has a plurality of wells including a first well 1 and a second well 2 formed therein, and a positioning recess 5 for joining with a member D constituting a part of the well bottom is provided on the bottom surface. The first well 1 has a first recess 4 for seeding a cell mass of nerve cells N formed therein. The bottom surface of the first recess 4 is open and forms a part of the flow path 3. The flow path 3 is formed by the first recess 4, the bottom surface of the well body B, and a groove on the upper surface of the member D constituting a part of the well bottom, by joining the well body B with the member D constituting a part of the well bottom.

[0026] FIG. 2 is a cross-sectional view of the well body B in FIG. 1. FIG. 3 is a partial plan view of a member D constituting a part of the well bottom, which is joined to the well body B in FIG. 1, and includes a groove of a flow path 3 that communicates the first well 1 and the second well 2, and a positioning protrusion 6 for joining to the well body B. The planar shape of the member D constituting a part of the well bottom is made to match the planar shape of the well body B in FIG. 1, but is not limited thereto. FIG. 4 is a cross-sectional view of the member D constituting a part of the well bottom. By joining the positioning recess 5 of the well body B in FIGS. 1 and 2 to the positioning protrusion 6 of the member D constituting a part of the well bottom in FIGS. 3 and 4, a well strip is created in which a flow path 3 that communicates the first well 1 and the second well 2 is formed. Since member D constituting part of the plate bottom in Figures 3 and 4 is composed of two wells, four members D constituting part of the well bottom are joined to one well body B, but this is not limited to this, and one well strip may be produced by forming the same number of wells as the number of wells in one member D and joining one well body B to one member D.

[0027] 1 to 4 show a plate having the same size as a 96-well plate commonly used in analytical devices by connecting twelve 1×8 well strips via a holder (for example, manufactured by Sumitomo Bakelite Co., Ltd., model number: MS-8508M), but the present invention is not limited thereto, and as long as a plurality of wells are formed, the plate may be a well strip having any number of wells, such as 6 wells, 12 wells, 24 wells, 48 ​​wells, or may be in the shape of a well plate. The size of the plate is preferably a size that complies with the ANSI / SBS standard that is compatible with experimental devices, and is, for example, the same dimensions as a commonly available 96-well plate of about 8.5 (length) × 12.8 (width). The culture vessel of the present invention is not limited to a strip well and a well plate, but may be configured to have two or more culture sections connected via a flow path, such as, for example, two dishes connected via a flow path.

[0028] The culture vessel according to the present invention may be made of materials used in general culture vessels, such as polystyrene, polycarbonate, polyolefin, cycloolefin polymer, polyimide, fluorinated products thereof, PDMS (polydimethylsiloxane), glass, etc. COP (cycloolefin polymer) is preferred because it has low self-luminescence and is suitable for fluorescence observation.

[0029] 1 to 4, the flow path 3 connecting the first well 1 and the second well 2 is formed by a groove formed on the upper surface of the member D constituting a part of the well bottom, and the flat bottom surface of the well body B and the first recess 4, but is not limited thereto, and may be formed, for example, by the groove and the first recess 4 formed on the bottom surface of the well body B, and the flat upper surface of the member D constituting a part of the well bottom. Alternatively, grooves may be formed on both the bottom surface of the well body B and the upper surface of the member D constituting a part of the well bottom, and one flow path 3 may be formed by joining these.

[0030] The cross-sectional shape of the groove constituting the flow path 3 in a direction perpendicular to the longitudinal direction may be rectangular, or may have an arc-shaped bottom such as a semicircular shape. The flow path 3 formed may also be rectangular, circular, elliptical, or the like. The size of the flow channel 3 is such that the maximum width in the direction perpendicular to the longitudinal direction is 20 to 100 μm, and the depth of the flow channel is 20 to 100 μm, or the maximum width in the direction perpendicular to the longitudinal direction of the flow channel is 20 to 120 μm, the depth of the flow channel is 20 to 120 μm, and the length of the flow channel in the longitudinal direction is 2 to 6 mm. By controlling the flow channel communicating between the wells within the above range, the medium does not flow between the wells, so that only the medium in the second well can be discarded. This allows the culture of the tip of the axon in the second well to be cultured, or the co-culture of the axon and cells other than nerve cells, by filling the first well with a medium different from that in the first well. Preferably, from the viewpoint of further increasing the sealing property, the maximum width in the direction perpendicular to the longitudinal direction of the flow channel 3 is 20 to 80 μm, and the depth of the flow channel is 20 to 80 μm.

[0031] A culture method according to one embodiment of the present invention will be described below: A first well 1 is filled with a medium for culturing nerve cells, and a second well 2 is filled with a medium suitable for culturing cells C to be co-cultured. Nerve cells N formed into cell clusters by culture are seeded in a first recess 4 formed in the bottom of a first well 1. Cells C to be co-cultured are seeded in a second well 2. An axon a extends from the cell body s of the seeded nerve cell N, and the extended axon a is cultured until it passes through the flow path 3 and reaches the second well 2. In the second well 2, the tip of the extended axon a is co-cultured with a cell C, and the axon a is joined to the co-cultured cell C. A culture method according to another embodiment of the present invention will be described below. A first well 1 and a second well 2 are filled with a medium for culturing nerve cells, and nerve cells N formed into a cell mass by culture are seeded in a first recess 4 formed in the bottom of the first well 1. An axon a extends from a cell body s of the seeded nerve cells N, and the extended axon a reaches the second well 2 through the flow channel 3, and is cultured until it blocks the inside of the flow channel 3. Since the extended axon a completely blocks the flow channel 3, the medium exchange of the first well 1 and the second well 2 can be performed independently, and only the medium of the second well 2 in which the tip of the extended axon a is cultured is discarded by suction or the like.

