Cell culture device and method for using the same

Abstract

To provide a cell culture device capable of setting culture conditions suitable for each cell before contacting multiple cells, and to provide a method for using the cell culture device.SOLUTION: A cell culture device including a bottom and a main body joined to the bottom consists of a through-hole penetrating the main body and comprises: a first well and a second well formed in positions adjacent to each other in a direction parallel to a main surface of the bottom; a microchannel formed in a part of a joining surface between the bottom and the main body, and mutually communicating the first well and the second well; and an opening / closing unit configured so that its position in a direction perpendicular to the main surface of the bottom can be changed, and adjusting the opening degree of the microchannel.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture device, and more particularly to a cell culture device used for culturing a plurality of cells in contact with each other. The present invention also relates to a method for using the cell culture device. [Background technology]

[0002] In vivo, neurites growing from nerve cells come into contact with other cells such as cardiac muscle, skeletal muscle, or skin cells. In order to observe the interactions between these cells, a method of culturing nerve cells in vitro (outside the body) in a state where they are in contact with other cells is known (see Patent Document 1 below). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6430680 Summary of the Invention [Problem to be solved by the invention]

[0004] Conventionally, as exemplified by Patent Document 1, cell culture devices have been proposed that have multiple wells and flow channels connecting the multiple wells to each other, with the aim of culturing nerve cells in contact with other cells.

[0005] However, the inventors realized that with conventional cell culture devices, it is difficult to set appropriate culture conditions for each cell before bringing the cells into contact with each other. If appropriate culture conditions cannot be set for each cell, it becomes difficult to culture each cell to the desired state. Even if such cells are cultured in contact with each other, the interaction between the cells cannot be reproduced, resulting in reduced observation accuracy.

[0006] In view of the above-mentioned problems, the present invention aims to provide a cell culture device that allows culture conditions suitable for each of a plurality of cells to be set before contacting the cells, and a method for using the cell culture device. [Means for solving the problem]

[0007] The cell culture device according to the present invention comprises: 1. A cell culture device comprising: a bottom portion; and a body portion joined to the bottom portion, a first well and a second well, each of which is a through hole that penetrates the main body and is formed adjacent to each other in a direction parallel to a main surface of the bottom; a microchannel formed in a part of the joining surface between the bottom and the main body, the microchannel connecting the first well and the second well; and an opening / closing unit configured to be able to change its position in a direction perpendicular to the main surface of the bottom portion and to adjust the opening of the microchannel.

[0008] In this specification, the term "main surface" refers to a surface that is much larger in area than the other surfaces of a plate-like object.

[0009] Fig. 17 is a cross-sectional view showing the structure of a conventional cell culture device disclosed in Patent Document 1. As shown in Fig. 17, cell culture device 80 is configured by joining an upper plate 80a and a lower plate 80b. Cell culture device 80 has a plurality of wells (81, 82) formed by through-holes penetrating upper plate 80a, and a microchannel 83 connecting wells 81 and 82 with each other. As an example, the width and height of microchannel 83 are approximately 100 µm to 200 µm.

[0010] When culturing cells, neuron 91 is placed on the bottom surface of well 81, and a culture medium is poured into well 81 so as to cover neuron 91. Furthermore, in well 82, target cell 92 to be brought into contact with neuron 91 is placed on the bottom surface, and a culture medium is poured into well 82 so as to cover cell 92. As neuron 91 is cultured, neurite 91a grows from neuron 91. Neurite 91a reaches well 82 via microchannel 83 and binds with cell 92. This makes it possible to observe the interaction between neuron 91 and cell 92 while they are in contact with each other.

[0011] However, the present inventors have noticed that the cell culture device having the structure shown in FIG. 17 has the following problems.

[0012] Before contacting the neuron 91 with the cell 92, the neuron 91 and the cell 92 need to be cultured to a desired state. Here, the required culture conditions vary depending on the individual cell. However, in the cell culture device 80, the well 81 and the well 82 are connected by the microchannel 83. Therefore, before the neuron 91 and the cell 92 come into contact with each other, a physiologically active substance released by the neuron 91 diffuses into the well 82 and affects the culture of the cell 92. Similarly, a physiologically active substance released by the cell 92 diffuses into the well 81 and affects the culture of the neuron 91.

[0013] Furthermore, because individual cells have different growth rates, the required culture period for each cell varies. For example, it is assumed that culture of neuron 91 is started in well 81, and then, after a predetermined period of time has elapsed, culture of cell 92 is started in well 82. However, because well 82 is connected to well 81 via microchannel 83, it is conceivable that the culture medium for neuron 91 and the physiologically active substance released by neuron 91 will have diffused into well 82 when cell 92 is placed in well 82. Even when culture of neuron 91 and cell 92 is started simultaneously, it is difficult for an operator to simultaneously introduce the respective cells and culture medium into each well.

[0014] As described above, it has been difficult to set culture conditions suitable for each cell before the cells come into contact with each other in the cell culture device 80. If culture conditions suitable for each cell cannot be set, it becomes difficult to culture each cell in the desired state.

[0015] It is also possible to culture each cell separately and then move them with a pipette or other device to bring them into contact with each other, but there is a risk that the cells may be damaged when they are moved in and out with the pipette, and this method is not considered appropriate from the perspective of observation accuracy.

[0016] The present inventors have conducted extensive research to solve the above problems and have come up with the present invention.

