CELL CULTURE CHIP, CELL CULTURE DEVICE, AND CELL CULTURE METHOD

Abstract

A cell culture chip (100) comprising a body part provided with a first flow channel (33) and a second flow channel (41), said second flow channel (41) at least partly overlapping the first flow channel (33) when viewed from a definite direction, and a first main face (13a) and a second main face (13b) opposed to each other. The cell culture chip (100) is provided with: a cell separation membrane (13) which is disposed between the first flow channel (33) and the second flow channel (41) in such a manner that the first flow channel (33) is positioned above the first main face (13a) and the second flow channel (41) is positioned above the second main face (13b); a first electrode (21) which is in contact with the first flow channel (33) and extends in the first flow channel (33) along the first flow channel (33); and a second electrode (22) which is in contact with the second flow channel (41) and extends in the second flow channel (41) along the second flow channel (41).

Description

[Technical field]

[0001] The present disclosure relates to a cell culture chip, and a cell culture device and a cell culture method using the cell culture chip. [Background technology]

[0002] 2. Description of the Related Art In recent years, biofunctional chips (Organ On a Chip: OoC) have been actively developed as cell culture chips (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2018-189474 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, the conventional OoC disclosed in the above Patent Document 1 and the like has a problem in that the evaluation of cells performed using the OoC lacks accuracy.

[0005] The present disclosure has been made in view of the above, and aims to provide a cell culture chip and the like that enables more accurate cell evaluation. [Means for solving the problem]

[0006] In order to achieve the above-mentioned object, one aspect of the cell culture chip according to the present disclosure comprises a main body having a first flow path and a second flow path at least a portion of which overlaps with the first flow path when viewed from a predetermined direction, a cell separation membrane having first and second main surfaces facing each other and arranged between the first flow path and the second flow path such that the first flow path is located on the first main surface and the second flow path is located on the second main surface, a first electrode in contact with the first flow path and extending along the first flow path within the first flow path, and a second electrode in contact with the second flow path and extending along the second flow path within the second flow path.

[0007] Moreover, one aspect of the cell culture device according to the present disclosure includes the cell culture chip described above and a measuring instrument that measures the electrical resistance between the first electrode and the second electrode.

[0008] Moreover, one aspect of the cell culture method according to the present disclosure includes a culture step of culturing a predetermined cell using the cell culture chip described above, and a measurement step of measuring the electrical resistance of the cultured cell using the first electrode and the second electrode. Effect of the Invention

[0009] According to the present disclosure, more accurate cell evaluation is possible. [Brief description of the drawings]

[0010] [Figure 1A] FIG. 1A is a diagram illustrating a cell culture chip according to a comparative example. [Figure 1B] FIG. 1B is a diagram showing distributions of inter-electrode potential and current density according to a comparative example. [Figure 1C] FIG. 1C is a simulation diagram showing theoretical and measured values ​​of electrical resistance according to a comparative example. [Figure 2A] FIG. 2A is a first conceptual diagram of a cell culture chip according to an embodiment. [Figure 2B] FIG. 2B is a second conceptual diagram of the cell culture chip according to the embodiment. [Diagram 3] FIG. 3 is a block diagram showing the cell culture device according to the embodiment. [Figure 4] FIG. 4 is a flowchart showing a cell culture method according to an embodiment. [Figure 5A] FIG. 5A is a perspective view of a cell culture chip according to an embodiment. [Figure 5B] FIG. 5B is an exploded perspective view of the cell culture chip according to the embodiment. [Figure 6] FIG. 6 is a plan view of an area A1 enclosed by a dashed dotted line in FIG. 1, as viewed from the stacking direction. [Figure 7A] FIG. 7A is a first diagram showing a result of a simulation regarding the length of the electrodes according to the embodiment. [Figure 7B] FIG. 7B is a second diagram showing the results of a simulation regarding the length of the electrodes according to the embodiment. [Figure 8] FIG. 8 is a diagram showing the results of a simulation regarding the electrode width of the embodiment. [Figure 9] FIG. 9 is a diagram showing the results of a simulation regarding the flow channel height according to the embodiment. [Figure 10A] FIG. 10A is a first diagram illustrating a configuration of an electrode according to an embodiment. [Figure 10B] FIG. 10B is a second diagram illustrating the configuration of the electrode according to the embodiment. [Figure 10C] FIG. 10C is a third diagram illustrating the configuration of the electrode according to the embodiment. [Figure 10D] FIG. 10D is a fourth diagram illustrating the configuration of the electrodes according to the embodiment. [Figure 11A] FIG. 11A is a plan view showing a first step in the production of a cell culture chip. [Figure 11B] FIG. 11B is a cross-sectional view taken along line XI-XI in FIG. 11A. [Figure 12A] FIG. 12A is a plan view showing a second step in the production of the cell culture chip. [Figure 12B] FIG. 12B is a cross-sectional view taken along line XII-XII in FIG. 12A. [Figure 13A] FIG. 13A is a cross-sectional view illustrating a first insulating film and a second insulating film according to the embodiment. [Figure 13B] FIG. 13B is an exploded perspective view illustrating the first insulating film and the second insulating film according to the embodiment. [Figure 14A] FIG. 14A is a plan view showing a third step in the production of a cell culture chip. [Figure 14B] FIG. 14B is a cross-sectional view taken along line XIV-XIV in FIG. 14A. [Figure 15A] FIG. 15A is a plan view showing a fourth step in the production of the cell culture chip. [Figure 15B] FIG. 15B is a cross-sectional view taken along line XV-XV in FIG. 15A. [Figure 16A] FIG. 16A is a plan view showing a fifth step in the production of a cell culture chip. [Figure 16B] FIG. 16B is a cross-sectional view taken along line XVI-XVI in FIG. 16A. [Figure 17A] FIG. 17A is a plan view showing a sixth step in the production of the cell culture chip. [Figure 17B] FIG. 17B is a cross-sectional view taken along line XVII-XVII in FIG. 17A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (Summary of disclosure) In order to achieve the above-mentioned object, one embodiment of the cell culture chip disclosed herein comprises a main body having a first flow path and a second flow path at least a portion of which overlaps the first flow path when viewed from a predetermined direction, a cell separation membrane having first and second main surfaces facing each other and arranged between the first flow path and the second flow path such that the first flow path is located on the first main surface and the second flow path is located on the second main surface, a first electrode in contact with the first flow path and extending along the first flow path within the first flow path, and a second electrode in contact with the second flow path and extending along the second flow path within the second flow path.

[0012] According to this, the electrical resistance of the cell sheet cultured on the cell separation membrane arranged between the first flow path and the second flow path can be measured by the first electrode and the second electrode extending along the first flow path and the second flow path. When measuring the electrical resistance of the cell sheet, the conductive distance for conducting the fluid inside the first flow path and the second flow path between the first electrode and the second electrode is reduced. Therefore, the influence of the potential drop when conducting the above-mentioned fluid is reduced, and the electrical resistance of the cell sheet can be measured more accurately. Therefore, the cell state can be estimated based on the more accurate electrical resistance of the cell sheet, and the cells can be evaluated more accurately.

[0013] Furthermore, for example, the first flow path and the second flow path may include an electric field region that is a region spanning the first flow path and the second flow path at a position where the first flow path and the second flow path extend and overlap when viewed from a predetermined direction, and within which the first electrode and the second electrode extend, and the length in the extension direction of the first electrode and the second electrode may be 25% or more of the length in the extension direction of the electric field region.

[0014] According to this, for example, when the electrical resistance of the measurement target is 1000Ω or more, the electrical resistance can be measured with a measurement level error rate of ±20% or less. Therefore, a cell culture chip that enables more accurate cell evaluation based on more accurate measured electrical resistance values ​​can be realized.

[0015] Furthermore, for example, the length of the first electrode and the second electrode in the extension direction may be 70% or more of the length of the electric field region in the extension direction.

[0016] According to this, for example, when the electrical resistance of the measurement target is 100Ω or more, the electrical resistance can be measured with a measurement level error rate of ±20% or less. Therefore, a cell culture chip that enables more accurate cell evaluation based on more accurate measured electrical resistance values ​​can be realized.

[0017] Furthermore, for example, the length of the first electrode and the second electrode in the extension direction may be 75% or more of the length of the electric field region in the extension direction.

[0018] According to this, for example, when the electrical resistance of the measurement target is 50Ω or more, the electrical resistance can be measured with a measurement level with an error rate of ±20% or less. Therefore, a cell culture chip that can more accurately measure the electrical resistance of 50Ω, which is the approximately minimum expected for cultured cells, and more accurately evaluate the cells can be realized.

[0019] Furthermore, for example, in the electric field region, the total length of the first flow path and the second flow path in a predetermined direction may be 0.2 mm or more and 1.5 mm or less.

[0020] This makes it possible to realize a cell culture chip that enables more accurate cell evaluation under specified conditions based on more accurate measured values ​​of electrical resistance.

[0021] Furthermore, for example, the first electrode and the second electrode may have a width of 0.1 mm in a direction intersecting the extension direction of the first electrode and the second electrode.

[0022] This makes it possible to realize a cell culture chip that enables more accurate cell evaluation under specified conditions based on more accurate measured values ​​of electrical resistance.

[0023] Furthermore, for example, the main body portion may have a laminated structure in which a first substrate having a first electrode formed on its main surface, a first partition layer, a second partition layer, and a second substrate having a second electrode formed on its main surface are laminated in this order along a predetermined direction, the cell separation membrane is sandwiched between the first partition layer and the second partition layer, the first partition layer has a first through hole penetrating the first partition layer in the thickness direction corresponding to the first electrode formed on the first substrate, and the second partition layer has a second through hole penetrating the second partition layer in the thickness direction corresponding to the second electrode formed on the second substrate, the first flow path has a first main flow path defined by the main surface on which the first electrode is formed, the first through hole, and the first main surface, the second flow path has a second main flow path defined by the main surface on which the second electrode is formed, the second through hole, and the second main surface, and the first main flow path and the second main flow path may overlap when viewed in the predetermined direction.

[0024] According to this, the first flow path can be formed by a laminated structure of the first substrate, the first partition layer, the cell separation membrane, and the second partition layer, and the second flow path can be formed by a laminated structure of the second substrate, the second partition layer, the cell separation membrane, and the first partition layer. In addition, the first main flow path defined by the first substrate, the first partition layer, and the cell separation membrane of the first flow path overlaps with the second main flow path defined by the second substrate, the second partition layer, and the cell separation membrane of the second flow path. With this configuration, the first main flow path and the second main flow path are in contact with both sides of the cell separation membrane. The electrical resistance of the cell sheet cultured on the cell separation membrane can be measured by the first electrode on the first main flow path side and the second electrode on the second main flow path side. Since the cell culture chip has a microstructure, it is required to be easily processed. Therefore, by forming the cell culture chip as a laminated structure as described above, a cell culture chip with a microscopic and complicated structure can be easily formed.

