Nerve cell culture chip

Abstract

Provided is a nerve cell culture chip enabling appropriate measuring of respective signals of nerve cells and neurites extending from the nerve cells.　The nerve cell culture chip comprises: a first well in which nerve cells can be cultured; a tubular microchannel that has one end connected to the first well and into which neurites extending from the nerve cells enter; a second well to which the other end of the microchannel is connected; a reference chamber that is adjacent to the first well with a partition therebetween; a shared part that allows the first well and the reference chamber to communicate with each other above the partition; a plurality of measurement electrodes that measure the potentials of the nerve cells and the neurites; and a reference electrode that provides a reference point for the measurement electrodes to measure the potentials. The plurality of measurement electrodes are independently disposed respectively on the bottom surfaces of the first well, the microchannel, and the second well. The reference electrode is disposed in the reference chamber.

Description

Nerve Cell Culture Chip

[0001] The present invention relates to a nerve cell culture chip (also referred to as a microfluidic device) for culturing nerve cells.

[0002] Neurodegenerative diseases (NDDs) are a type of neurological disorder (ND) involving degeneration of the central nervous system (CNS) and / or peripheral nervous system (PNS), affecting tens of millions of people worldwide, and this number continues to increase every year. Examples of neurodegenerative diseases (NDDs) include Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies (DLB), and amyotrophic lateral sclerosis (ALS).

[0003] In drug discovery for neurodegenerative diseases, in vitro studies use two-dimensional (2D) and three-dimensional (3D) culture of nerve cells or nerve tissues to perform neurocytotoxicity screening, thereby improving the drug discovery process.

[0004] Microfluidic devices are cutting-edge research tools that can reconstruct minimal human neural circuits for in vitro research. By co-culturing cells constituting a desired neural network in a flow channel, the cell bodies of seeded nerve cells can be separated from neurites, the directionality of neuronal connections can be controlled, and complex neuronal networks can be constructed. The device can also be used as a model for the association between nerves and tissues. For example, the neuromuscular junction (NMJ) is formed by synaptic interaction between nerve cells of the central nervous system and muscle cells, and plays a role in inducing muscle contraction. Analysis of such interactions helps to understand the etiology of diseases such as ALS.

[0005] Incidentally, electrophysiological methods are used to measure the functional activity of nerve cells (for example, Patent Document 1 below). When nerve cells are activated, ion pumps become active, and the potential difference generated therefrom is acquired by measurement probes such as patch clamps (microelectrode needles) or MEAs (microelectrode arrays). An MEA (microelectrode array) is a device in which electrodes are placed on a planar substrate. In measurement, the potential difference generated between the measurement electrode and the reference electrode is amplified and acquired as a signal. At this time, if the electrical resistance between the measurement electrode and the reference electrode is high, a sufficiently large signal can be obtained.

[0006] Patent No. 3101122

[0007] The two technologies can be integrated by using an MEA substrate as the base material for the microfluidic device. This allows for the measurement of desired functional activity using microelectrodes that directly contact nerve cells cultured in the microchannel. While the fabrication itself is easy, the microfluidic device and MEA must be properly designed for actual drug discovery testing. However, publicly available reports and products merely add an MEA to existing microfluidic devices and are not properly designed for their final applications.

[0008] For example, cultured models of iPS cells (normal models derived from healthy individuals and disease models derived from patients) and muscle cells that constitute NMJ require a culture period of about four weeks to express function. In long-term cell culture in a microfluidic channel, not only is the culture unstable due to the accumulation of culture medium, dead cells, and waste products, but cells also detach, resulting in a sparse cell culture. As a result, the cells do not express sufficient function. In addition, the measurement electrode is not sufficiently covered with cells, the electrical resistance between it and the reference electrode becomes small, and the measured signal becomes small. Because the signal is small, it becomes difficult to separate it from noise. Consequently, stable results cannot be obtained in drug discovery trials, and practical results cannot be obtained.