[0017] According to the above configuration, the opening degree of the microchannel can be adjusted by the opening / closing unit. As will be described in detail later, by closing the opening / closing unit, it is possible to culture cells in each of the first well and the second well while blocking the microchannel. In other words, blocking the microchannel suppresses the diffusion of the culture solution and the like between the first well and the second well. This makes it possible to set culture conditions suitable for each individual cell in the first well and the second well.

[0018] Furthermore, after the culture of each cell is completed, the opening and closing section can be opened to connect the first well and the second well. With this configuration, when bringing the cells into contact with each other, it is not necessary to remove the cells using, for example, a pipette, and the cells are less likely to be damaged.

[0019] The cell culture device is When viewed from the main body side, a slit may be formed at a position sandwiched between the first well and the second well, and into which an opening adjustment mechanism can be introduced.

[0020] In the cell culture device, The opening / closing portion may have a partition plate inserted into the slit.

[0021] In addition, the cell culture device Further provided is a fluid supply unit that introduces or discharges a fluid into the slit, The opening and closing portion may have a film that covers the opening on the bottom side of the slit.

[0022] The detailed structure of the opening and closing section will be described in detail in the section entitled "Forms for Carrying Out the Invention."

[0023] In the cell culture device, The height of the microchannel may be smaller than the diameter of a nerve cell and larger than the diameter of a neurite.

[0024] According to the above configuration, when neurons are cultured in the first well or the second well, the neurons are prevented from invading the microchannel. On the other hand, neurites growing from the neurons can be introduced into the microchannel. The above configuration is advantageous in that it makes it easy to evaluate the junction between the neurites growing from the neurons and the cells.

[0025] In the cell culture device, The microchannel may be composed of a plurality of grooves formed in the bottom portion.

[0026] A plurality of neurites grow from a nerve cell. With the above configuration, the neurites growing from the nerve cell can be made to infiltrate into the respective grooves, which makes it easier to observe the neurites.

[0027] The cell culture device is a plurality of said second wells; The first well and the plurality of second wells may be connected to each other by different microchannels.

[0028] The above configuration makes it possible to evaluate interactions between cells in more complex systems. For example, when evaluating interactions between multiple cells, such as in a system within the brain, it becomes more important to set culture conditions suitable for each cell. In other words, the more complex the system, the more susceptible the culture conditions of a cell are to being affected by the culture conditions of other cells. In contrast, the above configuration is advantageous in that it allows for the setting of culture conditions suitable for each cell by adjusting the opening of the microchannel.

[0029] The method for using the cell culture device according to the present invention includes the steps of: A step (a) of operating the opening / closing unit to close the flow path; After the step (a), a step (b) of placing first cells together with a culture medium in the first well and culturing them; Simultaneously with or after the step (b), a step (c) of placing second cells together with a culture medium in the second well and culturing them; and (d) operating the opening / closing unit to open the flow channel and bring the first cells into contact with the second cells.

[0030] In the above usage method, at least one of the first cell and the second cell is a neuron; The step (d) may include contacting the first cell with the second cell via a neurite.

[0031] In addition, in the above method of use, the microchannel of the cell culture device is composed of a plurality of grooves formed on the bottom, The step (d) may include the step of infiltrating neurites into each of the plurality of grooves. [Effects of the Invention]

[0032] According to the present invention, there are provided a cell culture device that allows culture conditions suitable for each of a plurality of cells to be set before contacting the cells, and a method for using the cell culture device. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a perspective view of a cell culture device according to a first embodiment. [Figure 2A] 2 is a cross-sectional view taken along the line AA in FIG. 1. [Figure 2B] 2B is a diagram in which the opening and closing portion is omitted from FIG. 2A. [Figure 3] FIG. 2C is a plan view of FIG. 2B when viewed in the −Z direction. [Figure 4] 2C is a diagram of the microchannel as viewed from the first well in the state of FIG. 2B. [Figure 5] FIG. 10 is a perspective view showing how the partition plate is inserted into the slit. [Figure 6] 2B is a diagram showing a state in which the position of the partition plate has been changed from the state in FIG. 2A. [Figure 7] 10 is a diagram showing a state in which cells that are nerve cells are placed in a first well. [Figure 8] 10 is a diagram showing the state in which cells are placed in a second well. [Figure 9] 1 is a diagram showing a state in which cells are in contact with each other. [Figure 10] 10 is a diagram showing an example different from that of FIG. 9. [Figure 11A] 10 is a diagram showing another example of the configuration of a microchannel. [Figure 11B] 11B is a diagram showing the microchannel according to FIG. 11A in a closed state. [Figure 12A] FIG. 10 is a perspective view of a cell culture device according to a second embodiment. [Figure 12B] 2B is a cross-sectional view of a cell culture device according to a second embodiment, similar to FIG. 2A. [Figure 12C] 12C is a diagram showing a state in which the microchannel is closed, following the state of FIG. 12B. [Figure 13A] 2B is a cross-sectional view of a cell culture device according to a third embodiment, similar to FIG. 2A. [Figure 13B] FIG. 13B is an enlarged view of the vicinity of the hinge in FIG. 13A. [Figure 14A] FIG. 10 is a perspective view showing a configuration example of a cell culture device according to a fourth embodiment. [Figure 14B] 14B is a schematic diagram showing the positional relationship between each well of the cell culture device shown in FIG. 14A and a microchannel. FIG. [Figure 15A] FIG. 10 is a schematic diagram showing another configuration example of the cell culture device according to the fourth embodiment. [Figure 15B] 15B is a schematic diagram showing the positional relationship between each well of the cell culture device shown in FIG. 15A and a microchannel. FIG. [Figure 16A] FIG. 10 is a perspective view showing yet another configuration example of the cell culture device according to the fourth embodiment. [Figure 16B] FIG. 16B is a schematic diagram showing the positional relationship between each well of the cell culture device shown in FIG. 16A and a microchannel. [Figure 17] FIG. 1 is a cross-sectional view showing the structure of a conventional cell culture device. DETAILED DESCRIPTION OF THE INVENTION

[0034] The cell culture device and cell culture method according to the present invention will be described with reference to the drawings. Note that the drawings are merely schematic illustrations. That is, the dimensional ratios in the drawings do not necessarily match the actual dimensional ratios, and the dimensional ratios between the drawings do not necessarily match.