[0032] Thereafter, the second well 2 is filled with a medium suitable for culturing the cells C to be co-cultured, and the cells C to be co-cultured are seeded and co-cultured to bond the axons a with the cells C to be co-cultured. Here, as shown in Fig. 5, a recess (second recess 7) may be formed in the second well 2 and in the bottom of the well, and the cells C to be co-cultured in the form of a cell mass may be seeded in this second recess 7.

[0033] Alternatively, instead of filling the second well 2 with a medium suitable for culturing the co-cultured cell C, the second well 2 may be filled with a medium suitable for culturing the tip portion of the axon a, and the culturing of only the axon a may be continued. In this way, neurotransmitters such as acetylcholine, glutamate, dopamine, serotonin, adrenaline, and noradrenaline secreted from the cord terminal (presynaptic terminal) can be detected in the culture medium of the second well 2 using an enzyme reaction system, and can be used for neurotoxicity evaluation, drug discovery screening, diagnosis, etc.

[0034] By carrying out cell culture by co-culture in the above-described steps, a neural network is formed in the second well 2. By carrying out the above-described steps in multiple sets of wells, multiple such neural networks can be produced in the multiwell plate.

[0035] Specifically, when co-culture is performed, as shown in FIG. 6, a plate can be used in which a first well 1 and a maximum of four second wells 2 are formed as a set, with a maximum of four adjacent second wells 2 for one first well 1. Cell clusters of nerve cells N are seeded in the first recesses 4 formed corresponding to each of the channels 3, and the axons a of the nerve cells N are extended into each of the second wells 2 to block a maximum of four channels 3 extending from the first well 1. Then, the medium in each of the second wells 2 is aspirated, and the medium suitable for the culture of the cells to be co-cultured is filled in each well. Then, the cells to be co-cultured are seeded in each of the second wells 2. In this way, a maximum of four types of cells can be co-cultured with the axon a portion of the nerve cells N in the set of wells. By performing such a process in multiple sets of wells, a multi-well plate in which multiple neural networks are formed can be produced. The multi-well plate produced in this way can be used for high-throughput drug screening.

[0036] 6 is a partial plan view of a 96-well plate, showing 32 of the 96 wells. The number of wells is not limited to 96, and multiwell plates with the same size as commercially available multiwell plates and the number of wells appropriate for the purpose, such as 6 wells, 12 wells, 24 wells, 48 ​​wells, etc., may be prepared and used.

[0037] In addition, the wells shown in FIGS. 1 to 6 have a circular shape in plan view, but are not limited thereto, and may have a square (rectangle) shape in plan view as shown in FIG. 7. When the wells have a circular shape in plan view, the number of channels 3 extending from one first well 1 is theoretically a maximum of eight in order to communicate between wells with the same channel length. However, since there is usually a wall between wells required for manufacturing design, the number of channels is a maximum of four per well, as shown in FIG. 6. By making the wells have a square shape in plan view, it is also possible to form a plurality of channels 3 that can communicate between a pair of adjacent wells with the same channel length. For example, if three first recesses 4 are formed per side on the side of the first well 1 adjacent to the second well 2 among the sides of the square of the first well 1, and there are four second wells 2 adjacent to the first well 1, a maximum of 12 channels 3 extending from one first well 1 can be formed. Furthermore, by making the wells have a rectangular shape in plan view, the length of the flow channel 3 can be made shorter than when the wells have a circular shape in plan view.

[0038] Fig. 7 is a partial plan view of well body B of a 1 × 8 strip well for a 96-well plate according to another embodiment of the present invention, and includes a first well 1, a second well 2, a first recess 4, and a positioning recess 5. Figs. 8 and 9 are plan and cross-sectional views, respectively, of member D constituting a part of the well bottom, which is used in conjunction with well body B in Fig. 7, and includes a groove portion of flow channel 3 that communicates between first well 1 and second well 2, and a positioning protrusion 6 for joining with well body B.

[0039] The embodiment shown in the above-mentioned Figures 7 to 9 has a similar configuration to the strip well shown in Figures 1 to 4, but is different in that three channels 3 and first recesses 4 are formed from one first well 1 to one second well 2. By making the well shape rectangular in plan view, it is possible to form a plurality of channels 3 of the same length for a pair of the first well 1 and the second well 2. Furthermore, due to the structure of the well shape, the length of the formed channel 3 can be made shorter than when the well shape is circular in plan view. This makes it possible to shorten the culture period until the axon a reaches the second well 2 and blocks the channel 3.

[0040] In the above embodiment, three first recesses 4 and flow paths 3 are formed on one side of the rectangular well, but this is not limited to this, and any number of first recesses 4 and flow paths 3 can be formed on each side depending on the well size and the size of the first recesses 4.

[0041] The first recess 4 preferably has a diameter of 0.5 to 2 mm in order to facilitate insertion of the cell aggregates. Moreover, the first recess 4 may have the same diameter at its opening and the same diameter at its bottom. From the viewpoint of operability and from the viewpoint of culturing the cell clusters more stably, it is preferable that the diameter of the opening is equal to or greater than the diameter of the bottom. Specifically, from the viewpoint of making it easier to insert the cell clusters, it is preferable that the first recess 4 has a tapered shape in which the diameter becomes smaller from the opening to the bottom. More preferably, the first recess 4 has a Y-shape in which the diameter is tapered from the opening to the middle of the bottom and the diameter is the same from the middle of the bottom to the bottom.