[0025] Furthermore, for example, the semiconductor device may further include a sheet-like first insulating film arranged between the first substrate and the first partition layer, the first insulating film having a first opening penetrating the first insulating film in a thickness direction corresponding to the first electrode, and a sheet-like second insulating film arranged between the second substrate and the second partition layer, the second insulating film having a second opening penetrating the second insulating film in a thickness direction corresponding to the second electrode.

[0026] When the first substrate and the first partition layer are made of a hard material, and when the second substrate and the second partition layer are made of a hard material, the fluid flowing through the first flow path or the second flow path may leak from these gaps. In particular, when a conductor pattern extending from the first electrode or the second electrode is formed, if the thickness of such a conductor pattern cannot be absorbed, the fluid will leak from the gap. If the fluid leaks from the gap, it may affect the cell culture, and may also affect the electrical resistance of the cell measured by the first electrode and the second electrode. Therefore, by arranging the first insulating film and the second insulating film so as to fill such a gap, the leakage of the fluid from the gap is suppressed. Therefore, the influence of the fluid leaking from the gap on the estimation of the cell state is reduced, and the cell can be evaluated more accurately.

[0027] Also, for example, the first flow path may have a first main flow path, and a first inlet flow path and a first outlet flow path connected to the first main flow path, and the second flow path may have a second main flow path, and a second inlet flow path and a second outlet flow path connected to the second main flow path, and when viewed from a predetermined direction, the first inlet flow path and the second inlet flow path do not overlap, and the first outlet flow path and the second outlet flow path do not overlap.

[0028] According to this, an inlet for injecting a fluid into the first flow path and an inlet for injecting a fluid into the second flow path can be formed at different positions in a plan view seen from the stacking direction. Similarly, an outlet for discharging a fluid from the first flow path and an outlet for discharging a fluid from the second flow path can be formed at different positions in a plan view seen from the stacking direction. It is desirable that the inlet and the outlet are opened in a direction opposite to the direction of gravity. By opening the inlet in a direction opposite to the direction of gravity, the fluid can be injected along the direction of gravity. Also, by opening the outlet in a direction opposite to the direction of gravity, the fluid in the flow path can be maintained until a certain water level is reached. As described above, by forming the openings at different positions in a plan view seen from the stacking direction, a configuration in which the inlet and the outlet of the first flow path and the second flow path are all opened in a direction opposite to the direction of gravity can be realized.

[0029] Furthermore, for example, if the region where the first main flow path and the second main flow path overlap when viewed from a predetermined direction is defined as the cell culture region, the first electrode may extend to the outside of the cell culture region in a direction along the first main flow path, and the second electrode may extend to the outside of the cell culture region in a direction along the second main flow path.

[0030] The first electrode is formed on the main surface of the first substrate, and the second electrode is formed on the main surface of the second substrate. That is, the first electrode and the second electrode have a step protruding from the main surface. When such a protruding structure exists in the first flow path and the second flow path, a minute turbulence occurs. In particular, in the case of a step protruding in a direction intersecting with the flow direction of the fluid, the scale of the turbulence generated is large and may affect the electrical resistance of the measured cells. By extending the first electrode and the second electrode to the outside of the cell culture region, it is possible to eliminate the step protruding from the first electrode and the second electrode in a direction intersecting with the flow direction of the fluid, at least in the cell culture region. Therefore, the generation of turbulence is suppressed, and the cell can be evaluated more accurately by the more accurately measured electrical resistance.

[0031] Furthermore, for example, the device may further include a first lead wire that electrically connects, on the first substrate, a first contact and one end of the first electrode extending to the outside of the cell culture region, and a second lead wire that electrically connects, on the second substrate, a second contact and one end of the second electrode extending to the outside of the cell culture region.

[0032] According to this, the first lead wire electrically connecting the first electrode and the first contact, and the second lead wire connecting the second electrode to the second contact can be arranged outside the cell culture region. As described above, a step protruding intersecting the direction of fluid flow may affect the electrical resistance of the cells being measured, but at least within the cell culture region, the steps of the first lead wire and the second lead wire protruding intersecting the direction of fluid flow can be eliminated. Thus, the generation of turbulence is suppressed, and the more accurately measured electrical resistance allows for more accurate evaluation of the cells.

[0033] Furthermore, for example, the liquid crystal display may further include a third electrode that is in contact with the first flow path and extends within the first flow path along the first flow path while being spaced apart from the first electrode, and a fourth electrode that is in contact with the second flow path and extends within the second flow path along the second flow path while being spaced apart from the second electrode.

[0034] According to this, the electrical resistance of the cell can be measured by a four-terminal method using four electrodes, the first electrode to the fourth electrode. By subtracting the resistance component that may be included as an error, the electrical resistance can be measured more accurately. Therefore, the more accurately measured electrical resistance allows the cell to be evaluated more accurately.

[0035] Also, for example, the first electrode and the third electrode may be flat plates having a main surface in contact with the first flow path, the second electrode and the fourth electrode may be flat plates having a main surface in contact with the second flow path, the third electrode may be wider than the first electrode, and the fourth electrode may be wider than the second electrode.

[0036] According to this, when the first electrode and the third electrode are used separately for current measurement and potential measurement, the current can be measured more accurately, and therefore the electrical resistance can be measured more accurately, and the cells can be evaluated more accurately.

[0037] Furthermore, for example, at least one of the first substrate and the first electrode, and the second substrate and the second electrode may be transparent.

[0038] This allows the cultured cells to be visually observed via the first substrate and the first electrode, or via the second substrate and the second electrode, and since visual changes can also be evaluated when evaluating the cells, the cells can be evaluated more accurately.

[0039] Moreover, one aspect of a cell culture device according to the present disclosure includes the cell culture chip described above and a measuring instrument that measures the electrical resistance between the first electrode and the second electrode.

[0040] According to this, the electrical resistance of the cell sheet cultured on the cell separation membrane arranged between the first flow path and the second flow path can be measured using a measuring device with the first electrode and the second electrode extending along the first flow path and the second flow path. When measuring the electrical resistance of the cell sheet, the conductive distance for conducting the fluid inside the first flow path and the second flow path between the first electrode and the second electrode is reduced. Therefore, the influence of the potential drop when conducting the above-mentioned fluid is reduced, and the electrical resistance of the cell sheet can be measured more accurately. Therefore, the cell state can be estimated based on the more accurate electrical resistance of the cell sheet, thereby making it possible to more accurately evaluate the cells.

[0041] Furthermore, one aspect of the cell culture method according to the present disclosure includes a culture step of culturing a specific cell using the cell culture chip described above, and a measurement step of measuring the electrical resistance of the cultured cell using the first electrode and the second electrode.

[0042] According to this, in the measurement step, the electrical resistance of the cell sheet cultured on the cell separation membrane arranged between the first flow path and the second flow path can be measured by the first electrode and the second electrode extending along the first flow path and the second flow path. When measuring the electrical resistance of the cell sheet, the conductive distance for conducting the fluid inside the first flow path and the second flow path between the first electrode and the second electrode is reduced. Therefore, the influence of the potential drop when conducting the above-mentioned fluid is reduced, and the electrical resistance of the cell sheet can be measured more accurately. Therefore, the cell state can be estimated based on the more accurate electrical resistance of the cell sheet, thereby making it possible to more accurately evaluate the cells.

[0043] Furthermore, these comprehensive or specific aspects may be realized by a system, a method, an integrated circuit, a computer program, or a computer-readable recording medium such as a CD-ROM, or may be realized by any combination of the system, the method, the integrated circuit, the computer program, and the recording medium.

[0044] (Knowledge on which the disclosure is based) As there is a demand for reducing the cost of drug development and for reducing non-clinical and clinical trials from an ethical perspective, development of a new technology that contributes to such demands, for example, an organ on a chip (OoC) as shown in Patent Document 1, is actively underway. OoC is a device that reproduces in vivo tissue functions on a microscale by culturing cells in an artificial extremely small space made of a combination of resin, glass, etc. It is expected that by adding a drug to cells cultured using such a device, it will be possible to evaluate the efficacy and toxicity of the drug, as well as its absorption, distribution, metabolism, excretion, and other tests that previously had to be evaluated using animal tests, within the artificial device.

[0045] The above evaluation system requires high accuracy due to its nature of targeting pharmaceuticals. In the OoC currently being developed, the state of the cells cultured in the device is visually confirmed, and there is room for improvement. In other words, in conventional OoC, the state of the target cells in drug evaluation tests varies, making it difficult to distinguish whether the test results are due to the drug or the variation in the cell state. In other words, there was a problem of lack of accuracy in cell tests using conventional OoC.

[0046] A more detailed explanation will be given with reference to Figures 1A to 2. Figure 1A is a schematic diagram of a cell culture chip according to a comparative example. In OoC, cells are cultured in a sheet form to form a cell sheet that reproduces the tissue function of cells.

[0047] FIG. 1A shows such a cell sheet (a row of rounded rectangles containing an ellipse located at the upper center of the figure), and a first flow path (a space above the cell sheet) and a second flow path (a space below the cell sheet) formed so as to sandwich the cell sheet. The first flow path is filled with a liquid medium used for cell culture, and the first and third electrodes are arranged so as to be immersed in the medium. Similarly, the second flow path is filled with a liquid medium used for cell culture, and the second and fourth electrodes are arranged so as to be immersed in the medium. The first and second flow paths are defined by a cell separation membrane located at the center of the figure, which has a plurality of through holes communicating between the first and second flow paths, so that medium components can be exchanged. The term "defined" refers to a predetermined space being formed by a wall surface formed between the bottom surface and the top surface.

[0048] For example, as shown in Fig. 1A, the electrical resistance between the first electrode and the second electrode is measured in a cell sheet formed on the OoC by using a cell culture chip according to a comparative example. This makes it possible to observe the state of cell sheet culture as an increase in electrical resistance due to the formation of tight junctions between cells in the cell sheet, from a state in which no electrical resistance is shown due to the through-holes in the cell culture chip.

[0049] As described above, when measuring the electrical resistance between the first electrode and the second electrode, the electrical resistance is measured in the path between the first electrode and the second electrode through which electricity flows via the culture medium of the first flow path and the culture medium of the second flow path in addition to the cell sheet. In other words, noise is included in the electrical resistance of the cell sheet to be measured due to the electrical resistance of the culture medium of the first flow path and the culture medium of the second flow path.