[0009] In view of the above problems, the present invention aims to provide a nerve cell culture chip that can appropriately measure the signals of nerve cells and neurites extending from nerve cells.

[0010] The nerve cell culture chip according to the present invention comprises: a first well in which nerve cells can be cultured; a tubular microchannel, one end of which is connected to the first well and into which neurites extending from the nerve cells enter; a second well to which the other end of the microchannel is connected; a reference chamber adjacent to the first well separated by a partition; a shared section above the partition that connects the first well and the reference chamber; a plurality of measuring electrodes for measuring the potential of the nerve cells and neurites; and a reference electrode that provides a reference point for the measuring electrodes to measure the potential. The measuring electrodes are arranged independently in a plurality on the bottom surfaces of the first well, the microchannel, and the second well, and the reference electrode is arranged in the reference chamber. Alternatively, the reference electrode may be arranged on the bottom surface of the reference chamber.

[0011] With this configuration, nerve cells can be cultured in the first well and neurites in the microchannel, meaning that nerve cells and neurites can be cultured separately. Furthermore, with this configuration, multiple measurement electrodes are placed on the bottom surfaces of the first well and the microchannel, allowing for appropriate measurement of the signals from the cultured nerve cells and neurites on the measurement electrodes.

[0012] In the nerve cell culture chip according to the present invention, the measuring electrode may be configured such that it is positioned at a first separation distance in the microchannel, and at a second separation distance that is an integer multiple of the first separation distance in the first well and the second well.

[0013] With such a configuration, the calculation of the signal propagation speed becomes easier.

[0014] In the nerve cell culture chip according to the present invention, a liquid storage section communicating with the shared section is provided above the shared section, and in a plan view, the area of ​​the liquid storage section is larger than the area of ​​the shared section, and the area of ​​the shared section is larger than the area of ​​the first well.

[0015] With this configuration, sufficient culture medium for culturing nerve cells and neurites can be stored.

[0016] The nerve cell culture chip according to the present invention may also be configured such that the first well, the reference chamber, and the shared section are provided in multiple sets, and the liquid storage section is in communication with the multiple shared sections.

[0017] With this configuration, the culture media in multiple first wells can be replaced simultaneously through the liquid storage section.

[0018] The nerve cell culture chip according to the present invention may also be configured to include a second microchannel, one end of which is connected to the first well and into which the neurites enter, and a third well to which the other end of the second microchannel is connected.

[0019] With this configuration, three types of cells, including nerve cells, can be cultured simultaneously.

[0020] In the nerve cell culture chip according to the present invention, the microfluidic channel may be configured to include a branched channel that branches off midway, and the branched channel may be connected to a third well.

[0021] With this configuration, three types of cells, including nerve cells, can be cultured simultaneously.

[0022] Figure 2 shows a perspective view of the appearance of a nerve cell culture chip according to the first embodiment. Figure 3 shows a plan view of the nerve cell culture chip according to the first embodiment. Figure 4 shows a cross-sectional view of the nerve cell culture chip shown in Figure 2, taken from line III-III. Figure 5 shows a plan view of the bottom plate of the nerve cell culture chip according to the first embodiment. Figure 6 shows a perspective view of the appearance of a nerve cell culture chip according to the second embodiment. Figure 6 shows a cross-sectional view of the nerve cell culture chip shown from line VII-VII. Figure 6 shows a plan view of the bottom plate of the nerve cell culture chip according to the second embodiment. Figure 6 shows a plan view of the arrangement of measurement electrodes in a nerve cell culture chip according to another embodiment. Figure 7 shows a plan view of the arrangement of measurement electrodes in a nerve cell culture chip according to another embodiment.

[0023] The nerve cell culture chip according to the present invention will be described with reference to the drawings. It should be noted that the drawings disclosed herein are for illustrative purposes only. That is, the dimensional ratios in the drawings do not necessarily correspond to the actual dimensional ratios, and the dimensional ratios do not necessarily correspond between the drawings.