[0035] [First embodiment] (Structure of cell culture device) A cell culture device according to a first embodiment will be described with reference to the drawings. Fig. 1 is a perspective view of the cell culture device according to this embodiment. Fig. 2A is a cross-sectional view taken along line AA in Fig. 1.

[0036] As shown in FIG. 1, the cell culture device 1 includes a bottom portion 2, a main body portion 3 joined to the upper surface of the bottom portion 2, and an opening / closing portion 6, which will be described later.

[0037] The bottom part 2 is formed of, for example, a rectangular substrate. As shown in Fig. 2A, the bottom part 2 is joined to the main body part 3 at a main surface 2a on the +Z side.

[0038] In the following description, an XYZ coordinate system will be referred to where appropriate, with the direction perpendicular to the main surface 2a of the bottom 2 being the Z direction and a plane parallel to the main surface 2a being the XY plane (see FIG. 1, etc.). In the following description, when a positive or negative direction is to be distinguished when expressing a direction, the direction will be described with a positive or negative sign, such as "+X direction" and "-X direction." In addition, when a direction is to be expressed without distinguishing between positive and negative directions, the direction will be simply described as "X direction." In other words, in this specification, when simply referring to the "X direction," both the "+X direction" and the "-X direction" are included. The same applies to the Y direction and the Z direction.

[0039] The main body 3 is bonded to the main surface 2a of the bottom 2. As shown in Fig. 2A, a bonded surface 15 is formed by bonding the bottom 2 and the main body 3 together.

[0040] 2B is a diagram in which the opening / closing unit 6 is omitted from the diagram in FIG. 2A. As shown in FIGS. 2A and 2B, the main body 3 has a first well 11, a second well 12 formed adjacent to and spaced apart from the first well 11 in the X direction, and a slit 7.

[0041] 2B, the first well 11 and the second well 12 are configured as through-holes that penetrate the main body 3 in the Z direction. As a result, the first well 11 and the second well 12 each expose the main surface 2a of the bottom 2 when viewed from the +Z side to the -Z direction.

[0042] 3 is a plan view of FIG. 2B when viewed in the −Z direction. Similar to FIG. 2B, the opening / closing portion 6 is not shown in FIG. 3. In this embodiment, the first well 11 and the second well 12 have a rectangular shape when viewed in the −Z direction. However, the shapes of the first well 11 and the second well 12 are arbitrary, and may be circular when viewed in the −Z direction, for example.

[0043] As shown in FIG. 2A, the first well 11 and the second well 12 are connected to each other in the X direction by a microchannel 4. The microchannel 4 is formed in a part of the joining surface 15 between the bottom portion 2 and the main body portion 3. FIG. 4 is a diagram of the microchannel 4 as viewed from the first well 11 side in the state of FIG. 2B. As shown in FIG. 4, in this embodiment, the width (dimension in the Y direction) of the microchannel 4 is set to be equal to the width of the first well 11 in the Y direction. In each drawing such as FIG. 4, the height H1 (dimension in the Z direction) of the microchannel 4 is schematically shown.

[0044] As shown in Fig. 3, the slit 7 is formed at a position sandwiched between the first well 11 and the second well 12. As shown in Fig. 2B, the slit 7 penetrates the main body 3 in the Z direction.

[0045] In this embodiment, the slit 7 is configured to allow the introduction of a partition plate 20 (see FIG. 2A). As shown in FIG. 2A, the partition plate 20 is inserted into the slit 7, and its position in the Z direction is changeable. FIG. 5 is a perspective view showing the partition plate 20 being inserted into the slit 7. FIG. 6 is a diagram showing a state in which the position of the partition plate 20 has been changed from the state shown in FIG. 2A.

[0046] 2A and 6, the cross-sectional area of ​​the microchannel 4 in the Y direction is changed by moving the partition plate 20 in the Z direction. In other words, the partition plate 20 functions as an opening adjustment mechanism that adjusts the opening of the microchannel 4. In this embodiment, the partition plate 20 corresponds to the "opening / closing unit 6."

[0047] (How to use the cell culture device) Next, a description will be given of a cell culture method using the cell culture device 1. As an example, the following describes a method in which nerve cells are placed in the first well 11 and cells that constitute skeletal muscle are placed in the second well 12, and these cells are cultured in a state where they are in contact with each other.

[0048] (Step S1: Closing of microchannel 4) First, the position of the partition plate 20 in the Z direction is changed so that the partition plate 20 is in contact with the main surface 2a of the bottom portion 2, thereby closing the microchannel 4 (see FIG. 6).

[0049] Step S1 corresponds to step (a).