[0050] Moreover, the electrical resistance in such a medium differs depending on the position of the cell culture chip. Fig. 1B is a diagram showing the distribution of inter-electrode potential and current density in a comparative example. Fig. 1B shows the potential drop in the flow path from one end (first electrode end) to the other end (second electrode end) of the cell sheet shown in Fig. 1A. Note that in Fig. 1B, a potential of 10 mV is applied between the first and second electrodes, the electrical resistance of the cell sheet is set to 310 Ω, and one end side is shown as the origin (0 mm) of the flow path position and the other end side is shown as 10 mm of the flow path position.

[0051] In the first flow path, a potential of about 7.0 mV is measured at one end, and a potential of about 4.3 mV is measured at the other end. In the second flow path, a potential of about 6.3 mV is measured at one end, and a potential of about 3.6 mV is measured at the other end. The distribution of current density between the first and second flow paths calculated based on the distribution of these potentials shows a downward convex distribution in which the current density is high at one and the other ends and low in the center. This type of distribution indicates that electricity flows easily at one and the other ends, but does not flow easily in the center.

[0052] FIG. 1C is a simulation diagram showing theoretical and measured values ​​of electrical resistance according to a comparative example. In FIG. 1C, the theoretical value of electrical resistance of the cell sheet and the value calculated by simulating the measured value in the case of actual measurement are plotted. It is desirable to draw a straight line with a slope of 1 as shown by the dashed line between the theoretical value and the measured value, but in reality, they draw a curve as shown by the solid line. When the cell sheet shows high electrical resistance, the difference between the theoretical value and the measured value is small. On the other hand, when the cell sheet shows low electrical resistance (for example, when the cell sheet shows electrical resistance of 10 kΩ or less), the measured value shows an electrical resistance lower than the theoretical value due to the uneven distribution of ease and difficulty of electrical conduction between the first flow path and the second flow path described in FIG. 1B. For example, the electrical resistance of the cell sheet is assumed to be 310Ω, but according to FIG. 1C, this value falls within a range with a large error. In this way, the measured value may be inaccurate in the cell culture chip in the comparative example because an electrical resistance lower than the electrical resistance to be measured is measured.

[0053] Therefore, the present disclosure provides a cell culture chip or the like capable of solving the above problems. FIG. 2A is a first conceptual diagram of a cell culture chip according to an embodiment. This diagram shows a cell culture chip according to an embodiment corresponding to the conceptual diagram of the cell culture chip according to the comparative example described in FIG. 1. As shown in FIG. 2A, in the cell culture chip according to the present embodiment, unlike the cell culture chip according to the comparative example, each electrode extends along the direction in which the flow path extends, and the inter-electrode distance between the first electrode and the second electrode is kept approximately constant at any point on the flow path. In this way, the influence of the electrical resistance of the culture medium is reduced, and more accurate measurement of the electrical resistance of the cell sheet is possible.

[0054] FIG. 2B is a second conceptual diagram of the cell culture chip according to the embodiment. The cell culture chip shown in FIG. 2B further includes a third electrode and a fourth electrode in addition to the same configuration as that of FIG. 2A. The distance between the third electrode and the fourth electrode is kept substantially constant at any point on the flow path. Therefore, in the cell culture chip shown in FIG. 2B, the influence of the electrical resistance of the culture medium between the first electrode and the second electrode and between the third electrode and the fourth electrode is reduced, and the electrical resistance of the cell sheet can be measured more accurately using a four-terminal method.

[0055] In the embodiments described below, a cell culture chip having four electrodes, a first electrode to a fourth electrode, as shown in FIG. 2B will be used as an example, but the present disclosure can also be realized using a cell culture chip having two electrodes, a first electrode and a second electrode, as shown in FIG. 2A.

[0056] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings.

[0057] The embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component arrangement positions, connection forms, steps, and order of steps shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. Furthermore, among the components in the following embodiments, components that are not described in an independent claim showing a top concept are described as optional components.

[0058] Note that each figure is not necessarily a strict illustration. In each figure, substantially the same configuration is given the same reference numeral, and duplicated explanations are omitted or simplified. In the following explanation, the X-axis, Y-axis, and Z-axis that are perpendicular to each other are used, but they do not specify the directions when using the cell culture chip, etc.

[0059] Furthermore, in this specification, terms indicating the relationship between elements, such as "parallel," terms indicating the shape of an element, such as "rectangle," as well as numerical values ​​and numerical ranges, are not expressions that only express a strict meaning, but are expressions that also include a substantially equivalent range, for example, differences such as an error of a few percent.

[0060] (Embodiment) [Cell culture equipment] First, the cell culture device in the embodiment will be described. Fig. 3 is a block diagram showing the cell culture device according to the embodiment.

[0061] As shown in FIG. 3, the cell culture device 500 in this embodiment includes a measuring device 300, a control device 301, a switch box 302, and the cell culture chip 100.

[0062] The cell culture chip 100 is a device that serves as a culture tank when culturing cells using the cell culture device 500. The cell culture device 500 calculates the electrical resistance of the cells cultured in the cell culture chip 100 by measuring the electrical resistance using electrodes, and estimates the state of the cells based on the calculated electrical resistance of the cells. Therefore, the cell culture chip 100 is provided with measurement electrodes for calculating the electrical resistance of the cells being cultured. Specifically, the cell culture chip 100 includes a first electrode 21, a second electrode 22, a third electrode 23, and a fourth electrode 24 as electrodes. A detailed configuration of the cell culture chip 100 will be described later.

[0063] The measuring instrument 300 is a measuring device for calculating the electrical resistance of the cells being cultured. Specifically, the measuring instrument 300 is a resistance meter having terminals T1 and T2 to which two electrodes to be measured for electrical resistance are connected, and measuring the electrical resistance between the two electrodes electrically connected to the terminals T1 and T2. The measuring instrument 300 measures, for example, the AC impedance and phase difference between the terminals T1 and T2, and calculates the electrical resistance between the terminals T1 and T2 based on the measurement results. In this case, the calculated electrical resistance is the measured electrical resistance.

[0064] The control device 301 is a processing device that calculates the electrical resistance of the cell based on the measured electrical resistance between the electrodes. The control device 301 is also a processing device that transmits a control signal to the switch box 302 to switch the electrical connection between the measuring device 300 and the electrodes provided on the cell culture chip. The control device 301 is realized as a computer that executes a program for implementing the above functions by a processor and a memory connected to the processor, for example. The control device 301 may be a dedicated circuit capable of executing the above processes.

[0065] The switch box 302 receives a control signal transmitted from the control device 301, and switches the connection between the terminal T1 and the connection terminal S1 to which the first electrode 21 is connected or the connection terminal S3 to which the third electrode 23 is connected based on the control signal. The control device 301 also switches the connection between the terminal T2 and the connection terminal S2 to which the second electrode 22 is connected or the connection terminal S4 to which the fourth electrode 24 is connected. In other words, the switch box 302 includes switches corresponding to the terminals T1 and T2, respectively, and switches the respective switches based on the control signal. In this way, in this embodiment, the measurement of electrical resistance is performed while switching the combination of electrodes to be measured.

[0066] Although not shown, the cell culture device 500 may further include a fluid control device such as a storage tank and a pump for a culture medium used in the culture, an environmental control device such as a gas chamber and a temperature control device for adapting the cell culture chip to a predetermined culture condition, etc. Alternatively, the cells may be cultured using these devices prepared separately from the cell culture device 500.

[0067] Moreover, the cell culture using the above-mentioned cell culture device 500 is performed as follows. FIG. 4 is a flow chart showing a cell culture method in the embodiment. As shown in FIG. 4, first, a predetermined cell is cultured using the cell culture chip 100 (culture step S11). Here, the predetermined cell cultured using the cell culture chip 100 may be any cell. Although details will be described later, the cell culture chip 100 has two flow paths, a first flow path and a second flow path, and it is possible to culture different cells in each flow path. For example, a respiratory system organ model may be constructed by combining the first flow path and the second flow path by culturing lung cells in the first flow path and culturing vascular cells in the second flow path. For example, a brain-blood barrier passage test model may be constructed by culturing cells of the brain-blood barrier system in the first flow path and culturing brain nerve cells in the second flow path.

[0068] Moreover, the cells cultured in each flow channel may not be one type, but may be a combination of multiple types. The cells cultured in each flow channel may be mixed and cultured with different types of cells at an appropriate ratio according to the cell composition of an organ or the like to be reproduced in the flow channel. The cultured cells form a sheet-like structure by intercellular bonds such as tight junctions. Due to such a sheet-like structure, the cultured cells have resistance (i.e., membrane resistance) from one main surface of the sheet to the other main surface. In addition, a fluid such as a culture medium may be passed through the first flow channel and the second flow channel. In a living body, fluids such as blood and lymph are constantly circulating, and cell growth and decline depending on the circulation are repeated. Therefore, by passing a fluid through such a flow channel, it is possible to reproduce conditions closer to those in a living body and perform cell culture and cell evaluation.

[0069] Next, the measuring device 300 measures the electrical resistance between the combination of electrodes that are electrically connected as a result of switching the switches of the switch box 302 based on the control signal of the control device 301 (step S12). The measured electrical resistance data is stored in a storage device such as a memory by the control device 301.

[0070] When the data of the electrical resistance required to calculate the membrane resistance of the cell sheet is stored in the storage device, the control device 301 calculates the membrane resistance of the cell sheet (step S13). For example, in this embodiment, the data of the electrical resistance required to calculate the membrane resistance of the cell sheet is the electrical resistance between the first electrode and the second electrode (R 12 ), between the first and third electrodes (R 13 ), between the second and fourth electrodes (R 24 ), and between the third and fourth electrodes (R 34 In this embodiment, the electrical resistance of the cell sheet is calculated based on the above four pieces of data and the following formula (1).

[0071]

number

[0072] In the above formula 1, a resistance component that may be included as an error in the electrical resistance between the electrodes is subtracted to calculate the electrical resistance of the cell sheet more accurately. Note that, depending on the accuracy of the electrical resistance required by the user of the cell culture device 500, the calculation of the electrical resistance of the cell sheet using the above formula 1 is not essential. In such a case, the electrical resistance of the cell sheet may be the electrical resistance measured between the first electrode and the second electrode as it is. That is, in this embodiment, the cell culture chip 100 may be provided with only the first electrode and the second electrode, and the third electrode and the fourth electrode may be provided only when a more accurate electrical resistance of the cell sheet is required.

[0073] In this manner, the electrical resistance of the cell sheet formed by the cultured cells is calculated, and the state of the cells can be estimated. According to the present disclosure, the calculated electrical resistance of the cells is more accurate, and therefore the state of the cells can be more accurately estimated. Note that steps S12 and S13 are collectively referred to as a measurement step for measuring the electrical resistance of the cells.