[0042] 10 and 11 are a plan view and a cross-sectional view of a 6-well plate according to yet another embodiment of the present invention. The embodiment shown in FIGS. 10 and 11 has a similar configuration to the strip well shown in FIGS. 1 to 4, but is different in that, whereas the strip well shown in FIGS. 1 to 4 has a groove forming a part of the flow channel 3 formed on the upper surface of a member D constituting a part of the well bottom, the groove is formed on the lower surface of the well body B in this embodiment. Regardless of the method of forming the groove, the bottom side of the flow channel becomes the joint, so that the overflow of the plate forming material into the flow channel 3 that occurs when the well body B is joined to the member D constituting a part of the well bottom does not hinder the proliferation of nerve cells N.

[0043] The size (thickness) of the flow channel 3 is approximately the same as the thickness of the axon bundle formed by bundling multiple axons a of the cultured nerve cells N. The thickness of the axon bundle is determined by the type and number of nerve cells N. For motor nerve cells, the number of cells is 1×10 4 ~2×10 4 The thickness of the axon bundle is about 80 μm for glutamatergic neurons with a cell count of 3 × 10 4 ~5×10 4 In the case of cells, the thickness of the axon bundle is about 80 μm. The number of cells to be cultured is adjusted so that the thickness of the axon bundle is approximately the same as the thickness at the inlet of the flow channel 3. Since there is a difference in the speed at which each axon extends, the thickness of the axon bundle tends to decrease toward the tip of the axon bundle. By doing this, the inside of the inlet of the flow channel 3 can be blocked by the axon bundle.

[0044] In view of the above, the size of the flow channel 3 of the co-culture vessel according to one embodiment of the present invention is such that the maximum width in the direction perpendicular to the longitudinal direction of the flow channel 3 is 20 to 100 μm, and the depth of the flow channel is 20 to 100 μm. From the viewpoint of more reliably increasing the sealing property of the flow channel, when the cells to be seeded are peripheral nerve cells, the number of peripheral nerve cells to be seeded in the first recess 4 is set to 1×10 4 ~4×10 4 cells, and the cross-sectional area of ​​channel 3 is 4×10 -6 ~1×10 -4 cm2 In addition, when the cells to be seeded are central nerve cells, the number of central nerve cells to be seeded in the first well 4 is preferably 4×10 4 ~8×10 4 cells, and the cross-sectional area of ​​channel 3 is 4×10 -6 ~1×10 -4 cm 2 It is preferable that:

[0045] Examples of the cells C to be co-cultured include, but are not limited to, skeletal muscle cells, skin cells, etc. In addition, together with or instead of the cells C to be co-cultured, an artificial object such as microbeads covered with Lrp4 described in Yumoto et al. "Lrp4 Is A Retrograde Signal For Presynaptic Differentiation At Neuromuscular Synapses" Nature, 489, 438-442 (2012) may be used for co-culture. When such an artificial object is used for co-culture, the medium may be the same as or different from the medium for the nerve cells placed in the first well.

[0046] The cultured nerve cells may be central nerve cells or peripheral nerve cells. Specifically, the central nerve cells include cells derived from glutamatergic nerves, dopaminergic nerves, and GABAergic nerves. The peripheral nerve cells include cells derived from motor nerves and sensory nerves. The above cells may be obtained by direct collection from animals, or may be cell lines thereof. The above cells may also be cells obtained by differentiation and culture of various stem cells.

[0047] The thickness of the axon bundle extending from a nerve cell is determined by the type and number of nerve cells. For motor nerve cells, the number of cells is 1×10 4 ~2×10 4 The thickness of the axon bundle is about 80 μm for glutamatergic neurons with a cell count of 3 × 10 4 ~5×10 4In the case of cells, the thickness of the axon bundle is approximately 80 μm.

[0048] In a culture vessel according to another embodiment of the present invention, a flow path connecting a first well and a second well has a maximum width in a direction perpendicular to the longitudinal direction of the flow path in the range of 20 to 120 μm, a depth of the flow path in the range of 20 to 120 μm, and a longitudinal length of the flow path in the range of 2 to 6 mm. If either the width or depth of the channel is less than 20 μm, it is difficult to form the channel and the axons are less likely to extend through the channel, whereas if either the width or depth of the channel is more than 120 μm, the media in both wells tend to mix through the channel. When the longitudinal length of the channel is less than 2 mm, the media in the first and second wells tend to mix through the channel, whereas when the longitudinal length of the channel is more than 6 mm, it becomes difficult for the axons to reach the second well through the channel.

[0049] By controlling the width and length of the flow channel within the above range, it is possible to stably prevent the liquid in the first well from mixing with the liquid in the second well through the flow channel even when there are no cells in the flow channel. Therefore, from the start of culture, the second well can be filled with a medium different from that of the first well for culture. Therefore, it is possible to prepare in advance for culture of the tip portion of the axon in the second well, or for co-culture of the axon with cells other than nerve cells.

[0050] To allow the thickness of the axon to fill the flow channel and make it easier for the tip of the axon to reach the second well, it is preferable that the maximum width of the flow channel in a direction perpendicular to the longitudinal direction is 60 to 80 μm, the depth of the flow channel is 60 to 80 μm, and the longitudinal length of the flow channel is 2 to 3 mm.

[0051] In the culture vessel according to the above aspect of the present invention, the water contact angle of at least the surface of the part in contact with the nerve cells is preferably 90 to 15°. By controlling the water contact angle within such a range, the nerve cells can be more stably attached to the culture surface of the culture vessel and cultured. More preferably, the water contact angle is 90 to 60° so that the adhesive strength of the cells is neither too strong nor too weak. The water contact angle can be controlled within the above range by a surface modification treatment such as atmospheric pressure plasma treatment, reduced pressure plasma treatment, vacuum ultraviolet treatment, corona treatment, or ozone treatment. Here, the water contact angle was measured using a fully automatic contact angle meter (Kyowa Interface Science Co., Ltd. "LCD-400S"), and the bottom surface of the culture vessel (well) was cut out with a Φ30 mm circle cutter to measure the center of the sample and the four vertices of a square with sides of 20 mm around the center. The measurement points were five points in total, and the radius r and height h of the droplet were calculated, and tan θ1 = h / r, θ = 2θ 1 →θ is calculated by θ = 2arctan(h / r) (θ / 2 method).