[0024] [First Embodiment] Figure 1 is a perspective view showing the external appearance of a nerve cell culture chip 1 according to the first embodiment. As shown in Figure 1, the nerve cell culture chip 1 comprises a bottom plate 2 and a base portion 3 that is positioned in partial contact with the upper surface of the bottom plate 2.

[0025] The base portion 3 is made of a transparent thermoplastic resin. Examples of materials that make up the base portion 3 include polymethyl methacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC), cycloolefin polymer (COP), and polystyrene (PS), among which medical-grade COP resin is particularly preferred. COP is a thermoplastic resin that exhibits high transparency, low autofluorescence, and low drug adsorption.

[0026] In the following explanation, the X-Y-Z coordinate system shown in Figure 1 will be referenced as appropriate. When expressing direction, positive and negative directions are distinguished, and these are indicated with a sign, such as "+X direction" and "-X direction". When expressing direction without distinguishing between positive and negative directions, it is simply written as "X direction". In other words, in this specification, when simply written as "X direction", both "+X direction" and "-X direction" are included. The same applies to the Y direction and Z direction. Note that the nerve cell culture chip 1 is usually used with the Z direction as the up and down direction, and the +Z direction corresponds to the up direction.

[0027] Figure 2 is a plan view of the nerve cell culture chip 1 according to the first embodiment. Figure 3 is a cross-sectional view taken along line III-III in Figure 2.

[0028] The nerve cell culture chip 1 has two first and second wells 4 and 5 formed at positions spaced apart in the X direction. The nerve cell culture chip 1 also has a microchannel 6 whose ends are connected to the bottom of the first and second wells 4 and 5, respectively.

[0029] Furthermore, the nerve cell culture chip 1 includes a first reference chamber 7 adjacent to the first well 4, separated by the first partition 1a, and a second reference chamber 8 adjacent to the second well 5, separated by the second partition 1b.

[0030] Furthermore, the nerve cell culture chip 1 includes a first shared section 9 that connects the first well 4 and the first reference chamber 7 above the first partition 1a, and a second shared section 10 that connects the second well 5 and the second reference chamber 8 above the second partition 1b.

[0031] The first shared section 9 and the second shared section 10 are spaced apart in the X direction. The first shared section 9 and the second shared section 10 are separated by a third partition 1c. The first and second shared sections 9 and 10 are recesses that open to the upper surface of the base section 3. The bottom surface of the first shared section 9 is made up of the first partition 1a, and a part of the side surface of the first shared section 9 is made up of the third partition 1c. The bottom surface of the second shared section 10 is made up of the second partition 1b, and a part of the side surface of the second shared section 10 is made up of the third partition 1c.

[0032] The first and second shared sections 9 and 10 may each have a substantially rectangular shape in plan view. In one example of this embodiment, the dimensions of the first and second shared sections 9 and 10 are X × Y × Z = 5 mm × 12 mm × 5 mm, respectively. The area of ​​the first shared section 9 is larger than the area of ​​the first well 4 in plan view. The area of ​​the second shared section 10 is larger than the area of ​​the second well 5 in plan view. The volume of the first shared section 9 is preferably 10 times or more the volume of the first well 4. The volume of the second shared section 10 is preferably 10 times or more the volume of the second well 5.

[0033] Appropriate amounts of culture medium are supplied to the first and second shared sections 9 and 10, respectively. Since the first shared section 9 is in communication with the first well 4 and the first reference chamber 7, the first well 4 and the first reference chamber 7 are filled with culture medium. Similarly, since the second shared section 10 is in communication with the second well 5 and the second reference chamber 8, the second well 5 and the second reference chamber 8 are filled with culture medium.

[0034] The first well 4 and the second well 5 are spaced apart in the X direction. The first well 4 and the second well 5 are separated by a third partition 1c. The first well 4 and the second well 5 may each have a substantially rectangular shape when viewed from above.