[0074] [Cell culture chip] The cell culture chip 100 according to the present embodiment will be described in more detail below. Fig. 5A is a perspective view of the cell culture chip according to the embodiment. Fig. 5B is an exploded perspective view of the cell culture chip according to the embodiment.

[0075] The cell culture chip 100 in this embodiment includes a main body, a cell separation membrane 13, a first electrode 21, a second electrode 22, a third electrode 23, and a fourth electrode 24. The main body has a laminated structure in which a first substrate 11, each of which has a main surface parallel to the XY plane and has the first electrode 21 and the third electrode 23 formed on its main surface on the negative side of the Z axis, a first partition layer 12, a second partition layer 14, and a second substrate 15, each of which has the second electrode 22 and the fourth electrode 24 formed on its main surface on the positive side of the Z axis, are laminated in this order along a predetermined direction (the Z-axis direction in the figure). The cell separation membrane 13 is sandwiched between the first partition layer 12 and the second partition layer 14 of the main body.

[0076] The first substrate 11 is a plate-like member formed using a material such as glass. The material of the first substrate 11 is not limited to glass, and any material such as resin or ceramics may be used. The first substrate 11 is formed from a material that does not have cytotoxicity, since it comes into contact with the cells when the cells are cultured. This is the same for all of the components constituting the cell culture chip 100 described below. In the embodiment, the first substrate 11 is a plate-like member having a rectangular main surface, and a first electrode 21 and a third electrode 23 are formed on the main surface.

[0077] Furthermore, on the first substrate 11, for example, a first contact 21b for connecting to the switch box 302 is formed, and a first lead wire 21a for electrically connecting the first electrode 21 and the first contact 21b is also formed. The first electrode 21, the first lead wire 21a, and the first contact 21b are integrally formed by patterning an indium tin oxide (ITO) film formed on the first substrate 11 by sputtering using photolithography. The first electrode 21, the first lead wire 21a, and the first contact 21b may be formed using other known techniques. The first electrode 21, the first lead wire 21a, and the first contact 21b may be made of a conductive thin film of gold, platinum, or the like, instead of an ITO film. The first electrode 21 and the third electrode 23 are flat plates having a main surface formed by pattern processing, but may be wire-like wiring materials arranged on the first substrate 11.

[0078] Furthermore, on the first substrate 11, for example, a third contact 23b for connecting to the switch box 302 is formed, and a third lead wire 23a for electrically connecting the third electrode 23 and the third contact 23b is also formed. The third electrode 23, the third lead wire 23a, and the third contact 23b are integrally formed by patterning an indium tin oxide (ITO) film formed on the first substrate 11 by sputtering using photolithography. The third electrode 23, the third lead wire 23a, and the third contact 23b may be formed using other known techniques. Also, the third electrode 23, the third lead wire 23a, and the third contact 23b may be formed using a conductive thin film of gold, platinum, or the like, instead of an ITO film.

[0079] The first electrode 21, the first lead 21a, and the first contact 21b, and the third electrode 23, the third lead 23a, and the third contact 23b are formed in a substantially line-symmetrical state while being spaced apart from each other with respect to a center line that bisects the first substrate 11 in the Y-axis direction. The first contact 21b and the third contact 23b are disposed on the negative side in the X-axis direction of the center line that bisects the first substrate 11 in the X-axis direction.

[0080] The first substrate 11 may be made of an insulating material so as to prevent short-circuiting between the conductive electrodes and the like. The first substrate 11 is provided with holes 31 penetrating the first substrate 11 along a predetermined direction so as to communicate with the first partition layer 12 to be laminated thereon. In the present embodiment, four holes 31 are provided, but the number is not limited to this. In a case where a part of the first partition layer 12 is exposed without overlapping with the first substrate 11, for example, the holes 31 may directly communicate with the first partition layer 12 without passing through the holes 31 of the first substrate 11.

[0081] The first partition layer 12 is a plate-like member formed of a silicone resin. The first partition layer 12 has a first through hole penetrating the first partition layer 12 in the thickness direction (Z-axis direction) corresponding to the first electrode 21 and the third electrode 23 formed on the first substrate 11, at least a part of which will be described later in detail. The first through hole corresponds to the first flow path 33. Both ends of the first through hole correspond to two of the holes 31 formed on the first substrate 11. In addition, the first partition layer 12 is provided with holes 32 penetrating the first partition layer 12 in the thickness direction so as to correspond to the remaining two holes 31 excluding the two holes corresponding to the first through holes and to communicate with the second partition layer 14 to be laminated. The number of holes 32 may be less than two, similar to the holes 31.

[0082] The cell separation membrane 13, which is generally called a membrane, is a membranous member having a first main surface 13a on the first partition layer 12 side and a second main surface 13b on the second partition layer 14 side. The cell separation membrane 13 is made of a porous resin material, and has a large number of through-holes with a predetermined hole diameter penetrating the first main surface 13a and the second main surface 13b facing each other. Here, the predetermined hole diameter is the average value of the hole diameters of a large number of through-holes having non-uniform hole diameters. In addition, the predetermined hole diameter is set to be sufficiently smaller than the cell diameter of the cells cultured using the cell culture chip 100.

[0083] Therefore, the cell separation membrane 13 is a semi-permeable membrane that prevents cells sufficiently larger than a predetermined pore size from passing from the first main surface 13a to the second main surface 13b, or from the second main surface 13b to the first main surface 13a, and allows solution components (e.g., medium components, etc.) smaller than a predetermined pore size to pass through. In addition, when the cells cultured in the cell culture chip 100 are adhesive cells, the cell separation membrane 13 also functions as a scaffold for the cells. Therefore, the cell separation membrane 13 may be made of a material that allows the cultured cells to adhere thereto. The cell separation membrane 13 is disposed at a position corresponding to the first through-hole and the second through-hole described later, and is sandwiched between the first partition layer 12 and the second partition layer 14 on the outside of the first through-hole and the second through-hole in a plan view seen from the stacking direction.

[0084] In this manner, the first through-hole and the second through-hole are partitioned by cell separation membrane 13 at the location where the first through-hole and the second through-hole overlap.

[0085] In this manner, a first flow path 33 is formed having a first main flow path 36 defined by the main surface of the first substrate 11 on which the first electrode 21 and the third electrode 23 are formed, the first through-hole, and the first main surface 13a. In other words, the first main flow path 36 is formed between the first substrate 11 and the cell separation membrane 13 by the first through-hole. The first electrode 21 and the third electrode 23 are in contact with the first flow path 33 defined in this manner, particularly the first main flow path 36, and extend within the first main flow path 36 along the first main flow path 36. In addition, the first flow path 33 has a first inlet 34 at one end corresponding to the hole 31 and a first outlet 38 at the other end, each of which communicates with the outside of the cell culture chip 100 via the hole 31. The first flow path 33 has a first inlet flow path 35 connecting from the first inlet 34 to the first main flow path 36, and a first outlet flow path 37 connecting from the first outlet 38 to the first main flow path 36. The first inlet flow path 35 and the first outlet flow path 37 are defined by the second partition layer 14 instead of the cell separation membrane 13 with respect to the first main flow path 36.

[0086] The second partition layer 14 is a plate-like member formed of a silicone resin. The second partition layer 14 has a second through hole penetrating the second partition layer 14 in the thickness direction (Z-axis direction) at least a part of which corresponds to the second electrode 22 and the fourth electrode 24 formed on the second substrate 15. The second through hole corresponds to the second flow path 41, as will be described in detail later. Both ends of the second through hole correspond to the hole 31 formed in the first substrate 11 and the hole 32 formed in the first partition layer 12.

[0087] The second substrate 15 is a plate-like member formed using a material such as glass. The material of the second substrate 15 is not limited to glass, and any material such as resin or ceramics may be used. In the embodiment, the second substrate 15 is a plate-like member having a rectangular main surface, and the second electrode 22 and the fourth electrode 24 are formed on the main surface.

[0088] Further, on the second substrate 15, for example, a second contact 22b for connecting to the switch box 302 is formed, and a second lead wire 22a for electrically connecting the second electrode 22 and the second contact 22b is also formed. The second electrode 22, the second lead wire 22a, and the second contact 22b are integrally formed by patterning an indium tin oxide (ITO) film formed on the second substrate 15 by sputtering using photolithography. The second electrode 22, the second lead wire 22a, and the second contact 22b may be formed using other known techniques. The second electrode 22, the second lead wire 22a, and the second contact 22b may be made of a conductive thin film of gold, platinum, or the like, instead of an ITO film. The second electrode 22 and the fourth electrode 24 are flat-shaped with a main surface formed by pattern processing, but may be wire-like wiring material arranged on the second substrate 15.

[0089] Furthermore, on the second substrate 15, for example, a fourth contact 24b for connecting to the switch box 302 is formed, and a fourth lead wire 24a for electrically connecting the fourth electrode 24 and the fourth contact 24b is also formed. The fourth electrode 24, the fourth lead wire 24a, and the fourth contact 24b are integrally formed by patterning an indium tin oxide (ITO) film formed on the second substrate 15 by sputtering using photolithography. The fourth electrode 24, the fourth lead wire 24a, and the fourth contact 24b may be formed using other known techniques. Also, the fourth electrode 24, the fourth lead wire 24a, and the fourth contact 24b may be made of a conductive thin film of gold, platinum, or the like, instead of an ITO film.

[0090] The second electrode 22, the second lead 22a, and the second contact 22b, and the fourth electrode 24, the fourth lead 24a, and the fourth contact 24b are formed in a substantially line-symmetrical state while being spaced apart from each other with respect to the center line that bisects the second substrate 15 in the Y-axis direction. The second contact 22b and the fourth contact 24b are disposed on the positive side of the X-axis direction relative to the center line that bisects the second substrate 15 in the X-axis direction. As a result, the first contact 21b and the fourth contact 24b do not overlap in a plan view seen from the stacking direction, and the second contact 22b and the third contact 23b do not overlap in a plan view seen from the stacking direction.

[0091] The second substrate 15 may be made of an insulating material so as to prevent short-circuiting of the conductive electrodes and the like.

[0092] In this manner, the second flow path 41 is formed, which has a second main flow path 44 defined by the main surface of the second substrate 15 on which the second electrode 22 and the fourth electrode 24 are formed, the second through-hole, and the second main surface 13b. In other words, the second main flow path 44 is formed between the second substrate 15 and the cell separation membrane 13 by the second through-hole. The second main flow path 44 is a part of the second flow path 41 formed by the second through-hole. The second electrode 22 and the fourth electrode 24 are in contact with the second flow path 41 defined in this manner, particularly the second main flow path 44, and extend within the second main flow path 44 along the second main flow path 44. In addition, the first main flow path 36 and the second main flow path 44 overlap each other in a plan view seen from the stacking direction, and each flow path is defined via the cell separation membrane 13.