[0052] The material of the culture vessel according to the above aspect of the present invention can be any material commonly used for culture vessels, such as polystyrene, polycarbonate, polyolefin, cycloolefin polymer, polyimide, fluorinated products thereof, PDMS (polydimethylsiloxane), glass, etc. The material of the culture vessel is preferably an alicyclic structure-containing polymer, since it has low green autoluminescence, is suitable for fluorescence observation, is highly transparent, and has low toxicity, and it is preferable that at least the surface of the part that comes into contact with the nerve cells is composed of an alicyclic structure-containing polymer.

[0053] The alicyclic structure-containing polymer is a resin having an alicyclic structure in the main chain and / or side chain, and preferably has an alicyclic structure in the main chain from the viewpoint of mechanical strength, heat resistance, etc., and more preferably has no polar group from the viewpoint of differentiation induction efficiency. Here, the polar group refers to a polar atomic group. Examples of the polar group include an amino group, a carboxyl group, a hydroxyl group, and an acid anhydride group.

[0054] Examples of the alicyclic structure include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. From the viewpoints of mechanical strength, heat resistance, and the like, a cycloalkane structure or a cycloalkene structure is preferred, and among these, those having a cycloalkane structure are most preferred.

[0055] The number of carbon atoms constituting the alicyclic structure is not particularly limited, but is usually 4 to 30, preferably 5 to 20, and more preferably 5 to 15. When the number of carbon atoms constituting the alicyclic structure is within this range, the mechanical strength, heat resistance, and moldability are highly balanced, which is preferable.

[0056] The proportion of repeating units having an alicyclic structure in the alicyclic structure-containing polymer may be appropriately selected according to the intended use, but is usually 30% by weight or more, preferably 50% by weight or more, more preferably 70% by weight or more. If the proportion of repeating units having an alicyclic structure in the alicyclic structure-containing polymer is too small, the heat resistance is poor, which is not preferable. The remainder other than the repeating units having an alicyclic structure in the alicyclic structure-containing polymer is not particularly limited and may be appropriately selected according to the intended use.

[0057] Specific examples of the alicyclic structure-containing polymer include (1) norbornene-based polymers, (2) monocyclic olefin-based polymers, (3) cyclic conjugated diene-based polymers, (4) vinyl alicyclic hydrocarbon-based polymers, and hydrogenated products of (1) to (4), etc. Among these, norbornene-based polymers and hydrogenated products thereof are preferred from the viewpoints of heat resistance, mechanical strength, etc.

[0058] (1) Norbornene-based polymers Norbornene polymers are obtained by polymerizing norbornene monomers, which are monomers having a norbornene skeleton, and are roughly classified into those obtained by ring-opening polymerization and those obtained by addition polymerization.

[0059] Examples of the products obtained by ring-opening polymerization (cycloolefin polymers; COPs) include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers copolymerizable therewith, and hydrogenated products thereof. Examples of the products obtained by addition polymerization (cycloolefin copolymers; COCs) include addition polymers of norbornene-based monomers, and addition polymers of norbornene-based monomers and other monomers copolymerizable therewith. Among these, hydrogenated ring-opening polymers of norbornene-based monomers are preferred from the viewpoints of heat resistance, mechanical strength, etc.

[0060] Norbornene monomers that can be used to synthesize norbornene polymers include bicyclo[2.2.1]hept-2-ene (commonly known as norbornene), 5-methyl-bicyclo[2.2.1]hept-2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, and 5-ethylidene-bicyclo[2.2.1]hept-2-ene. Bicyclic monomers such as hept-2-ene, 5-vinyl-bicyclo[2.2.1]hept-2-ene, 5-propenylbicyclo[2.2.1]hept-2-ene, 5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, 5-cyanobicyclo[2.2.1]hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene; Tricyclo[4.3.0 1,6 .1 2,5 ] Tricyclic monomers such as deca-3,7-diene (commonly known as dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, and 2,3-dihydroxydicyclopentadiene; Tetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene (tetracyclododecene), tetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-methyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethyltetracyclo[4.4.0.1 2,5 .17,10 ]-3-dodecene, 8-ethylidenetetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8,9-dimethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethyl-9-methyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethylidene-9-methyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-methyl-8-carboxymethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 7,8-benzotricyclo[4.3.0.1 2,5 ]dec-3-ene (commonly known as methanotetrahydrofluorene: also known as 1,4-methano-1,4,4a,9a-tetrahydrofluorene), 1,4-methano-8-methyl-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene, and other tetracyclic monomers;

[0061] Other monomers capable of ring-opening copolymerization with norbornene monomers include monocyclic cycloolefin monomers such as cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, 1,5,9-cyclododecatriene, and 1,5,9,13-cyclohexadecatetraene. These monomers may have one or more kinds of substituents, such as an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

[0062] Other monomers that can be addition copolymerized with norbornene monomers include α-olefin monomers having 2 to 20 carbon atoms, such as ethylene, propylene, 1-butene, 1-pentene, and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo[9.2.1.0 2,10 .0 3,8 ]Cycloolefin monomers such as tetradeca-3,5,7,12-tetraene (also called 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); non-conjugated diene monomers such as 1,4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene; and the like.