[0035] The first well 4 is a recess that opens at the bottom surface of the first shared portion 9. The first well 4 is formed to penetrate the base portion 3 in the Z direction and expose the bottom plate 2. For example, nerve cells are seeded in the first well 4 and cultured on the bottom plate 2. The first well 4 may have an inclined surface 4a that is inclined with respect to the Z direction, as shown in Figure 3. By having an inclined surface 4a in the first well 4, the inner wall of the first well 4 narrows toward the bottom plate 2, making it easier to collect the nerve cells seeded in the first well 4 toward the bottom surface (upper surface of the bottom plate 2). In one example of this embodiment, the dimensions of the first well 4 are X × Y × Z = 1.5 to 2.5 mm × 4.3 mm × 3 mm.

[0036] The second well 5 is a recess that opens at the bottom surface of the second shared portion 10. The second well 5 is formed to penetrate the base portion 3 in the Z direction and expose the bottom plate 2. Cells such as nerve cells, muscle cells, and epithelial cells are seeded in the second well 5 and cultured on the bottom plate 2. The second well 5 may have an inclined surface 5a that is inclined with respect to the Z direction, as shown in Figure 3. By having an inclined surface 5a in the second well 5, the inner wall of the second well 5 narrows toward the bottom plate 2, making it easier to collect the cells seeded in the second well 5 toward the bottom surface (upper surface of the bottom plate 2). In one example of this embodiment, the dimensions of the second well 5 are X × Y × Z = 1.5 to 2.5 mm × 4.3 mm × 3 mm.

[0037] The microchannel 6 is a tubular channel whose ends are connected to the first well 4 and the second well 5, respectively. Here, a tubular channel means a channel that is closed on all sides (top, bottom, left, and right) and has a rectangular or circular cross-section when viewed in the direction of flow. The microchannel 6 in this embodiment has a rectangular cross-section. Nerve processes extending from nerve cells cultured in the first well 4 can enter the microchannel 6.

[0038] The microchannel 6 is formed by a groove extending in the X direction formed on the bottom surface of the base portion 3, more specifically on the bottom surface of the third partition 1c, and the upper surface of the bottom plate 2. Multiple microchannels 6 may be formed to connect the bottom of the first well 4 and the bottom of the second well 5. Each microchannel 6 has an extremely fine structure. More specifically, the channel depth (length in the Z direction) of each microchannel 6 is preferably 3 μm to 80 μm, more preferably 3 μm to 60 μm, and particularly preferably 3 μm to 20 μm. If the channel depth is lower than 3 μm, it is difficult for neurites to penetrate, and even if neurites penetrate, their growth may be inhibited. On the other hand, if the channel depth is higher than 80 μm, nerve cells also penetrate the microchannel 6, making it impossible to separate nerve cells from neurites. In this embodiment, the channel depth of the microchannel 6 is 5 μm.

[0039] Furthermore, the channel width (length in the Y direction) of the microchannel 6 is preferably 15 μm to 1000 μm, more preferably 50 μm to 500 μm, and particularly preferably 100 μm to 300 μm. In this embodiment, the channel width of the microchannel 6 is 200 μm. Also, in this embodiment, the distance between adjacent microchannels 6 is 250 μm.

[0040] The first reference chamber 7 is positioned adjacent to the first well 4, with the first partition 1a in between. Multiple first reference chambers 7 (for example, two) may be provided for each first well 4. In a plan view, the first reference chamber 7 is positioned at the corner of the first common area 9.

[0041] The first reference chamber 7 opens at the bottom surface of the first common section 9. The first reference chamber 7 is formed to penetrate the base section 3 in the Z direction and expose the bottom plate 2. The first reference chamber 7 may have a frustoconical side wall that narrows toward the bottom plate 2. In one example of this embodiment, the first reference chamber 7 has an inner diameter of 1.5 to 2 mm and a depth of 2 to 3 mm.

[0042] The first reference chamber 7 is partitioned from the first well 4 by the first partition 1a, and is arranged independently of the first well 4. Accordingly, the first reference chamber 7 is clearly separated from the culture area (the first well 4) where nerve cells are seeded and cultured, so no nerve cells are seeded into the first reference chamber 7.