[0050] (Step S2: Culturing cells 31) Next, cells are placed in the first well 11 together with a predetermined culture solution. FIG. 7 is a diagram showing the state in which cells 31, which are nerve cells, have been placed in the first well 11. Specifically, a culture solution 32 containing the cells 31 is poured into the first well 11, so that the cells 31 are seeded on the bottom surface of the first well 11. Thereafter, the cells 31 are cultured in the first well 11 for a predetermined period (e.g., several days), so that neurites 31a grow from the cells 31. The cells 31 correspond to the "first cells."

[0051] 7, the microchannel 4 is closed by the partition plate 20. Therefore, while the cells 31 are being cultured, the culture solution 32 and the physiologically active substances released by the cells 31 are prevented from diffusing into the second well 12.

[0052] The "closed state" in which the microchannel 4 is closed includes not only a state in which the partition plate 20 is in contact with the bottom 2, but also a state in which the partition plate 20 is close to the bottom 2 and the microchannel 4 is substantially closed. Here, "substantially closed" may mean that the distance between the partition plate 20 and the bottom 2 is 1.0 μm or less. In consideration of the surface tension of the liquid, etc., a distance of 1.0 μm or less between the partition plate 20 and the bottom 2 prevents the culture solution or the like from passing through the microchannel 4.

[0053] Step S2 corresponds to step (b).

[0054] (Step S3: Cultivation of Cells 33) In the second well 12, target cells to be contacted with the cells 31 are placed together with a predetermined culture solution. FIG. 8 is a diagram showing the state in which the cells 33 are placed in the second well 12. Specifically, a culture solution 34 containing the cells 33 is poured into the second well 12, so that the cells 33 are seeded on the bottom surface of the second well 12. Thereafter, the cells 33 are cultured in the second well 12 for a predetermined period (for example, several days). The cells 33 correspond to "second cells."

[0055] As described above, since the microchannel 4 is closed by the partition plate 20, the diffusion of the culture solution 32 and the like into the second well 12 is suppressed while the cells 31 are being cultured. Therefore, even when the cells 33 are seeded into the second well 12 several days or several weeks after the cells 31 are seeded into the first well 11, the culture conditions of the cells 31 are suppressed from affecting the culture conditions of the cells 33.

[0056] Furthermore, since the microchannel 4 is closed by the partition plate 20, diffusion of the culture solution (32, 34) and the like between the first well 11 and the second well 12 is suppressed. This makes it possible to set culture conditions suitable for the respective cells (31, 33) in the first well 11 and the second well 12.

[0057] In the above description, the cells 31 are cultured to a certain extent before the cells 33 are seeded in the second well 12, but this is not a limitation. For example, even when the culture of the cells 31 and 33 is started simultaneously, the microchannel 4 is closed with the partition plate 20, so that culture conditions suitable for each of the cells (31, 33) can be set.

[0058] Step S3 corresponds to the step (c).

[0059] (Step S4: Contact between cell 31 and cell 33) After steps S2 and S3 are performed and cells 31 and 33 are cultured to the desired state, partition plate 20 is moved in the +Z direction to open microchannel 4. In this state, as culture of cell 31, which is a nerve cell, progresses, neurite 31a reaches second well 12 via microchannel 4. Then, neurite 31a binds to cell 33, and cell 31 and cell 33 come into contact with each other. This makes it possible to culture cell 31 and cell 33 in a state of contact. FIG. 9 is a diagram showing the state in which cell 31 and cell 33 come into contact with each other.

[0060] The partition plate 20 can be moved in the Z direction while being held with, for example, tweezers.

[0061] Step S4 corresponds to step (d).

[0062] Furthermore, with the cell culture device 1, it is also possible to adjust the aperture of the microchannel 4 by moving the partition plate 20 while the cells 31 and 33 are in contact with each other. This makes it possible to adjust, for example, the amount of a physiologically active substance released from each cell that diffuses between the first well 11 and the second well 12. In other words, by adjusting the aperture of the microchannel 4, it is possible to vary the conditions for the transfer of a physiologically active substance between cells. This makes it possible to observe the interaction between cells under various conditions.

[0063] Furthermore, by intentionally bringing the partition plate 20 into contact with the neurites 31a, it is possible to damage a part of the neurites 31a and observe the progress of each cell (31, 33) containing the neurites 31a.

[0064] In the above description, cell 31 is a nerve cell, and neurite 31a binds to cell 33, thereby bringing cell 31 into contact with cell 33. However, both cell 31 and cell 33 may be nerve cells. Furthermore, the cell culture device 1 is not limited to the example in which cells are brought into contact with each other by neurite 31a.

[0065] Fig. 10 is a drawing showing an example different from Fig. 9. As shown in Fig. 10, after the microchannel 4 is opened (step S4), the cells 31 placed in the first well 11 may be allowed to migrate. As a result, the cells 31 move into the second well 12, and come into contact with the cells 33. As an example, the cells 31 are immune cells, and the cells 33 are cancer cells. In other words, the method of using the cell culture device 1 is not limited to whether the cells 31 are nerve cells or not.

[0066] Next, an example of a method for manufacturing the cell culture device 1 will be described below.

[0067] (Preparation of plate-shaped components) The bottom 2 and main body 3 that make up the cell culture device 1 are prepared. At this point, for example, both are rectangular flat plate-shaped members. The dimensions of the bottom 2 and main body 3 are arbitrary, but for example, the dimension in the X direction is 10 mm to 50 mm, and the dimension in the Y direction is 5 mm to 30 mm. The thickness of the bottom 2 (dimension in the Z direction) is, for example, 0.1 mm to 2 mm. The thickness of the main body 3 is, for example, 2 mm to 20 mm.