[0093] In other words, the cell separation membrane 13 is disposed between the first flow path 33 and the second flow path 41 such that the first main flow path 36 of the first flow path 33 is located on the first main surface 13a, and the second main flow path 44 of the second flow path 41 is located on the second main surface 13b. Therefore, the first main flow path 36 and the second main flow path 44 can exchange components smaller than a predetermined pore size, such as medium components, flowing through each flow path, via the cell separation membrane 13. Furthermore, since electrolytes can also be exchanged between the first main flow path 36 and the second main flow path 44, similar to the medium components, the first main flow path 36 and the second main flow path 44 are electrically connected by the medium components, etc., flowing through each flow path.

[0094] The second flow path 41 has a second inlet 42 at one end corresponding to the holes 31 and 32, and a second outlet 46 at the other end, which are connected to the outside of the cell culture chip 100 via the holes 31 and 32, respectively. The second flow path 41 also has a second inlet flow path 43 connecting from the second inlet 42 to the second main flow path 44, and a second outlet flow path 45 connecting from the second outlet 46 to the second main flow path 44. The second inlet flow path 43 and the second outlet flow path 45 are defined by the first partition layer 12 instead of the cell separation membrane 13 with respect to the second main flow path 44.

[0095] That is, in a plan view seen from the stacking direction, the first inlet flow path 35 and the second inlet flow path 43 do not overlap, and the first outlet flow path 37 and the second outlet flow path 45 do not overlap. As a result, in the first inlet flow path 35 and the first outlet flow path 37, a part of the first flow path 33 is formed by the main surface of the second partition layer 14 where the second through hole is not formed. Also, in the second inlet flow path 43 and the second outlet flow path 45, a part of the second flow path 41 is formed by the main surface of the first partition layer 12 where the first through hole is not formed.

[0096] Fig. 6 is a plan view of area A1 enclosed by a dashed line in Fig. 1, as viewed from the stacking direction. In Fig. 6, the positions of first electrode 21 to fourth electrode 24, first lead wire 21a to fourth lead wire 24a, first contact point 21b to fourth contact point 24b, first flow path 33, second flow path 41, and cell separation membrane 13 are shown by dashed lines when viewed through the film to indicate their respective positional relationships.

[0097] As shown in the figure, the first flow path 33 and the second flow path 41 overlap each other in the first main flow path 36 and the second main flow path 44. The cell separation membrane 13 has a first main surface 13a and a second main surface 13b larger than the entire first main flow path 36 or the entire second main flow path 44, and extends to the outside of the first main flow path 36 and the second main flow path 44 when viewed from a predetermined direction. As a result, in the region where the first main flow path 36 and the second main flow path 44 overlap, the cell separation membrane 13 suppresses contact between the first main flow path 36 and the second main flow path 44 and separates them from each other. The configuration of the cell separation membrane 13 defines each flow path as described above. Here, the cells separated into each flow path by the cell separation membrane 13 are cultured. In this way, the region where the first main flow path 36 and the second main flow path 44 overlap when viewed from the stacking direction (i.e., the region where the first flow path 33 and the second flow path 41 overlap) is defined as a cell culture region 50.

[0098] The first electrode 21 to the fourth electrode 24 extend along the cell culture region 50 within the cell culture region 50. The first electrode 21 to the fourth electrode 24 extend along the cell culture region 50 over a range that is longer than 50%, longer than 75%, and longer than 90% of the length of the cell culture region 50, respectively. The length of the cell culture region 50 is the length between the longest vertices of a flat hexagon in the figure. Furthermore, only one of each of the first electrode 21 to the fourth electrode 24 is disposed within the cell culture region 50. Furthermore, in the figure, the first electrode 21 and the fourth electrode 24 are disposed so as to overlap, and the second electrode 22 and the third electrode 23 are disposed so as to overlap.

[0099] As described above, the first electrode 21 and the third electrode 23 are made of indium tin oxide (ITO) and are transparent. The first substrate 11 is also made of a transparent material such as glass. With this configuration, the first substrate 11, the first electrode 21, and the third electrode 23 are transparent, and the first partition layer 12 is visible through the first substrate 11, the first electrode 21, and the third electrode 23. This allows the cells cultured in the first flow path 33 to be visually observed. The first substrate 11, the first electrode 21, and the third electrode 23 are not necessarily made of a transparent material, and may be made of an opaque material. Similarly, the second substrate 15, the second electrode 22, and the fourth electrode 24 may be transparent or opaque.

[0100] Here, the arrangement and size of the first to fourth electrodes 21 to 24, including the lengths of the first to fourth electrodes 21 to 24 relative to the cell culture region 50, will be described in more detail with reference to the results of various simulations shown in Figures 7A to 9. In the following description, the first to fourth electrodes 21 to 24 may be referred to simply as "electrodes" when there is no particular distinction between them.

[0101] For example, a predetermined measurement level is required for measuring the electrical resistance of cultured cells using the cell culture device 500 described above. For example, the measurement level is set as an error condition such as an error rate of ±20% or less based on the reliability of general measurement values. As described above, in this embodiment, measurement is performed using electrodes formed along the direction in which the flow path extends, and various conditions derived from the results of simulations of the configuration of the cell culture chip 100 that satisfies the above-mentioned measurement level will be described. Note that, as an example, the measurement level is set to an error rate of ±20% or less. Therefore, the numerical values ​​used in the following description are merely examples, and when the measurement level required by the user of the cell culture chip 100 or the cell culture device 500 is different, numerical values ​​corresponding to the measurement level may be set.

[0102] Fig. 7A is a first diagram showing the results of a simulation regarding the length of an electrode according to an embodiment. Fig. 7B is a second diagram showing the results of a simulation regarding the length of an electrode according to an embodiment. Fig. 7A shows the error rate of a simulated measured value (or a simulated value) with respect to a theoretical value of the electrical resistance of a cultured cell to be measured. Fig. 7A also shows the results of a simulation of the relationship between this theoretical value and the error rate for electrodes of a plurality of lengths.

[0103] The length of the electrodes will be described. Here, the length of the electrodes as a relative size to the region (hereinafter also referred to as the electric field region) in which the electric field is formed relatively uniformly by the electrodes in the first flow path 33 and the second flow path 41 is shown as a percentage (hereinafter also referred to as the coverage rate). Specifically, the electric field region is a region having a space spanning the first flow path 33 and the second flow path 41 at a position where the first flow path 33 and the second flow path 41 overlap and extend. In the electric field region, the first electrode 21 and the third electrode 23 extending along the first flow path 33 are arranged, and the second electrode 22 and the fourth electrode 24 extending along the second flow path 41 are arranged. In addition, the first electrode 21, the second electrode 22, the third electrode 23, and the fourth electrode 24 extend along the first flow path 33 or the second flow path 41 in the electric field region. The electric field region here is treated simply as a region having a uniform cross-sectional shape (e.g., a succession of rectangular cross sections of the same area) when cut along a plane perpendicular to the extension direction of the first main flow path 36 and the second main flow path 44, which coincides with the extension direction of the first electrode 21 to the fourth electrode 24 used to form the electric field.

[0104] That is, it is a rectangular region in plan view from the stacking direction of the main body portion 10, consisting of four sides including the two longest sides of the hexagonal cell culture region 50 described in Fig. 6 above as opposite sides. The electric field region is in the shape of a quadrangular prism having a height spanning the first flow path 33 and the second flow path 41 as described above. This height corresponds to the total thickness of the first partition layer 12, the second partition layer 14, and the cell separation membrane 13, but since the thickness of the cell separation membrane 13 is sufficiently small, it can be said that it substantially corresponds to the thickness of the first partition layer 12 and the second partition layer 14.

[0105] In this embodiment, a simulation is performed on the simplified electric field region in this way, and an appropriate electrode length is derived while simplifying the calculation. In reality, an electric field is formed over the entire area of ​​the first flow path 33 and the second flow path 41. Therefore, the entire area of ​​the first flow path 33 and the second flow path 41 may be the electric field region, or the area corresponding to the hexagonal shape of the cell culture region 50 may be the electric field region, or the flow path shape of the cell culture chip 100 may be designed so that the main parts of the first flow path 33 and the second flow path 41 are covered by an electric field region of a simple shape.

[0106] In this way, the length of the electrodes constituting the cell culture chip 100 can be specified by using the above coverage rate as a numerical value that indicates the length of the electrodes relatively to the length of the electric field region that is specified in advance based on the error rate of the simulation value. The length of the electric field region is the length in the extension direction of the electric field region that extends in the same manner as the first flow path 33 and the second flow path 41 extend as flow paths. The length of the electrodes is the length in the extension direction of the first electrode 21 to the fourth electrode 24 that extends along the first flow path 33 and the second flow path 41. The extension direction of the electric field region and the extension direction of the first electrode 21 to the fourth electrode 24 are the same, and the lengths of the first electrode 21 to the fourth electrode 24 are the same.

[0107] Therefore, in Fig. 7A, the error rate of the simulation value with respect to the theoretical electrical resistance value is shown for electrodes with a coverage rate of 100%, 75%, 50%, 25%, and 10% as the coverage rate for defining the length of the electrode. In addition, in Fig. 7A, as a comparative example, the error rate of the simulation value with respect to the theoretical electrical resistance value for the electrodes in the cell culture chip with the configuration shown in Fig. 1A is also shown. Note that the electrodes in the cell culture chip with the configuration shown in Fig. 1A are not electrodes that extend along the flow paths as in the embodiment, but are electrodes that are arranged at the inlet or outlet of each flow path and are electrically connected to each other via a fluid flowing through the flow paths.

[0108] As shown in FIG. 7A, for any coverage, the smaller the electrical resistance of the cultured cells to be measured, the larger the error rate tends to be. A larger error rate means a larger absolute value of the error rate. In other words, the more the error rate moves away from 0% in the positive or negative direction, the larger the error rate. Here, the value of the electrical resistance of the cultured cells varies depending on the cell type and the cell state (or growth state), etc. Therefore, the coverage may be appropriately selected depending on the cell type and cell state, etc. of the cultured cells. For example, the case of the cell using the cell culture chip 100 with the electrical resistance value of the arrow shown in (b) in the figure (i.e., 100Ω) will be described with reference to FIG. 7B. In FIG. 7B, the electrical resistance value is fixed at 100Ω, and a graph showing the change in the error rate of the simulation value with respect to the change in the coverage is shown. Note that the dot hatching in the figure shows the range of the error rate of the simulation value that meets the measurement standard. This is the same in FIG. 7A, as well as in FIG. 8 and FIG. 9 described later.