[0043] The second reference chamber 8 is arranged adjacent to the second well 5 with the second partition 1b interposed therebetween. A plurality of (for example, two) second reference chambers 8 may be provided for the second well 5. The second reference chamber 8 is arranged at a corner of the second shared portion 10 in a plan view.

[0044] The second reference chamber 8 is open at the bottom surface of the second shared portion 10. The second reference chamber 8 is formed so as to penetrate the base portion 3 in the Z direction and expose the bottom plate 2. The second reference chamber 8 may have a truncated conical side wall that narrows toward the bottom plate 2. As an example of the present embodiment, the second reference chamber 8 has an inner diameter of 1.5 to 2 mm and a depth of 2 to 3 mm.

[0045] The second reference chamber 8 is partitioned from the second well 5 by the second partition 1b, and is arranged independently of the second well 5. Accordingly, the second reference chamber 8 is clearly separated from the culture area (the second well 5) where cells are seeded and cultured, so no cells are seeded into the second reference chamber 8.

[0046] As shown in FIG. 4, the bottom plate 2 may include an insulating substrate 21, a plurality of measurement electrodes 22 and a reference electrode 23 arranged on the insulating substrate 21, and lead wires (not shown) extending from the measurement electrodes 22 and the reference electrode 23. The lead wires are covered and insulated by an insulating layer (not shown).

[0047] The insulating substrate 21 is made of glass, for example. The measurement electrodes 22, the reference electrode 23, and the lead wires are made of indium tin oxide, for example. The insulating layer is made of polyimide resin, for example.

[0048] The plurality of measurement electrodes 22 are arranged on the respective bottom surfaces of the first well 4, the second well 5, and the microchannel 6. A plurality of the measurement electrodes 22 are respectively arranged on the respective bottom surfaces of the first well 4, the second well 5, and the microchannel 6.

[0049] Preferably, the measurement electrodes 22 are arranged at a first separation distance D1 in the microchannel 6, and are arranged at a second separation distance D2 that is an integer multiple of the first separation distance D1 in the first well 4 and the second well 5. As in the present embodiment, the second separation distance D2 may be one time the first separation distance D1. That is, the second separation distance D2 may be equal to the first separation distance D1, and all of the plurality of measurement electrodes 22 may be arranged at equal intervals. The signal propagation velocity can be calculated based on the arrival time of the signal at the measurement electrodes 22. By arranging the measurement electrodes 22 such that the second separation distance D2 is an integer multiple of the first separation distance D1, calculation of the signal propagation velocity is facilitated. Further, by arranging the plurality of measurement electrodes 22 at equal intervals, calculation of the signal propagation velocity is particularly facilitated. Note that the first separation distance D1 and the second separation distance D2 refer to the distance between the centers of adjacent measurement electrodes 22.

[0050] The plurality of measurement electrodes 22 may be arranged at respective intersection points on a grid. In the present embodiment, the plurality of measurement electrodes 22 are arranged at respective intersection points on an 8×8 grid. Accordingly, the plurality of measurement electrodes 22 are arranged at equal intervals. Twenty-four measurement electrodes 22 are respectively arranged on the bottom surfaces of the first well 4 and the second well 5, and two measurement electrodes 22 arranged linearly are arranged on the bottom surface of each microchannel 6.

[0051] The plurality of measurement electrodes 22 are insulated from each other and also insulated from the reference electrode 23. As an example of the present embodiment, the dimensions of the measurement electrode 22 are, for example, 50 μm × 50 μm. Further, the closest inter-electrode distance among the plurality of measurement electrodes 22 (in the present embodiment, the first separation distance D1 and the second separation distance D2) is 0.45 mm.