[0068] (Shape processing of main body 3) Through holes are formed in the main body 3 at locations where the first well 11, the second well 12, and the slit 7 are to be formed by cutting, injection molding, or other methods. As an example, the first well 11 has a rectangular shape when viewed in the -Z direction, and dimensions in the X and Y directions are 2 mm to 10 mm. Furthermore, when the first well 11 has a circular shape when viewed in the -Z direction, the diameter of the first well 11 may be 2 mm to 10 mm. The same discussion as for the first well 11 can be applied to the shape of the second well 12.

[0069] Moreover, the slit 7 has, as an example, a rectangular shape when viewed in the −Z direction, a dimension in the X direction of 0.5 mm to 2 mm, and a dimension in the Y direction of 2 mm to 10 mm.

[0070] Next, using lithography technology, etching is performed to a fine depth in the area where the microchannel 4 is to be formed. For example, on the -Z side surface of the main body 3, etching is performed so that the area where the microchannel 4 is to be formed recedes in the +Z direction, based on the area where the bonding with the bottom 2 is planned. This etching depth is set according to the height H1 of the microchannel 4. Thereafter, the main body 3 is bonded to the +Z side surface of the bottom 2, thereby forming the microchannel 4.

[0071] The height H1 of the microchannel 4 is preferably 1 μm to 100 μm, and more preferably 5 μm to 10 μm. The length of the microchannel 4 (the dimension in the X direction) is preferably 1 mm to 10 mm, and the width of the microchannel 4 (the dimension in the Y direction) is preferably 0.01 mm to 1 mm.

[0072] When the main purpose is to evaluate the interaction at the junction between the neurite 31a and the cell 33, it is preferable that the height H1 of the microchannel 4 is smaller than the diameter of the neuron and larger than the diameter of the neurite, thereby allowing the neurite 31a to reach the second well 12 while keeping the cell 31 within the first well 11 (see FIG. 9).

[0073] The diameter of a nerve cell is typically 10 μm to 100 μm. Furthermore, depending on the type of nerve cell, the diameter of a neurite is said to be 0.5 μm to 20 μm. In view of the above, it is preferable that the height H1 of the microchannel 4 is greater than 1 μm and less than 10 μm.

[0074] Furthermore, when cell 31 is caused to migrate and come into contact with cell 33 (see FIG. 10), it is preferable that height H1 of microchannel 4 is larger than the diameter of cell 31. Although it depends on the diameter of cell 31, typically, if height H1 of microchannel 4 is 10 μm or more, cell 31 can be caused to migrate.

[0075] As described above, the opening of the microchannel 4 is adjusted by inserting, for example, a partition plate 20 into the slit 7. Here, from the viewpoint of facilitating observation of the microchannel 4 from, for example, the bottom 2 side, the ratio of the dimension of the slit 7 in the X direction to the length of the microchannel 4 (the dimension in the X direction) is preferably 1:0.1 to 1:0.9. Furthermore, the ratio of the dimension of the slit 7 in the Y direction to the dimension of the microchannel 4 in the Y direction is preferably 1:1 to 1:20.

[0076] Furthermore, considering that the opening degree of the microchannel 4 can be adjusted by the opening / closing unit 6, the height H1 of the microchannel 4 may be made relatively large during manufacturing. For example, the first well 11 and the second well 12 may be connected with the partition plate 20 positioned lower than the height H1 of the microchannel 4. As an example, the height H1 of the microchannel 4 may be set to 50 μm or more. In other words, the opening / closing unit 6 can be said to ease the precision required for processing the microchannel 4.

[0077] (Joining of bottom 2 and main body 3) The bottom portion 2 and the main body portion 3 can be joined using a method that utilizes surface modification using light or plasma, thermal bonding, adhesion using an adhesive, or solvent bonding. One example is a method of irradiating ultraviolet light onto the area where the bottom portion 2 and the main body portion 3 are to be joined. More specifically, vacuum ultraviolet light (VUV) with a wavelength of 200 nm or less can be used as the ultraviolet light. A suitable ultraviolet light source is a Xe excimer lamp with a peak wavelength of approximately 172 nm. The bottom portion 2 and the main body portion 3 can then be joined by pressing them together using a press or the like while they are in contact with each other through the area irradiated with ultraviolet light.

[0078] For example, the bottom 2 and main body 3 are made of thermoplastic resins such as polymethyl methacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), cycloolefin polymer (COP), and polystyrene (PS). To facilitate observation of the cultured cells, the bottom 2 and main body 3 are typically made of a transparent material. Among the above materials, COP, designated as a medical-grade resin, is particularly preferred. COP is a thermoplastic resin that exhibits high transparency, low autofluorescence, and low drug adsorption.

[0079] The partition plate 20 can be made of the same material as that described for the bottom portion 2 and the main body portion 3. The partition plate 20 is preferably made of a flexible material. For example, the partition plate 20 may be made of a material such as a fluororesin or a styrene-butadiene copolymer resin.

[0080] As an example, the partition plate 20 has a rectangular flat plate shape. The dimensions of the partition plate 20 are preferably configured to be approximately the same as the dimensions of the slit 7 when viewed in the -Z direction. This allows the slit 7 to be blocked by inserting the partition plate 20 into the slit 7, thereby preventing the culture solution and the like from leaking from the microchannel 4 into the slit 7. The height of the slit 7 (the dimension in the Z direction) is preferably greater than the height of the main body part 3, from the viewpoint of facilitating manipulation with tweezers and the like.