[0063] Among these, as the other monomer capable of addition copolymerization with the norbornene monomer, an α-olefin monomer is preferable, and ethylene is more preferable. These monomers may have one or more kinds of substituents, such as an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

[0064] The ring-opening polymer of a norbornene monomer, or the ring-opening polymer of a norbornene monomer and another monomer capable of ring-opening copolymerization therewith, can be obtained by polymerizing the monomer components in the presence of a known ring-opening polymerization catalyst. As the ring-opening polymerization catalyst, for example, a catalyst consisting of a metal halide such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a catalyst consisting of a metal halide or an acetylacetone compound such as titanium, zirconium, tungsten, or molybdenum, and an organoaluminum compound can be used. The hydrogenated ring-opening polymer of a norbornene monomer can usually be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to a polymerization solution of the ring-opening polymer and hydrogenating the carbon-carbon unsaturated bonds.

[0065] The addition polymer of a norbornene-based monomer or the addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith can be obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst, such as a catalyst made of a titanium, zirconium or vanadium compound and an organoaluminum compound.

[0066] (2) Monocyclic olefin polymer As the monocyclic olefin polymer, for example, an addition polymer of a monocyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used. (3) Cyclic conjugated diene polymer As the cyclic conjugated diene polymer, for example, a polymer obtained by 1,2- or 1,4-addition polymerization of a cyclic conjugated diene monomer such as cyclopentadiene or cyclohexadiene, or a hydrogenated product thereof can be used. (4) Vinyl alicyclic hydrocarbon polymer Examples of vinyl alicyclic hydrocarbon polymers include polymers of vinyl alicyclic hydrocarbon monomers such as vinylcyclohexene and vinylcyclohexane, and hydrogenated products thereof; hydrogenated aromatic ring moieties of polymers of vinyl aromatic monomers such as styrene and α-methylstyrene; etc. The vinyl alicyclic hydrocarbon polymer may be a copolymer of these monomers with other copolymerizable monomers.

[0067] There is no particular restriction on the molecular weight of the alicyclic structure-containing polymer, but the weight average molecular weight, calculated as polyisoprene, measured by gel permeation chromatography of a cyclohexane solution (a toluene solution if the polymer is not soluble) is usually 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and particularly preferably 10,000 to 100,000. When the weight average molecular weight is within this range, the mechanical strength and moldability are well balanced, which is preferable.

[0068] The glass transition temperature of the alicyclic structure-containing polymer may be appropriately selected depending on the purpose of use, but is usually 50 to 300° C., preferably 80 to 280° C., particularly preferably 90 to 250° C., and further preferably 90 to 200° C. When the glass transition temperature is within this range, heat resistance and moldability are highly balanced, which is preferable. The glass transition temperature of the alicyclic structure-containing polymer in the present invention is measured based on JIS K 7121.

[0069] The above alicyclic structure-containing polymers can be used alone or in combination of two or more kinds. Furthermore, to the alicyclic structure-containing polymer, compounding agents that are usually used in thermoplastic resin materials, for example, soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, fluorescent brighteners, etc., can be added in usually employed amounts. The alicyclic structure-containing polymer may be mixed with other polymers (hereinafter simply referred to as "other polymers") other than the soft polymer. The amount of the other polymers mixed with the alicyclic structure-containing polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, and more preferably 100 parts by mass or less, based on 100 parts by mass of the alicyclic structure-containing polymer. If the ratio of various additives or other polymers to the alicyclic structure-containing polymer is too high, cells will be difficult to suspend, so it is preferable to add them in an amount that does not impair the properties of the alicyclic structure-containing polymer. The mixing method of the alicyclic structure-containing polymer with the compounding agent and other polymers is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer.In addition, there is no particular limit to the order of mixing.As the mixing method, for example, a method of kneading the resin in a molten state using a mixer, a single-screw kneader, a twin-screw kneader, a roll, a Brabender, an extruder, etc., a method of dissolving and dispersing the resin in a suitable solvent, and then removing the solvent by a solidification method, a casting method, or a direct drying method, etc. can be mentioned. When using a twin-screw kneader, after kneading, the material is usually extruded in a molten state into a rod shape, cut to an appropriate length with a strand cutter, and pelletized for use.

[0070] The method for forming the culture vessel used in the present invention can be selected arbitrarily depending on the desired shape of the culture vessel. Examples of the forming method include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendar molding, rolling molding, cutting molding, spinning, etc., and these forming methods can also be combined.

[0071] The culture vessels used in the present invention are preferably sterilized. There are no particular limitations on the sterilization method, and it can be selected from methods commonly used in the medical field, such as heating methods such as high-pressure steam and dry heat; radiation methods that irradiate with radiation such as gamma rays and electron beams, and irradiation methods that irradiate with high frequency waves; gas methods that involve contact with gases such as ethylene oxide gas (EOG); and filtration methods that use sterilizing filters, depending on the shape of the molded body and the cells to be used. EXAMPLES

[0072] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. (Example 1-1) The bottom of each well of a 1 × 8 strip-well shaped culture vessel according to the present invention, which was made of a cycloolefin polymer (ZEONEX (registered trademark) 690R, Zeon Corporation), and in which four pairs of first wells 1 and second wells 2 were formed as shown in Figure 1, was coated with a coating liquid (iMatrix (registered trademark)-211, manufactured by Nippi Corporation). After that, 400 μl / well of a neuronal culture medium (manufactured by Veritas, model number: ST-05790) that had been warmed to 37°C was added to each well, and 2 × 10 cells were cultured in a 96-well plate. 4A cell cluster (sphere) of motor neurons (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell (registered trademark) Motor Neuron) from 1990 cells was seeded one by one in each of the first recesses 4 having a diameter of 1 mm formed in the bottom of the first well 1. It was confirmed under a microscope that the cell cluster was placed in the first recess 4, and the cells were incubated at 37° C., 5% CO 2 The cells were cultured in an incubator. The medium was replaced once every 3 to 4 days. Specifically, the medium was gently sucked up with a 1000 μm pipette and discarded, and fresh medium for neuronal culture was added to each well at 400 μl / well.