[0052] The reference electrode 23 is positioned on the bottom surface of the first reference chamber 7 and the second reference chamber 8, respectively. The reference electrode 23 is positioned on the same plane as the measuring electrode 22. The reference electrode 23 may also be positioned approximately in the center of the bottom surfaces of the first reference chamber 7 and the second reference chamber 8, as in this embodiment.

[0053] Multiple reference electrodes 23 are insulated from each other and from the measuring electrode 22. In one example of this embodiment, the dimensions of the reference electrode 23 are, for example, 200 μm × 200 μm. Also, in a plan view, the reference electrode 23 is larger than the measuring electrode 22.

[0054] When nerves are active, ion pumps are activated by nerve activation, causing a change in electrical potential. By measuring the electrical potential changes of nerve cells and neurites using the measuring electrode 22, nerve activity can be detected. The reference electrode 23 provides a reference point for the measuring electrode 22 to measure the electrical potential. In measuring the electrical potential of nerve cells and neurites, the potential difference between the measuring electrode 22 and the reference electrode 23 is amplified and acquired as a signal. At this time, a higher electrical resistance between the measuring electrode 22 and the reference electrode 23 results in a sufficiently large signal.

[0055] For example, when culturing nerve cells in the first well 4, the first well 4 is filled with culture medium, and culture medium is also supplied to the first shared section 9 which is connected to the first well 4, so there is sufficient culture medium for culturing nerve cells. Furthermore, since both the first well 4 and the first shared section 9 are open at the top, it is easy to change the culture medium. As a result, sufficient nutrients are supplied to the nerve cells, and dead cells and waste products can be easily removed without accumulation. Consequently, the nerve cells cultured in the first well 4 have a dense cell layer and can express sufficient function. In addition, the dense cell layer increases the electrical resistance between the measurement electrode 22 placed on the bottom of the first well 4 and the reference electrode 23 placed on the bottom of the first reference chamber 7, reducing the decrease in the signal generated by the nerve cells and making measurement easier.

[0056] Furthermore, the neurites extend within the microchannel 6. Here, the cross-sectional area of ​​the conductive culture medium is restricted by the microchannel 6, which increases the electrical resistance, reducing the decrease in the signal generated from the neurites and making measurement easier.

[0057] As described above, the nerve cell culture chip 1 according to this embodiment allows for the culture of nerve cells in the first well 4 and the culture of neurites in the microchannel 6, that is, it allows for the culture of nerve cells and neurites in a separated state. Furthermore, the nerve cell culture chip 1 according to this embodiment allows for the appropriate measurement of signals from both nerve cells and neurites extending from nerve cells.

[0058] Furthermore, the nerve cell culture chip 1, which is made of a transparent material that allows for microscopic observation, can also be used for monitoring neuronal functional activity through microscopic imaging of living cells.

[0059] In bright-field imaging, motion imaging is one example, where the movement of muscle cells in a microscopic image within the second well (well 5) can be quantified as a motion vector. This allows for evaluation of whether muscle cells responded to stimuli from motor neurons.

[0060] Fluorescence-based calcium imaging allows for the visualization of intracellular calcium changes as indicators of neuronal functional activity, using fluorescent calcium probes such as Fluo-4 and calcium voltage indicators such as GCaMP6f.

[0061] By combining the above imaging method with the nerve cell culture chip 1, it becomes possible to accurately record signal transduction in in vitro neural circuits as follows: (1) Imaging analysis enables recording that complements the spatial resolution of the nerve cell culture chip 1. (2) The nerve cell culture chip 1 enables recording that complements the temporal resolution of the imaging analysis.

[0062] [Second Embodiment] The differences between the nerve cell culture chip 1 according to the second embodiment and the first embodiment will be described below. Note that the same reference numerals are used for components identical to those in the first embodiment, and their descriptions will be omitted as appropriate.

[0063] Figure 5 is a perspective view showing the external appearance of the nerve cell culture chip 1 according to the second embodiment. Figure 6 is a plan view of the nerve cell culture chip 1 according to the second embodiment. Figure 7 is a cross-sectional view taken along line VII-VII in Figure 6.