[0109] As shown in the figure, when the electrical resistance of the cultured cells is 100Ω, the error rate of the simulation value that satisfies the measurement standard can be obtained when the coverage value is in the range of 70% or more and 100% or less. Referring again to Figure 7A, considering that the error rate decreases as the electrical resistance of the measurement target increases, if the cell type and cell state are such that the electrical resistance of the cultured cells is expected to be 100Ω or more, good measurement can be performed using the cell culture chip 100 having electrodes with a coverage rate of 70% or more.

[0110] By the same judgment, for example, when measuring the electrical resistance of 50Ω (arrow indicated as (a) in the figure), which is the approximately minimum value expected for cultured cells, the measurement can be performed satisfactorily using a cell culture chip 100 having electrodes with a coverage rate of 75% or more. Also, for example, when measuring the electrical resistance of 1000Ω (arrow indicated as (c) in the figure), which is expected for cultured cells, the measurement can be performed satisfactorily using a cell culture chip 100 having electrodes with a coverage rate of 25% or more.

[0111] In the following, the regulation regarding the direction intersecting the extension direction of the electrodes will be described based on the results of a simulation with reference to Fig. 8. Fig. 8 is a diagram showing the results of a simulation regarding the electrode width of the embodiment. Fig. 8 shows the error rate of the simulation value that changes when the width of the electrode (hereinafter also referred to as the electrode width) is changed during pattern processing of the electrode coating formed on the main surface of the first substrate 11 or the second substrate 15.

[0112] The electrode width here refers to the size of the electrode in the direction intersecting the electrode extension direction, which is different from the electrode thickness, which is the size of the electrode in the stacking direction, and is determined by the film thickness at the time of film formation. In other words, the electrode width refers to the size of the electrode in the direction perpendicular to the extension direction and stacking direction. Here, the electrical resistance value of the cultured cells to be measured is set to 100 Ω.

[0113] As shown in Fig. 8, if the electrode thickness is constant, the larger the electrode width, the lower the electrical resistance in the electrode, and the smaller the error rate of the simulation value. As shown in Fig. 8, in order to measure an electrical resistance of 100 Ω while satisfying the measurement standard, the electrode width needs to be 0.1 mm or more. In addition, considering the result of Fig. 7A in which the error rate decreased as the electrical resistance of the cultured cells increased, the cell culture chip 100 having an electrode with an electrode width of 0.1 mm is sufficient to measure an electrical resistance of 100 Ω or more.

[0114] Hereinafter, the relationship between the channel height, which defines the distance between the first electrode 21 and the third electrode 23, and the second electrode 22 and the fourth electrode 24, and the error rate of the simulation value will be described with reference to FIG. 9. FIG. 9 is a diagram showing the results of a simulation regarding the channel height of the embodiment. FIG. 9 shows a graph showing the relationship between the size in the stacking direction of the first main channel 36 and the second main channel 44, which correspond to the electric field region, among the first channel 33 and the second channel 41, and the error rate of the simulation value. Note that the simulation here is performed by setting the channel width, which is the size in the direction intersecting the extension direction and the stacking direction among the sizes of the first main channel 36 and the second main channel 44, to 10 mm, which is the size that is most likely to cause the largest error within the designable range.

[0115] The first electrode 21 and the third electrode 23 formed on the main surface of the first substrate 11 and the second electrode 22 and the fourth electrode 24 formed on the main surface of the second substrate 15 are spaced apart in accordance with the thicknesses of the first partition layer 12 and the second partition layer 14. The thickness of the first partition layer 12 matches the size of the first main flow channel 36 in the stacking direction. The thickness of the second partition layer 14 matches the size of the second main flow channel 44 in the stacking direction. The distance between the first electrode 21 and the third electrode 23 and the second electrode 22 and the fourth electrode 24 is related to the resistance value of the fluid flowing through the flow channel, and is one of the factors that can cause errors.

[0116] As shown in Fig. 9, there is an appropriate range for the channel height to meet the measurement standard. For example, in the example shown in Fig. 9, in order to measure an electrical resistance of 100 Ω while meeting the measurement standard, the channel height needs to be set within a range of 0.2 mm or more and 1.5 mm or less. In other words, the electric field region spanning the first channel 33 and the second channel 41 has a height (size in the stacking direction) of 0.2 mm or more and 1.5 mm or less. If the cell culture chip 100 has a channel height within the above range, it is possible to satisfactorily measure the electrical resistance of cultured cells such as 100 Ω at a measurement level with an error rate of ±20% or less.

[0117] Here, another example of the electrode configuration in this embodiment will be described with reference to Figures 10A to 10D. Note that Figures 10A to 10D described below only show the cell culture region 50, the first electrode, and the third electrode in a plan view in area A1, which is the same viewpoint as in Figure 6. Also, the other example of the electrode configuration described below may be applied to only one of the first electrode and the third electrode, may be applied to both the first electrode and the third electrode, or may be applied to all of the first electrode, the second electrode, the third electrode, and the fourth electrode.

[0118] Fig. 10A is a first diagram for explaining the configuration of the electrodes of the embodiment. The first electrode 21c and the third electrode 23c shown in Fig. 10A extend along the cell culture region 50 over a range longer than 100% of the length of the cell culture region 50. In other words, the first electrode 21c and the third electrode 23c extend to the outside of the cell culture region 50 in a direction along the first main channel 36. The second electrode and the fourth electrode may be similar. In this way, the ends of the electrodes are not located within the cell culture region 50, so that the influence of minute turbulence generated at the ends of the electrodes on the measurement value can be reduced.

[0119] 10B is a second diagram for explaining the configuration of the electrodes in the embodiment. The first electrode 21d and the third electrode 23d shown in FIG. 10B extend along the cell culture region 50 over a range longer than 100% of the length of the cell culture region 50. The widths (lengths in the Y-axis direction) of the first electrode 21d and the third electrode 23d are larger than the first electrode 21 and the third electrode 23 in the above embodiment. The second electrode and the fourth electrode may be similar. This makes it easy to align the first electrode 21d and the third electrode 23d with the second electrode and the fourth electrode in the Y-axis direction, and to align the first electrode 21d and the third electrode 23d with the cell culture region 50 (i.e., with the first flow path 33 and the second flow path 41).

[0120] FIG. 10C is a third diagram for explaining the configuration of the electrodes of the embodiment. The first electrode 21e and the third electrode 23e shown in FIG. 10C extend along the cell culture region 50 over a range longer than 100% of the length of the cell culture region 50. In addition, the first electrode 21e is electrically connected to the first contact 21g at one end of the first electrode 21e extending to the outside of the cell culture region 50 by the first lead wire 21f outside the cell culture region 50. Similarly, the third electrode 23e is electrically connected to the third contact 23g at one end of the third electrode 23e extending to the outside of the cell culture region 50 by the third lead wire 23f outside the cell culture region 50. The second electrode and the fourth electrode may be similar. According to this, the lead wire connecting the electrodes to the terminals is not located in the cell culture region 50, so that the influence of minute turbulence generated in the lead wire on the measurement value can be reduced.

[0121] FIG. 10D is a fourth diagram for explaining the configuration of the electrodes of the embodiment. The third electrode 23h shown in FIG. 10D has a larger width (length in the Y-axis direction) (i.e., wider) than the first electrode 21h. The second electrode and the fourth electrode may have the same. For example, in measuring electrical resistance, when an electrode for measuring a current and an electrode for measuring a potential are separate, it is preferable that the electrode for measuring the electrode has as small an internal resistance as possible (a large cross-sectional area). Therefore, by doing as described above, the third electrode 23h is used as an electrode for measuring a current, and the first electrode 21h is used as an electrode for measuring a potential, and electrical resistance can be measured with higher accuracy.

[0122] 11A to 17B, examples of the embodiment will be described below. In the examples described below, a cell culture chip 100 will be produced, and the results of culturing model cells thereon will be described.

[0123] FIG. 11A is a plan view showing the first step in the fabrication of the cell culture chip. FIG. 11B is a cross-sectional view of the XI-XI section in FIG. 11A. As shown in FIG. 11A and FIG. 11B, in the first step in the fabrication of the cell culture chip 100, a second substrate 15 made of glass was prepared. The second substrate 15 had a main surface area of ​​1200 square millimeters, 30 millimeters in the Y-axis direction × 40 millimeters in the X-axis direction, and a thickness (length in the Z-axis direction) of 0.7 millimeters. Next, indium tin oxide (ITO) was formed on the main surface of this second substrate 15 by a sputtering method so as to have a thickness of 150 nanometers, and the second electrode 22, the second lead wire 22a, and the second contact point 22b, and the fourth electrode 24, the fourth lead wire 24a, and the fourth contact point 24b were formed by etching. At this time, the second electrode 22 and the fourth electrode 24 were designed to have a width of 0.1 millimeters and a length of 10 millimeters.

[0124] FIG. 12A is a plan view showing a second step in the production of the cell culture chip. FIG. 12B is a cross-sectional view of the XII-XII section in FIG. 12A. As shown in FIG. 12A and FIG. 12B, in the second step in the production of the cell culture chip 100, the second partition layer 14 formed of silicone resin was bonded to the main surface of the second substrate on which the second electrode 22 and the fourth electrode 24 were formed. The second partition layer 14 used had a main surface area of ​​800 square millimeters, 20 millimeters in the Y-axis direction × 40 millimeters in the X-axis direction, and a thickness (length in the Z-axis direction) of 1.0 millimeter. The second partition layer 14 used had a second through hole formed in advance. Specifically, the second through hole was formed by laser cutting so as to have a width of 1.0 millimeter.

[0125] When the second through hole was formed, a straight portion having a length of 13 millimeters along the X-axis direction and corresponding to the second main flow path 44 was formed. When the second through hole was formed, a slanted portion on one end side corresponding to the second inlet flow path 43 was formed, extending from the end of the straight portion on the negative X-axis side in a direction inclined by 30 degrees toward the negative X-axis and negative Y-axis sides with respect to the X-axis direction. When the second through hole was formed, a portion corresponding to the second inlet 42 was formed on an extension of the slanted portion on one end side and at a position not reaching the end of the second partition layer 14.