[0073] Axons extended from the motor neurons placed in the first recess 4, passed through the channel 3, which had a width of 80 μm in the direction perpendicular to the longitudinal direction and a depth of 80 μm, and reached the second well 2. The culture was continued until the axon bundle formed by bundling the axons completely blocked the inside of the channel 3. After that, the medium in the second well 2 was completely aspirated and removed, and a medium (manufactured by Takara Bio, Inc., model number: C-23160) for culturing human skeletal muscle cells (manufactured by Takara Bio, Inc., model number: C-12530) for co-culturing with the axons of the nerve cells was added to the second well 2 at 400 μl / well. Then, the above-mentioned human skeletal muscle cells were seeded in the second well 2 at a cell number of 14400 cells, and co-cultured with the axons of the nerve cells, and the axons and the human skeletal muscle cells were joined.

[0074] Whether the axon bundles completely blocked the inside of the flow channel 3 was confirmed by microscopic observation. As shown in the microscopic photograph of FIG. 12, it can be seen that the axon bundles fill the inside of the flow channel 3. Furthermore, even when the medium in the second well 2 was aspirated, the medium in the first well 1 did not flow in from the flow channel 3. This also shows that the axon bundles completely blocked the inside of the flow channel 3.

[0075] (Example 1-2) The same culture process as in Example 1 was carried out, with the only differences being that the type of neurons to be cultured was glutamatergic neurons, that the material of the 1×8 strip-well shaped culture vessel according to the present invention was polydimethylsiloxane (manufactured by Dow Corning Toray Co., Ltd., model number: SILPOT 184 W / C), and that co-culture cells were not used. 4 Glutamatergic neurons (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell (registered trademark) Gluta Neuron) were cultured in a neuronal culture medium (manufactured by Veritas, model number: ST-05790). As in Example 1, the axon bundle filled and blocked the inside of the flow channel 3, so that the medium in the first well 1 did not flow in from the flow channel 3 even when the medium in the second well 2 was aspirated. This shows that the axon bundle completely blocked the inside of the flow channel 3. As described above, the culture was continued until the axon bundle formed by bundling the axons of the glutamatergic neurons completely blocked the inside of the flow channel 3. Then, the medium in the second well 2 was completely removed by suction, and a glia-conditioned medium (KAC Corporation, product number: SBMBX9501D-3A) containing 100-fold diluted N2 supplement (Thermo Fisher Scientific, product number: 17502048) and 50-fold diluted B27 supplement (Thermo Fisher Scientific, product number: 17504044) was added, and the culture was continued further. As a result, the secretion of glutamate, a neurotransmitter secreted from the axon terminal, could be confirmed from the medium in the second well 2 by observing the change in absorbance of the coenzyme using glutamate dehydrogenase.

[0076] (Example 2-1) As shown in Fig. 1, a culture vessel was used, which was composed of a well body B made of PDMS (manufactured by Shin-Etsu Chemical Co., Ltd., product name: ultraviolet-curable liquid silicone rubber, product number: KER-4690-A / B, water contact angle 90°) in which four pairs of first wells 1 and second wells 2 were formed, and a member D constituting a part of the well bottom made of glass (manufactured by AGC Technoglass Co., Ltd., product name: borosilicate glass). The flow channel 3 formed between the first well 1 and the second well 2 was rectangular, with a width in the direction perpendicular to the longitudinal direction of 120 μm, a depth of 120 μm, and a length in the longitudinal direction (distance between wells) of 2 mm. A neuronal culture medium (manufactured by Veritas, product number: ST-05790) was added to each of the first wells 1 at 400 μl / well, and pure water was added to each of the second wells 2 communicating with the first wells 1 via a flow path 3 at 400 μl / well. It was visually confirmed that the neuronal culture medium in the first wells 1 and the pure water in the second wells 2 did not mix via the flow path 3 even after 24 hours.

[0077] Then, the neuronal culture medium in the first well 1 was gently sucked up with a 1000 μl pipette and discarded, and 400 μl / well of neuronal culture medium (Veritas, model number: ST-05790) that had been warmed to 37°C was added to each well. The number of cells cultured in the 96-well plate was 2 × 10 4 A cell cluster (sphere) of motor neuron cells (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell (registered trademark) Motor Neuron) was seeded in each of the first recesses 4 with a diameter of 1 mm formed on the bottom of the first well 1, and it was confirmed under a microscope that the cell cluster was placed in the first recess 4. Next, the pure water in the second well 2 was gently sucked up with a 1000 μ pipette and discarded, and a medium (manufactured by Takara Bio, Inc., model number: C-23160) for culturing human skeletal muscle cells (manufactured by Takara Bio, Inc., model number: C-12530) for co-culturing with the axons of the nerve cells was added to the second well 2 at 400 μl / well. Then, the human skeletal muscle cells were seeded in the second well 2 at a cell number of 14,400 cells. The culture vessel was incubated at 37 °C, 5% CO 2The cells were cultured in an incubator. The medium was replaced once every 3 to 4 days. Specifically, the medium was gently sucked up with a 1000 μm pipette and discarded, and fresh medium was added to each well at 400 μl / well. Cultivation was continued until axon a extended from the motor neuron placed in the first recess 4, as shown in Figure 12, passed through the flow channel 3 and reached the second well 2, and then co-cultured with the axon of the neuron to join the axon with the human skeletal muscle cell. Co-culture was possible without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2. Even when motor neurons were cultured alone, the neuronal culture medium in the first well 1 and the medium for culturing axons a in the second well 2 did not mix during the culture period of about one week.