[0064] The first well 4 and the second well 5 are circular in shape when viewed from above. One first reference chamber 7 and one second reference chamber 8 are provided for each of the first well 4 and the second well 5. The first reference chamber 7 and the second reference chamber 8 are also circular in shape when viewed from above. In one example of this embodiment, the first well 4 and the second well 5 have an inner diameter of 3.96 mm and a depth of 4 mm. In another example of this embodiment, the first reference chamber 7 and the second reference chamber 8 have an inner diameter of 1.5 mm and a depth of 4 mm.

[0065] The nerve cell culture chip 1 is equipped with a first liquid storage section 11 above the first shared section 9, which communicates with the first shared section 9. In a plan view, the area of ​​the first liquid storage section 11 is larger than the area of ​​the first shared section 9, and the area of ​​the first shared section 9 is larger than the area of ​​the first well 4. This allows for the storage of sufficient culture medium for cell culture.

[0066] The nerve cell culture chip 1 is equipped with a second liquid storage section 12 above the second shared section 10, which communicates with the second shared section 10. In a plan view, the area of ​​the second liquid storage section 12 is larger than the area of ​​the second shared section 10, and the area of ​​the second shared section 10 is larger than the area of ​​the second well 5. This allows for the storage of sufficient culture medium for cell culture.

[0067] Furthermore, the nerve cell culture chip 1 is provided with multiple sets of first wells 4, first reference chambers 7, and first shared sections 9, and the first liquid storage section 11 is in communication with multiple first shared sections 9. In this embodiment, the first liquid storage section 11 is in communication with four first shared sections 9. This allows the culture medium in multiple first wells 4 to be replaced simultaneously through the first liquid storage section 11.

[0068] Furthermore, by raising the liquid level of the culture medium to a height exceeding the first shared section 9, multiple first wells 4 can be cultured under the same conditions. After that, by raising the liquid level of the culture medium to a height midway through the first shared section 9, each group (first well 4, first reference chamber 7, and first shared section 9) can be separated, and different drug tests can be performed on each group.

[0069] The nerve cell culture chip 1 is provided with multiple sets of second wells 5, second reference chambers 8, and second shared sections 10, and the second liquid storage section 12 is in communication with multiple second shared sections 10. In this embodiment, the second liquid storage section 12 is in communication with four second shared sections 10. This allows the culture medium in multiple second wells 5 to be replaced simultaneously through the second liquid storage section 12.

[0070] Furthermore, by raising the liquid level of the culture medium to a height exceeding the second shared section 10, multiple second wells 5 can be cultured under the same conditions. After that, by raising the liquid level of the culture medium to a height midway through the second shared section 10, each group (second well 5, second reference chamber 8, and second shared section 10) can be separated, and different drug tests can be performed on each group.

[0071] As shown in Figure 8, the multiple measuring electrodes 22 are arranged on the bottom surfaces of the first well 4, the second well 5, and the microchannel 6, similar to the first embodiment. However, in the microchannel 6, the measuring electrodes 22 are arranged at a first spacing distance D1, while in the first well 4 and the second well 5, they are arranged at a second spacing distance D2 (D1 × 2 or D1 × 3), which is two or three times the first spacing distance D1. In other words, the measuring electrodes 22 in the microchannel 6 are arranged more densely than the measuring electrodes 22 in the first well 4 and the second well 5. This allows for even measurement of signals from nerve cells in the first well 4 and neurites in the microchannel 6, for example.

[0072] Furthermore, in the first well 4, it is preferable that the multiple measuring electrodes 22 be arranged at least on each point of the square centered on the center 4c of the first well 4. The same applies to the second well 5. As a result, the cells seeded in the first well 4 and the second well 5 are more easily cultured on the measuring electrodes 22 near the center of the wells because they are away from the well walls, resulting in a more uniform seeding density and improved nutrient supply, thus allowing for appropriate measurement of the cell potential.