[0081] Furthermore, a filler such as silicone oil may be placed between the slit 7 and the partition plate 20.

[0082] In the above description, the microchannel 4 is formed by etching the −Z side surface of the main body 3. However, the microchannel 4 may be formed by etching the regions of the bottom 2 where the first well 11, the second well 12, and the microchannel 4 are to be formed, and then bonding the bottom 2 and the main body 3 together.

[0083] Fig. 11A is a diagram showing another example of the configuration of the microchannel 4. Like Fig. 4, Fig. 11A shows the microchannel 4 as viewed from the first well 11. Fig. 11A shows an example in which the microchannel 4 is composed of a plurality of grooves 8 extending in the X direction. It is optional whether the microchannel 4 is composed of a plurality of grooves 8.

[0084] 11A is formed by etching the +Z side surface (main surface 2a) of the bottom portion 2 so that the regions where the first well 11, the second well 12, and the plurality of grooves 8 are to be formed are recessed in the -Z direction, based on the region where bonding with the main body portion 3 is planned. The etching depth is set according to the height H1 of the microchannel 4.

[0085] By joining the bottom part 2 and the main body part 3, the first well 11 and the second well 12 can be connected via the plurality of grooves 8. For example, by allowing neurites 31a growing from cells 31 (see FIG. 7, etc.) to enter each of the plurality of grooves 8, it becomes easy to observe the neurites 31a.

[0086] FIG. 11B is a diagram showing the microchannel 4 shown in FIG. 11A in a closed state. For convenience, the inner wall surface on the +X side of the first well 11 is not shown in FIG. 11B. As shown in FIG. 11B, a partition plate 20 is disposed in the microchannel 4, thereby closing off the plurality of grooves 8. In this case, from the viewpoint of facilitating closing off the plurality of grooves 8, it is preferable that the partition plate 20 be made of a flexible material. As a result, as shown in FIG. 11B, when the partition plate 20 is pressed against the bottom 2, the partition plate 20 deforms and enters the grooves 8, making it easier to substantially close off the grooves 8.

[0087] In view of the above, it is preferable that the partition plate 20 be made of a material such as fluororesin, particularly in the example of FIGS. 11A and 11B.

[0088] [Second embodiment] The second embodiment of the cell culture device 1 will be described, focusing on the differences from the first embodiment.

[0089] Fig. 12A is a perspective view of a cell culture device 1 according to a second embodiment. Fig. 12B is a drawing showing a cross section of the cell culture device 1 according to the second embodiment, following Fig. 2A. This embodiment is similar to the first embodiment in that the main body portion 3 has a first well 11, a second well 12, and a slit 7 that penetrate the main body portion 3 in the Z direction. However, this embodiment differs from the first embodiment in that the cell culture device 1 has a film 21 that closes the opening on the -Z side of the slit 7, as shown in Fig. 12B.

[0090] In this embodiment, a fluid such as water or air is supplied to space A1 on the +Z side of film 21 of slit 7. By supplying the fluid and increasing the pressure in space A1, it is possible to deform film 21 and close microchannel 4. FIG. 12C is a diagram showing a state in which microchannel 4 is closed, following the state of FIG. 12B. As shown in FIG. 12C, fluid L1 is supplied to space A1 from an arbitrary fluid supply unit 22. Fluid supply unit 22 can be configured, for example, by a pump or the like.

[0091] As the pressure in space A1 increases when fluid L1 is supplied, film 21 deforms to form a convex shape on the -Z side. Then, by continuing to supply fluid L1 and bringing film 21 into contact with main surface 2a of bottom portion 2, microchannel 4 can be closed. In this embodiment, film 21 corresponds to "opening / closing portion 6."

[0092] The fluid L1 may be any fluid as long as it can increase the pressure in the space A1. For example, the fluid L1 may be water or air. Although not shown, in order to facilitate increasing the pressure in the space A1, the opening on the +Z side of the slit 7 may be closed, and the pipe 22a may be drawn into the space A1, and the fluid L1 may be supplied to the space A1 via the pipe 22a.

[0093] Furthermore, by discharging the fluid L1 in the space A1 from the space A1, for example, via a pipe (not shown), the film 21 can be deformed, thereby changing the position of the film 21 in the Z direction. Specifically, when the fluid L1 is a liquid, the microchannel 4 can be opened by lowering the height of the liquid fluid L1 below the liquid level of the culture solution. Furthermore, the aperture of the microchannel 4 can be adjusted by adjusting the relationship between the height of the fluid L1 and the liquid level of the culture solution. In other words, the aperture of the microchannel 4 is adjusted by changing the pressure in the space A1. In this embodiment, the fluid L1 functions as an aperture adjustment mechanism that adjusts the aperture of the microchannel 4.

[0094] From the viewpoint of facilitating deformation due to changes in pressure within space A1, film 21 is preferably made of a material that is flexible when formed into a film. For example, film 21 may be made of a material such as silicone resin, styrene-butadiene copolymer resin, polyethylene terephthalate, or cycloolefin polymer (COP). As an example, film 21 has a thickness (dimension in the Z direction) of 5 μm to 15 μm. The thickness of film 21 can be adjusted as appropriate in consideration of the material of film 21 and the height H1 of microchannel 4.