[0126] In addition, in the formation of the second through hole, an inclined portion was formed on the other end side corresponding to the second outlet flow path 45, extending in a direction inclined by 30 degrees from the end of the straight portion on the positive side of the X axis toward the positive side of the X axis and the positive side of the Y axis with respect to the X axis direction. In addition, in the formation of the second through hole, a portion corresponding to the second outlet port 46 was formed on an extension line of the inclined portion on the other end side and at a position not reaching the end of the second partition layer 14. The lengths of the second partition layer 14 and the second substrate 15 in the Y axis direction were set to 20 mm and 30 mm, respectively, so that the second contact 22b and the fourth contact 24b formed on the second substrate 15 were exposed. In other words, the second partition layer 14 was bonded so that the end of the second substrate 15 in the Y axis direction was at least partially exposed.

[0127] The second partition layer 14 was aligned and bonded such that the straight portions of the second through holes were positioned to correspond to the second electrodes 22 and the fourth electrodes 24. The second partition layer 14 was bonded by applying a molten silicone resin material similar to that used for the second partition layer 14 to the main surface of the second substrate 15, bonding the second partition layer 14, and then leaving the material to stand in an atmosphere at 65° C. for two hours.

[0128] Here, the adhesion between the second substrate 15 and the second partition layer 14 will be described in more detail with reference to Fig. 13A and Fig. 13B. Fig. 13A is a cross-sectional view illustrating the first insulating film and the second insulating film according to the embodiment. Fig. 13B is an exploded perspective view illustrating the first insulating film and the second insulating film according to the embodiment. Fig. 13A (a) shows a cross-sectional view taken along line XIII-XIII in Fig. 5A, and Fig. 13A (b) shows the first substrate 11, the first partition layer 12, the first insulating film 16 disposed therebetween, and the second substrate 15, the second partition layer 14, and the second insulating film 17 disposed therebetween.

[0129] FIG. 13B illustrates the first substrate 11, the first partition layer 12, and the first insulating film 16 disposed therebetween. By inverting the diagram, FIG. 13B can also be considered to illustrate the second substrate 15, the second partition layer 14, and the second insulating film 17 disposed therebetween. Note that the reference symbols and the directional axes indicating the directions when inverted are shown in parentheses. When inverted, the holes 31 and 32 that are not present in the second substrate 15 and the second partition layer 14 are ignored, and similarly, the holes 16b that are present in the first insulating film 16 but not in the second insulating film 17 are also ignored. In the following description, the configuration of the first substrate 11 and the first partition layer 12 side will be described. Note that the same description can be made by replacing the first substrate 11 with the second substrate 15 and the first partition layer 12 with the second partition layer 14, and therefore the description of the configuration of the second substrate 15 and the second partition layer 14 side will be omitted.

[0130] 13A, in the embodiment described above, the first substrate 11 and the first partition layer 12 are bonded via the first electrode 21, the first lead wire 21a, and the first contact point 21b, as well as the second electrode 22, the second lead wire 22a, and the second contact point 22b. In this case, if the first substrate 11 and the first partition layer 12, or any of the adhesive layers formed by the adhesive for bonding them, is made of a flexible material, there is no need to consider the thickness of the first electrode 21, etc.

[0131] However, under certain conditions, such as when the first substrate 11 and the first partition layer 12 are made of a hard material and the adhesive forms a very thin adhesive layer, a gap is formed between the first substrate 11 and the first partition layer 12 due to the thickness of the first electrode 21, etc. In this case, when a fluid is passed through the first flow path 33, the fluid may leak from the gap, making it impossible to perform accurate measurement. In such a case, a cell culture chip 100a in which a sheet-like first insulating film 16 is disposed between the first substrate 11 and the first partition layer 12 may be used.

[0132] The first insulating film 16 is a material having a certain thickness and flexibility, and is formed of, for example, an acrylic resin. Due to its certain thickness and flexibility, the first insulating film 16 absorbs the thickness of the first electrode 21 and fills the gap formed between the first substrate 11 and the first partition layer 12. Here, the first electrode 21 formed on the main surface of the first substrate 11 needs to be exposed to the first through-hole of the first partition layer 12.

[0133] That is, it is desirable that no other members exist between the first partition layer 12 and a part of the main surface of the first substrate 11 on which the first electrode 21 exists. Therefore, a first opening 16a (a second opening 17a in the case of the second insulating film 17) penetrating the first insulating film 16 in the thickness direction is formed in the first insulating film 16 at a location corresponding to the first electrode 21. Similarly, it is desirable that no other members exist between the holes 31 and 32. Therefore, a hole 16b penetrating the first insulating film 16 in the thickness direction is formed in the first insulating film 16 at a location corresponding to the holes 31 and 32.

[0134] In this manner, in the cell culture chip 100a, a first insulating film 16 may be provided for filling the gap formed between the first substrate 11 and the first partition layer 12 without impairing functions such as cell culture and electrical resistance measurement.

[0135] Fig. 14A is a plan view showing a third step in the production of the cell culture chip. Fig. 14B is a cross-sectional view of the XIV-XIV section in Fig. 14A. As shown in Fig. 14A and Fig. 14B, in the third step in the production of the cell culture chip 100, the cell separation membrane 13 formed of a porous resin was bonded to the main surface of the second partition layer 14 such that the second main surface 13b was oriented toward the main surface side of the second partition layer 14.

[0136] The cell separation membrane 13 used had a main surface area of ​​70 square millimeters (5.0 mm in the Y-axis direction × 14 mm in the X-axis direction) and a thickness of 10 micrometers (length in the Z-axis direction). The cell separation membrane 13 used had through-holes with a predetermined hole diameter of 3.0 micrometers. The cell separation membrane 13 was bonded by aligning it with the second partition layer 14 so that it covered at least the straight part of the second through-hole, preferably so that the centers of the second partition layer 14 and the second partition layer 14 were approximately aligned in the XY plane. The cell separation membrane 13 was bonded in the same manner as the bonding between the second substrate 15 and the second partition layer 14, by applying the same silicone resin material as the second partition layer 14 in a molten state to the main surface of the second partition layer 14, bonding the cell separation membrane 13, and then leaving it to stand in a 65°C atmosphere for 2 hours.

[0137] Fig. 15A is a plan view showing a fourth step in the production of the cell culture chip. Fig. 15B is a cross-sectional view of the XV-XV section in Fig. 15A. As shown in Figs. 15A and 15B, in the fourth step in the production of the cell culture chip 100, the first partition layer 12 formed of silicone resin was bonded to the main surfaces of the cell separation membrane 13 and the second partition layer 14.

[0138] The first partition wall layer 12 used had a main surface area of ​​800 square millimeters (20 millimeters in the Y-axis direction × 40 millimeters in the X-axis direction) equivalent to that of the second partition wall layer 14, and a thickness (length in the Z-axis direction) of 1.0 millimeter. The first partition wall layer 12 used had the first through hole and hole 32 formed therein in advance. Specifically, the first through hole was formed by laser cutting so as to have a width of 1.0 millimeter, and the hole 32 was formed by laser cutting so as to have a diameter of 0.5 millimeters.

[0139] In forming the first through hole, a straight portion having a length of 13 mm along the X-axis direction and corresponding to the first main flow path 36 was formed. In addition, in forming the first through hole, an inclined portion on one end side corresponding to the first inlet flow path 35 was formed, extending from the end of the straight portion on the negative X-axis side in a direction inclined by 30 degrees toward the negative X-axis side and the positive Y-axis side with respect to the X-axis direction. In forming the first through hole, a portion corresponding to the first inlet 34 was formed on an extension line of the inclined portion on one end side and at a position not reaching the end of the first partition wall layer 12.

[0140] In addition, when the first through hole was formed, an inclined portion was formed on the other end side that corresponds to the first outlet flow path 37, extending from the end portion on the positive side of the X-axis of the straight portion in a direction inclined by 30 degrees toward the positive side of the X-axis and the negative side of the Y-axis with respect to the X-axis direction. In addition, when the first through hole was formed, a portion that corresponds to the first outlet 38 was formed on an extension line of the inclined portion on the other end side and at a position that does not reach the end of the first partition wall layer 12.

[0141] The first partition layer 12 was bonded in the same manner as the bonding between the second substrate 15 and the second partition layer 14, by applying a molten silicone resin material similar to that used for the first partition layer 12 to the main surfaces of the cell separation membrane 13 and the second partition layer 14, bonding the first partition layer 12, and then leaving the resulting mixture to stand in an atmosphere at 65°C for 2 hours.

[0142] FIG. 16A is a plan view showing a fifth step in the fabrication of the cell culture chip. FIG. 16B is a cross-sectional view of the XVI-XVI section in FIG. 16A. As shown in FIG. 16A and FIG. 16B, in the fifth step in the fabrication of the cell culture chip 100, a first substrate 11 made of glass was prepared. The first substrate 11 had a main surface area of ​​1200 square millimeters, 30 millimeters in the Y-axis direction × 40 millimeters in the X-axis direction, and a thickness (length in the Z-axis direction) of 0.7 millimeters. Next, indium tin oxide (ITO) was formed on the main surface of the first substrate 11 by a sputtering method so as to have a thickness of 150 nanometers, and the first electrode 21, the first lead wire 21a, and the first contact point 21b, the third electrode 23, the third lead wire 23a, and the third contact point 23b were formed by etching. At this time, the first electrode 21 and the third electrode 23 were designed to have a width of 0.1 millimeters and a length of 10 millimeters.

[0143] The first substrate 11 was bonded to the first partition layer 12 so that the main surface of the first substrate 11 on which the first electrode 21, the first lead 21a, and the first contact 21b, and the third electrode 23, the third lead 23a, and the third contact 23b were formed faced the first partition layer 12. The lengths of the first partition layer 12 and the first substrate 11 in the Y-axis direction were set to 20 mm and 30 mm, respectively, so that the first contact 21b and the third contact 23b formed on the first substrate 11 were exposed. In other words, the first substrate 11 was bonded so that at least a part of the end of the first substrate 11 in the Y-axis direction was exposed.

[0144] The first substrate 11 was aligned and bonded such that the straight line portions of the first through holes were positioned to correspond to the first electrode 21 and the third electrode 23. The first substrate 11 was bonded by applying a molten silicone resin material similar to that of the first partition layer 12 to the main surface of the first partition layer 12, bonding the first substrate 11, and then leaving the material to stand in an atmosphere at 65°C for 2 hours.

[0145] In addition, when the silicone resin material is applied in a molten state and left to stand for 2 hours in an atmosphere at 65° C. for adhesion, the step of leaving the material to stand for 2 hours in an atmosphere at 65° C. may be performed integrally. In this manner, the cell culture chip 100 similar to the above embodiment was produced.