[0073] Although embodiments of the present invention have been described above with reference to the drawings, it should be understood that the specific configuration is not limited to these embodiments. The scope of the present invention is indicated not only by the above-described embodiments but also by the claims, and further includes all modifications within the meaning and scope of equivalence to the claims.

[0074] The structures adopted in each of the above embodiments can be adopted in any other embodiment. The specific configuration of each part is not limited to the embodiments described above, and various modifications are possible without departing from the spirit of the present invention.

[0075] (1) The multiple measuring electrodes 22 may be arranged linearly and continuously on the bottom surfaces of the first well 4, the microchannel 6, and the second well 5, as shown in Figure 9.

[0076] (2) The nerve cell culture chip 1 may also be configured as shown in Figures 10 and 11, comprising a second microchannel 6a, one end of which is connected to the first well 4 and into which neurites enter, and a third well 13 to which the other end of the second microchannel 6a is connected. In the example shown in Figure 11, the nerve cell culture chip 1 is provided with a third microchannel 6b, one end of which is connected to the second well 5 and the other end of which is connected to the third well 13.

[0077] (3) In the nerve cell culture chip 1, as shown in Figure 12, the microchannel 6 may be configured to include a branched channel 6c that branches off midway, and the branched channel 6c is connected to the third well 13. For example, different parts of the brain have different functions, and by using a chip with multiple wells connected together, a model of the relationships between these brain regions can be created.

[0078] (4) The nerve cell culture chip 1 may also have four or more wells (not shown).

[0079] (5) The reference electrode 23 may be placed in a location other than the bottom surface of the first reference chamber 7 and the second reference chamber 8.

[0080] 1: Nerve cell culture chip 1a: First partition 1b: Second partition 1c: Third partition 2: Bottom plate 3: Base part 4: First well 4a: Inclined surface 4c: Center 5: Second well 5a: Inclined surface 6: Microchannel 6a: Second microchannel 6b: Third microchannel 6c: Branch channel 7: First reference chamber 8: Second reference chamber 9: First common part 10: Second common part 11: First liquid storage part 12: Second liquid storage part 13: Third well 21: Insulating substrate 22: Measuring electrode 23: Reference electrode D1: First separation distance D2: Second separation distance

Claims

1. A nerve cell culture chip comprising: a first well capable of culturing nerve cells; a tubular microchannel, one end of which is connected to the first well and into which neurites extending from the nerve cells enter; a second well to which the other end of the microchannel is connected; a reference chamber adjacent to the first well separated by a partition; a shared section above the partition that connects the first well and the reference chamber; a plurality of measuring electrodes for measuring the potential of the nerve cells and neurites; and a reference electrode that provides a reference point for the measuring electrodes to measure the potential, wherein the plurality of measuring electrodes are independently arranged on the bottom surfaces of the first well, the microchannel, and the second well, and the reference electrode is located in the reference chamber.

2. The nerve cell culture chip according to claim 1, wherein the reference electrode is placed on the bottom surface of the reference chamber.

3. The nerve cell culture chip according to claim 1 or 2, wherein the measuring electrode is arranged at a first separation distance in the microchannel, and at a second separation distance that is an integer multiple of the first separation distance in the first well and the second well.

4. The nerve cell culture chip according to claim 1 or 2, wherein a liquid storage section is provided above the shared section and communicates with the shared section, and in a plan view, the area of ​​the liquid storage section is larger than the area of ​​the shared section, and the area of ​​the shared section is larger than the area of ​​the first well.

5. The nerve cell culture chip according to claim 4, wherein the first well, the reference chamber, and the shared section are provided in multiple sets, and the liquid storage section communicates with the multiple shared sections.

6. A nerve cell culture chip according to claim 1 or 2, comprising a second microchannel, one end of which is connected to the first well, through which the neurites enter, and a third well to which the other end of the second microchannel is connected.

7. The nerve cell culture chip according to claim 1 or 2, wherein the microchannel comprises a branched channel that branches off midway, and the branched channel is connected to a third well.

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