[0095] Furthermore, for example, even if the microchannel 4 is composed of multiple grooves 8 (see Figure 11A), if the film 21 is made of a flexible material, the multiple grooves 8 can be substantially blocked, as described with reference to Figure 11B.

[0096] As an example, before bonding the bottom 2 and the main body 3, the film 21 is bonded to a region on the -Z side of the main body 3 where the microchannel 4 is to be formed. The method for bonding the main body 3 and the film 21 may be bonding using surface modification using light or the like, or may be thermal bonding or the like.

[0097] Regarding the method of using the cell culture device 1 of the second embodiment, the same discussion as that of the first embodiment can be applied, except that in steps S1 and S4, the opening degree of the microchannel 4 is adjusted by the pressure change in space A1.

[0098] [Third embodiment] Next, a third embodiment of the cell culture device 1 will be described, focusing on the differences from the first and second embodiments.

[0099] Fig. 13A is a diagram showing a cross section of a cell culture device 1 according to a third embodiment, following Fig. 2A. This embodiment differs from the first embodiment in that, as shown in Fig. 13A, a hinge 25 is disposed on the -Z side surface of the main body 3 that forms the microchannel 4. Fig. 13B is an enlarged view of the vicinity of the hinge 25 in Fig. 13A.

[0100] 13B, the hinge 25 has a base portion 25a, a movable plate 25b, and a shaft 25c that rotates the movable plate 25b relative to the base portion 25a. The hinge 25 has a shape that extends in the Y direction, and spans the entire area of ​​the microchannel 4 in the Y direction.

[0101] The opening degree of the microchannel 4 can be adjusted by changing the open / closed state of the movable plate 25b relative to the base part 25a. The microchannel 4 is closed by bringing the movable plate 25b into contact with the main surface 2a of the bottom part 2. Furthermore, the position of the movable plate 25b in the Z direction is changed and the movable plate 25b moves away from the main surface 2a, thereby opening the microchannel 4. In this embodiment, the hinge 25 corresponds to the "opening / closing part 6."

[0102] The open / closed state of the hinge 25 can be changed, for example, by using magnetic force. Specifically, a magnet (not shown) may be disposed on a part of the movable plate 25b, and another magnet (not shown) may be disposed on the −Z side of the bottom 2, thereby attracting the movable plate 25b by attractive force and bringing it into contact with the main surface 2a of the bottom 2. Alternatively, by reversing the magnet disposed on the −Z side of the bottom 2, the movable plate 25b can be moved away by repulsive force, thereby opening the microchannel 4. As another example, the movable plate 25b may be made of a metal material, and an opening penetrating the main body 3 may be formed on the +Z side of the hinge 25. In this case, the open / closed state of the movable plate 25b can be changed by appropriately disposing a magnet on the −Z side surface of the bottom 2 or on the +Z side surface of the hinge 25 via the opening.

[0103] The hinge 25 can be formed by, for example, microfabrication using a 3D printer or microfabrication using MEMS (Micro Electro Mechanical Systems). The constituent material of the hinge 25 can be selected arbitrarily in consideration of the processing method, etc., and examples include light-curing epoxy permanent photoresist SU-8, acrylic resin, silicon, silicone resin, etc. The hinge 25 is bonded to the region on the -Z side of the main body 3 where the microchannel 4 is to be formed, before the bottom 2 and the main body 3 are bonded. The method of bonding the main body 3 and the hinge 25 is appropriately selected from the bonding methods described above in consideration of the constituent materials of the main body 3 and the hinge 25.

[0104] [Fourth embodiment] In the above description, it has been explained that one second well 12 is connected to the first well 11 via the microchannel 4. However, the first well 11 may be connected to a plurality of second wells 12.

[0105] 14A is a perspective view showing a configuration example of a cell culture device 1 according to a fourth embodiment. As shown in Fig. 14A, a second well 12a may be formed on the +X side of a first well 11, and a second well 12b may be formed on the -X side. The same discussion as in the first embodiment applies to the fact that slits 7 are formed between the first well 11 and the second wells (12a, 12b) and partition plates 20 are inserted into each slit 7.

[0106] Fig. 14B is a schematic diagram showing the positional relationship between each well and a microchannel in the cell culture device 1 shown in Fig. 14A. As shown in Fig. 14B, the first well 11 and the second well (12a, 12b) are connected by different microchannels (4a, 4b). The opening degree of the microchannels (4a, 4b) can be adjusted by changing the position of the partition plate 20 in the Z direction (see Fig. 14A).

[0107] Furthermore, the opening degree of the microchannels (4a, 4b) may be adjusted by deforming the film 21, for example, as described in the second embodiment.

[0108] 15A is a schematic diagram showing another configuration example of the cell culture device 1 according to the fourth embodiment, and FIG. 15B is a schematic diagram showing the positional relationship between each well and a microchannel of the cell culture device 1 shown in FIG. 15A. As shown in FIGS. 15A and 15B, a second well 12a may be formed on the +X side of a first well 11, and a second well 12b may be formed on the +Y side. Furthermore, a third well 13 may be formed on the +Y side of the second well 12a, and the second wells (12a, 12b) may be connected to the third well 13 by microchannels (5a, 5b).

[0109] Fig. 16A is a perspective view showing yet another configuration example of the cell culture device 1 according to the fourth embodiment, and Fig. 16B is a schematic diagram showing the positional relationship between each well and a microchannel in the cell culture device shown in Fig. 16A. As shown in Fig. 16B, in the example of Fig. 15B, the first well 11 and the third well 13 may be connected to each other by a microchannel 4c. That is, in the examples of Figs. 16A and 16B, the third well 13 corresponds to the second well 12c connected to the first well 11.