[0078] (Example 2-2) The same test and cell culture process as in Example 1 were carried out, with the only difference being that the depth of the flow channel 3 in the culture vessel was 20 μm. As in Example 1, it was confirmed that even after 24 hours, the neuronal culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow path 3. In addition, co-culture could be performed without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2.

[0079] (Example 2-3) Tests and cell culture steps similar to those in Example 1 were carried out, with the only difference being that the width of the channel 3 of the culture vessel in the direction perpendicular to the longitudinal direction was 20 μm. As in Example 1, it was confirmed that even after 24 hours, the neuronal culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow path 3. In addition, co-culture could be performed without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2.

[0080] (Examples 2-4) Tests and cell culture processes similar to those in Example 1 were carried out, with the only differences being that the culture vessel was made of COP (manufactured by Zeon Corporation, product name: Zeonex, product number: 690R), the width of the flow path 3 in the direction perpendicular to the longitudinal direction was 80 μm, the depth of the flow path 3 was 80 μm, and the water contact angle was 80°. As in Example 1, it was confirmed that even after 24 hours, the neuronal culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow path 3. In addition, co-culture could be performed without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2.

[0081] (Examples 2-5) Tests and cell culture processes were carried out similarly to those in Example 1, with the only differences being that the culture vessel was formed of COP, the width of the flow path 3 in the direction perpendicular to the longitudinal direction was 80 μm, the depth of the flow path 3 was 20 μm, and the water contact angle was 80°. As in Example 1, it was confirmed that even after 24 hours, the neuronal culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow path 3. In addition, co-culture could be performed without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2.

[0082] (Examples 2-6) Tests and cell culture processes were carried out similarly to those in Example 1, with the only differences being that the culture vessel was formed of COP, the width of the flow path 3 in the direction perpendicular to the longitudinal direction was 20 μm, the depth of the flow path 3 was 80 μm, and the water contact angle was 80°. As in Example 1, it was confirmed that even after 24 hours, the neuronal culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow path 3. In addition, co-culture could be performed without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2.

[0083] (Examples 2-7) Tests and cell culture steps similar to those in Example 1 were carried out, with the only difference being that the culture vessel was made of COP and the length of the flow channel 3 in the longitudinal direction was 6 mm. As in Example 1, it was confirmed that even after 24 hours, the neuronal culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow path 3. In addition, co-culture could be performed without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2.

[0084] (Examples 2-8) The culture vessel was formed from PS (polystyrene) (manufactured by Toyo Styrene Co., Ltd., product name: Toyo Styrol GP, grade: MW1C), and the width of the flow path 3 in the direction perpendicular to the longitudinal direction was 60 μm, the depth of the flow path 3 was 60 μm, and the water contact angle was 40°. Tests and cell culture processes were carried out in the same manner as in Example 1. As in Example 1, it was confirmed that even after 24 hours, the neuronal culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow path 3. In addition, co-culture could be performed without mixing the neuronal culture medium in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2.

[0085] (Comparative Example 1-1) The same culture process as in Example 1 was carried out, except that the number of motor neuron cells to be cultured was different from that in Example 1. Specifically, the number of motor neuron cells was 5×10 3 The cells were cultured in the first recess 4 of the first well 1. Number of cells 5×10 3 In the case of cells, the axons of the cultured neurons were not able to sufficiently block the inside of the flow path 3, and when the medium in the second well 2 was aspirated, the medium in the first well 1 flowed into the second well 2 through the flow path 3, making it impossible to independently add medium for co-culture to the second well 2.

[0086] (Comparative Example 1-2) The same culture process as in Example 1 was carried out, except that the number of motor neuron cells to be cultured was different from that in Example 1. Specifically, the number of motor neuron cells was 5×10 4 The cells were cultured in the first recess 4 of the first well 1. Number of cells 5×10 4 In the case of cells, nutrients do not reach the inside of the cell mass shape, so the cells inside die and a sufficient number of axons cannot be cultured. When the medium in the second well 2 was aspirated, the medium in the first well 1 flowed into the second well 2 through the flow path 3, making it impossible to independently add medium for co-culture to the second well 2.

[0087] (Comparative Example 2-1) The same test and cell culture steps as in Example 1 were carried out, with the only difference being that the length of the flow channel 3 in the longitudinal direction was 1.5 mm. Immediately after the medium and pure water were added to each well, the neuronal culture medium in the first well 1 and the pure water in the second well 2 began to mix through the flow path 3, and after 24 hours, the mixing of the medium and pure water could be clearly confirmed with the naked eye. Therefore, without mixing the medium for culturing nerve cells in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2, co-culture could not be performed.

[0088] (Comparative Example 2-2) The same test and cell culture steps as in Example 1 were carried out, with the only difference being that the depth of the flow channel 3 was 18 μm. As in Example 1, it was confirmed that even after 24 hours, the nerve cell culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow channel 3. However, the flow channel 3 was too narrow, so the axon a extending from the motor nerve cell could not pass through it.

[0089] (Comparative Example 2-3) Preparations were made to carry out the same tests and cell culture steps as in Example 1, with the only difference being that the width of the flow channel 3 in the direction perpendicular to the longitudinal direction was 18 μm. However, it was not possible to create a mold for a culture vessel for forming a flow channel with the above-mentioned width, and it was not possible to mold the culture vessel.