[0146] Furthermore, in this embodiment, a storage tank for a medium to be used for culturing cells was attached. FIG. 17A is a plan view showing the sixth step in the production of the cell culture chip. FIG. 17B is a cross-sectional view of the XVII-XVII section in FIG. 17A. As shown in FIG. 17A and FIG. 17B, in the sixth step in the production of the cell culture chip, a cell culture chip 100b was produced in which a storage tank 200 was attached to the cell culture chip 100 produced up to the fifth step. The storage tank 200 is a cylindrical member formed of silicone resin and has a diameter of 6.0 mm and a height (length in the Z-axis direction) of 7.0 mm, and becomes a container with the main surface of the first substrate 11 as the bottom surface by being attached on the first substrate 11. The storage tank 200 was attached corresponding to each of the holes 31 provided in the first substrate. The storage tank 200 has a volume of about 200 microliters due to the above dimensions. The reservoir 200 was bonded in the same manner as above by applying a silicone resin material in a molten state to the main surface of the first substrate 11 and leaving it to stand in an atmosphere at 65° C. for 2 hours.

[0147] Next, using the cell culture chip 100b produced up to the sixth step, Caco-2 cells, a cell line derived from human colon cancer, which are model cells, were cultured. First, the cell culture chip 100b was sterilized by irradiating it with ultraviolet light for 30 minutes, and washed three times with phosphate buffered saline (PBS). Next, 10 microliters of medium was injected into the second flow path 41, and 10 microliters of cell suspension was injected into the first flow path 33. The cell suspension was previously diluted with Caco-2 cells at a concentration of 2.0×10 6 The cell culture chip 100b was incubated at 37° C., 5% CO2 Then, 150 microliters of medium was poured into each of the reservoirs 200 of the cell culture chip 100b, and the cells were cultured in a stationary atmosphere at 37° C. and 5% CO 2 During static culture in the atmosphere, the electrical resistance was measured once a day. In addition, once every two days, the entire medium in the storage tank 200 was removed and 150 microliters of new medium was injected.

[0148] The electrical resistance was measured using the measuring device 300, the control device 301, and the switch box 302 of the cell culture device 500 described above. The first electrode 21 was connected to the connection terminal S1 via the first contact 21b, and the third electrode 23 was connected to the connection terminal S3 via the third contact 23b. The second electrode 22 was connected to the connection terminal S2 via the second contact 22b, and the fourth electrode 24 was connected to the connection terminal S4 via the fourth contact 24b.

[0149] The control signal from the control device 301 causes R 12 , R 13 , R 24 , and R 34 was measured. In addition, the electrical resistance of the cell sheet of cultured Caco-2 cells was calculated using the above formula 1. From the calculated electrical resistance of the cell sheet, a transient increase in resistance was observed, and the process of formation and collapse of intercellular matrices such as tight junctions was estimated. Since such an estimation was not observed in the estimation of cell states by visual observation, the high accuracy of the estimation of cell states in the present disclosure was confirmed compared to the conventional estimation of cell states by visual observation.

[0150] (Other embodiments) Although the embodiments have been described above, the present disclosure is not limited to the above-described embodiments.

[0151] Further, although the components constituting the cell culture device have been exemplified in the above-mentioned embodiments, the functions of the components of the cell culture device may be allocated in any manner to multiple parts constituting the cell culture device.

[0152] In addition, the present disclosure also includes forms obtained by applying various modifications to the embodiments, etc. that a person skilled in the art would conceive, or forms realized by arbitrarily combining the components and functions of the embodiments, etc., within the scope that does not deviate from the spirit of the present disclosure.

[0153] For example, the cell culture chip described in the above embodiment as having a layered structure may be integrally formed using a technique such as a 3D printer.

[0154] Furthermore, for example, the first substrate and the first and second electrodes do not have to be transparent. When the purpose is only to measure the electrical resistance in the cell sheet, the cell state does not have to be visually confirmed.

[0155] Furthermore, a single cell culture chip may have multiple configurations similar to those of the above-described embodiment (i.e., a first flow path, a second flow path, a first electrode, and a second electrode), allowing cell culture under multiple conditions and testing using the cultured cells to be performed simultaneously.

[0156] In addition, in the above embodiment, the cell culture region has a linear shape in which the first main flow path extending in a straight line and the second main flow path extending in a straight line overlap, but the cell culture region may have a curved shape in which the first main flow path and the second main flow path extending in a curved line overlap, as long as the first flow path and the second flow path overlap.

[0157] In addition, for example, the present disclosure may be realized by a two-terminal system with only the first and second electrodes, without the third and fourth electrodes. In this case, the switch box and the control device may not be provided. The present disclosure can be realized with only a cell culture chip and a measuring device as the cell culture device. [Industrial Applicability]

[0158] INDUSTRIAL APPLICABILITY The present disclosure contributes to new developments in pharmaceutical development and the like, such as a cell culture device and the construction of a test system using cells cultured using the cell culture device. [Explanation of symbols]

[0159] 11 First board 12 1st partition layer 13 Cell separation membrane 13a First main surface 13b Second main surface 14 Second partition layer 15 Second board 16 First insulating film 16a 1st opening 16b, 31, 32 holes 17 Second insulating film 17a 2nd opening 21, 21c, 21d, 21e, 21h 1st electrode 21a, 21f First lead wire 21b, 21g 1st contact 22 2nd electrode 22a 2nd lead wire 22b 2nd contact 23, 23c, 23d, 23e, 23h 3rd electrode 23a, 23f 3rd lead wire 23b, 23g 3rd contact 24 4th electrode 24a 4th lead 24b 4th contact 33 First Channel 34 1st inlet 35 1st inlet channel 36 1st main channel 37 1st outlet flow path 38 1st outlet 41 Second Stream 42 2nd inlet 43 2nd inlet channel 44 2nd main channel 45 2nd outlet flow path 46 2nd outlet 50 Cell culture area S1, S2, S3, S4 connection terminals T1, T2 terminals 100, 100a, 100b Cell Culture Chip 200 Reservoir 300 Measuring Instruments 301 Control device 302 Switch Box 500 cell culture equipment

Claims

1. a main body portion having a first flow path and a second flow path at least a portion of which overlaps with the first flow path when viewed from a predetermined direction; a cell separation membrane having a first main surface and a second main surface facing each other, the cell separation membrane being disposed between the first flow path and the second flow path such that the first flow path is located on the first main surface and the second flow path is located on the second main surface; a potential measuring electrode that is in contact with the first flow path and extends along the first flow path within the first flow path; A first electrode that is an electrode; a second electrode in contact with the second flow path and extending along the second flow path within the second flow path; a third electrode that is a current measuring electrode and is in contact with the first flow path and extends along the first flow path in a state separated from the first electrode; a fourth electrode that is in contact with the second flow path and extends along the second flow path within the second flow path while being spaced apart from the second electrode; the first electrode and the third electrode are flat plates each having a main surface in contact with the first flow path, the second electrode and the fourth electrode are flat plates each having a main surface in contact with the second flow path, The third electrode is wider than the first electrode, The fourth electrode is wider than the second electrode. Cell culture chip.

2. the first flow path and the second flow path include an electric field region that is a region that spans the first flow path and the second flow path at a position where the first flow path and the second flow path extend while overlapping each other as viewed from the predetermined direction, and is a region in which the first electrode and the second electrode extend within the region; The length of the first electrode and the second electrode in the extension direction is 25% or more of the length of the electric field region in the extension direction. The cell culture chip according to claim 1 .

3. The length of the first electrode and the second electrode in the extension direction is 70% or more of the length of the electric field region in the extension direction. The cell culture chip according to claim 2 .

4. The length of the first electrode and the second electrode in the extension direction is 75% or more of the length of the electric field region in the extension direction. The cell culture chip according to claim 3 .

5. In the electric field region, the total length of the first flow path and the second flow path in the predetermined direction is 0.2 mm or more and 1.5 mm or less. The cell culture chip according to any one of claims 2 to 4.

6. The first electrode and the second electrode have a width of 0.1 mm in a direction intersecting with an extension direction of the first electrode and the second electrode. The cell culture chip according to any one of claims 1 to 5.

7. the main body portion has a laminated structure in which a first substrate having the first electrode formed on a main surface thereof, a first partition layer, a second partition layer, and a second substrate having the second electrode formed on a main surface thereof are laminated in this order along the predetermined direction; The cell separation membrane is sandwiched between the first barrier layer and the second barrier layer, the first partition layer has a first through hole penetrating the first partition layer in a thickness direction in correspondence with the first electrode formed on the first substrate, the second partition layer has a second through hole penetrating the second partition layer in a thickness direction in correspondence with the second electrode formed on the second substrate, the first flow path has a first main flow path defined by a main surface on which the first electrode is formed, the first through hole, and the first main surface, The second flow path has a second main flow path defined by a main surface on which the second electrode is formed, the second through hole, and the second main surface, and the first main flow path and the second main flow path overlap when viewed from the predetermined direction. The cell culture chip according to any one of claims 1 to 6.

8. a first insulating film in a sheet form disposed between the first substrate and the first partition layer, the first insulating film having a first opening penetrating the first insulating film in a thickness direction in correspondence with the first electrode; a sheet-like second insulating film disposed between the second substrate and the second partition layer, the second insulating film having a second opening penetrating the second insulating film in a thickness direction in correspondence with the second electrode; The cell culture chip according to claim 7 .

9. The first flow path includes the first main flow path, and a first inlet flow path and a first outlet flow path connected to the first main flow path, The second flow path includes the second main flow path, and a second inlet flow path and a second outlet flow path connected to the second main flow path, When viewed from the predetermined direction, the first inlet flow path and the second inlet flow path do not overlap, and the first outlet flow path and the second outlet flow path do not overlap. The cell culture chip according to claim 7 or 8.

10. When a region where the first main channel and the second main channel overlap as viewed from the predetermined direction is defined as a cell culture region, The first electrode extends to an outside of the cell culture region in a direction along the first main channel, The second electrode extends to the outside of the cell culture region in a direction along the second main channel. The cell culture chip according to any one of claims 7 to 9.

11. a first lead wire electrically connecting, on the first substrate, a first contact point provided on the first substrate and one end of the first electrode extending to the outside of the cell culture region; a second lead wire electrically connecting, on the second substrate, a second contact provided on the second substrate and one end of the second electrode extending to the outside of the cell culture region; The cell culture chip according to claim 10.

12. At least one of the first substrate and the first electrode and the second substrate and the second electrode is transparent. The cell culture chip according to any one of claims 7 to 11.

13. The cell culture chip according to any one of claims 1 to 12, a measuring instrument for measuring the electrical resistance between the first electrode and the second electrode. Cell culture equipment.

14. A culturing step of culturing a predetermined cell using the cell culture chip according to any one of claims 1 to 12; and a measuring step of measuring the electrical resistance of the cultured cells using the first electrode and the second electrode. Cell culture method.

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