[0110] 16B, second well 12a and second well 12b may be connected to each other by a microchannel. In this case, microchannel 4c merges with this microchannel, forming an X-shape when viewed in the Z direction. Microchannel 4c is configured so that its opening degree can be adjusted by inserting a partition plate 20 also having an X-shape into slit 7 having an X-shape (see FIG. 16A). Furthermore, by forming a film 21 in the region where each microchannel is formed, the opening degree of each microchannel can be adjusted, as described in the second embodiment.

[0111] According to the cell culture device 1 of the fourth embodiment, by culturing cells in the first well 11 and the plurality of second wells (12a, 12b, 12c), it is possible to evaluate the interaction between cells in a more complex system than in the first embodiment. For example, in the example of Fig. 16A, assuming a system within the brain, brainstem-derived cells can be cultured in the first well 11, cerebellum-derived cells in the second well 12a, midbrain-derived cells in the second well 12b, and thalamus-derived cells in the second well 12c, and brought into contact with each other.

[0112] The same discussion as in the first embodiment can be made about the fact that suitable culture for each cell can be performed by appropriately adjusting the opening degree of the micro-flow paths 4a to 4c using the partition plate 20.

[0113] In the above description, the opening degree of the microchannels 4a to 4c and the microchannels 5a to 5b is adjusted by the partition plate 20. However, in each configuration example, as described above, the opening degree of each microchannel may be adjusted by the film 21. Similarly, as described in the third embodiment, the opening degree of each microchannel may be adjusted by the hinge 25.

[0114] Furthermore, each of the micro-flow paths 4a to 4c and the micro-flow paths 5a to 5b may be configured with a plurality of grooves 8 (see FIG. 11A).

[0115] [Variations] Modified examples of the cell culture device 1 will be described below.

[0116] <1> In the above, the cell culture device 1 has been described as having one set of a first well 11 and a second well 12 connected to the first well 11. However, the cell culture device 1 may have multiple sets of first wells 11 and second wells 12 on the XY plane. This also applies to the case where multiple second wells (12a to 12c) are connected to the first well 11.

[0117] <2> To observe the activity of the cells 31 and 33 in more detail, a multi-electrode array (MEA) sensor may be disposed on the main surface 2a of the bottom 2. This allows, for example, the action potential of the neurites 31a to be detected as an electrical signal.

[0118] <3> The configurations of the above-described embodiments can be realized by appropriately combining them.

[0119] <4> The configuration of the cell culture device 1 described above is merely an example, and the present invention is not limited to the illustrated configurations. [Explanation of symbols]

[0120] 1: Cell culture device 2 : Bottom 3: Main body 4, 4a to 4c, 5a, 5b: Microchannel 6: Opening and closing section 7: Slit 8: Groove 11: First well 12, 12a to 12c: Second well 13: Third well 15: Joint surface 20: Partition 21: Film 22: Fluid supply section 22a: Piping 25: Hinge 25a: Base 25b: Movable plate 25c: Axis 31,33 : Cell 31a: neurite 32,34 : Culture solution 80: Cell culture device 81,82: Well 83: Microchannel 91,92 :Cell

Claims

1. 1. A cell culture device comprising: a bottom portion; and a body portion joined to the bottom portion, a first well and a second well, each of which is a through hole that penetrates the main body and is formed adjacent to each other in a direction parallel to a main surface of the bottom; a microchannel formed in a part of the joining surface between the bottom and the main body, the microchannel connecting the first well and the second well; an opening / closing unit configured to be able to change its position in a direction perpendicular to the main surface of the bottom portion and to adjust the opening degree of the microchannel.

2. The cell culture device described in claim 1, characterized in that, when viewed from the main body side, it has a slit formed at a position sandwiched between the first well and the second well, and into which an opening adjustment mechanism can be introduced.

3. The cell culture device according to claim 2 , wherein the opening and closing section has a partition plate inserted into the slit.

4. Further provided is a fluid supply unit that introduces or discharges a fluid into the slit, The cell culture device according to claim 2 , wherein the opening and closing portion has a film that covers the opening on the bottom side of the slit.

5. 5. The cell culture device according to claim 1, wherein the height of the microchannel is smaller than the diameter of a nerve cell and larger than the diameter of a neurite.

6. 5. The cell culture device according to claim 1, wherein the microchannel is composed of a plurality of grooves formed in the bottom portion.

7. a plurality of said second wells; The cell culture device according to any one of claims 1 to 4, wherein the first well and the plurality of second wells are connected by different microchannels.

8. A method for using the cell culture device according to any one of claims 1 to 4, comprising: a step (a) of operating the opening / closing unit to close the microchannel; After the step (a), a step (b) of placing first cells together with a culture medium in the first well and culturing them; a step (c) of placing second cells together with a culture medium in the second well and culturing them simultaneously with or after the step (b); and (d) operating the opening / closing portion to open the microchannel and bring the first cell into contact with the second cell.

9. at least one of the first cell and the second cell is a neuron; The method for using the cell culture device according to claim 8 , wherein the step (d) comprises the step of contacting the first cells and the second cells by neurites.

10. the microchannel of the cell culture device is composed of a plurality of grooves formed on the bottom, The method for using the cell culture device according to claim 8 , wherein the step (d) includes a step of infiltrating neurites into each of the plurality of grooves.

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