[0090] (Comparative Example 2-4) Tests and cell culture processes were carried out similarly to those in Example 1, with the only differences being that the culture vessel was formed of COP, the width perpendicular to the longitudinal direction of the flow path 3 was 80 μm, the depth of the flow path 3 was 80 μm, and the length of the flow path 3 in the longitudinal direction was 1.5 mm. Immediately after the medium and pure water were added to each well, the neuronal culture medium in the first well 1 and the pure water in the second well 2 began to mix through the flow path 3, and after 24 hours, the mixing of the medium and pure water was clearly visible. Therefore, without mixing the medium for culturing nerve cells in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2, co-culture could not be performed.

[0091] (Comparative Example 2-5) Tests and cell culture processes were carried out in the same manner as in Example 1, with the only differences being that the culture vessel was made of COP, the width perpendicular to the longitudinal direction of the flow path 3 was 80 μm, the depth of the flow path 3 was 18 μm, and the length of the flow path 3 in the longitudinal direction was 1.5 mm. As in Example 1, it was confirmed that even after 24 hours, the nerve cell culture medium in the first well 1 and the pure water in the second well 2 did not mix through the flow channel 3. However, the flow channel 3 was too narrow, so the axon a extending from the motor nerve cell could not pass through it.

[0092] (Comparative Example 2-6) Tests and cell culture steps similar to those in Example 1 were carried out, with the only differences being that the culture vessel was made of COP, the width of the channel 3 in the direction perpendicular to the longitudinal direction was 150 μm, and the depth of the channel 3 was 150 μm. Immediately after the medium and pure water were added to each well, the neuronal culture medium in the first well 1 and the pure water in the second well 2 began to mix through the flow path 3, and after 24 hours, the mixing of the medium and pure water was clearly visible. Therefore, without mixing the medium for culturing nerve cells in the first well 1 and the medium for culturing human skeletal muscle cells in the second well 2, co-culture could not be performed. [Industrial Applicability]

[0093] According to the culture method and culture vessel of the present invention, in a culture vessel having multiple wells, the cell body of a nerve cell and the tip part of the axon extending from the cell body can be cultured under separate and suitable medium conditions. Also, the cell body of a nerve cell, the tip part of the axon and another cell such as a skeletal muscle cell connected thereto can be cultured under separate and suitable medium conditions. This allows culture to be performed by using different media for each well depending on the cultured cells without complicating the culture process. This also allows a neural network of multiple types of cells to be efficiently formed between multiple wells. [Explanation of symbols]

[0094] 1. First well 2 Second well 3. Flow Path 4 First recess 5 Positioning recess 6 Positioning protrusion 7 Second recess B Well Body D. Part of the well bottom N nerve cell s cell body a Axon C. Cells to be co-cultured

Claims

1. A culture method for co-culturing a plurality of types of cells including neural cells in a culture vessel having a plurality of wells, comprising: Filling a first well of the culture vessel and a second well communicating with the first well via a flow path with a medium for neural cells, and seeding neural cells in the first well; Cultivating the axons of the nerve cells until the axons of the nerve cells reach the second well through the channel and block the channel, and then A culture method comprising removing the medium from the second well and filling it with medium for co-cultured cells to be co-cultured with neural cells, seeding the cells to be co-cultured into the second well and culturing them together with the axons of the neural cells.

2. A culture method for culturing nerve cells in a culture vessel having a plurality of wells, comprising: Filling a first well of the culture vessel and a second well communicating with the first well via a flow path with a medium for neural cells, and seeding neural cells in the first well; Cultivating the axons of the nerve cells until the axons reach the second well through the channel and block the channel, and then The culture method comprises removing the medium from the second well and filling it with a medium for axon culture, and culturing the axons.

3. 3. The culture method according to claim 1 or 2, wherein the first well has a first recess in a bottom portion capable of accommodating a cell cluster, and the cell cluster of neural cells is seeded in the first recess.

4. The culture method according to claim 1 or 2, wherein the nerve cells are central nerve cells or peripheral nerve cells.

5. A culture vessel for carrying out the culture method of claim 1 or 2, A first well having a first recess in a bottom portion capable of accommodating a cell cluster; a second well communicating with the first well via a flow path; Equipped with A culture vessel, wherein the maximum width of the flow channel in a direction perpendicular to the longitudinal direction is 20 to 100 μm, and the depth of the flow channel is 20 to 100 μm.

6. the nerve cell is a peripheral nerve cell, The number of peripheral nerve cells seeded in the first well is 1×10 ４ ~4×10 ４ cells, and the maximum cross-sectional area of ​​the flow channel is 4×10 -６ ~1×10 -４ cm ２ The culture vessel according to claim 5 ,

7. the nerve cell is a central nerve cell, The number of central nerve cells seeded in the first well is 4×10 ４ ~8×10 ４ cells, and the maximum cross-sectional area of ​​the flow channel is 4×10 -６ ~1×10 -４ cm ２ The culture vessel according to claim 5 ,

8. The culture vessel according to claim 5 , further comprising a second recess in the bottom of the second well capable of accommodating a cell cluster.

9. A culture vessel for carrying out the culture method of claim 1 or 2, A first well having a first recess in a bottom portion capable of accommodating a cell cluster; a second well communicating with the first well via a flow path; Equipped with One end of the flow path is connected to the bottom of a first well, and the other end of the flow path is connected to the bottom of a second well; The maximum width of the flow channel in a direction perpendicular to the longitudinal direction is 20 to 120 μm, and the depth of the flow channel is 20 to 120 μm; A culture vessel, wherein the length of the flow channel in the longitudinal direction is 2 to 6 mm.

10. 10. The culture vessel according to claim 9, wherein at least the surface of the portion in contact with the nerve cells has a water contact angle of 90 to 15°.

11. The culture vessel according to claim 9, wherein at least the surface of the portion in contact with the nerve cells is made of an alicyclic structure-containing polymer.

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