Cell Imaging Device

Abstract

To provide an electrical impedance tomography sensor for cell imaging and a cell imaging device, which enable low-invasive, efficient evaluation of ion channels even when cells are anisotropic.SOLUTION: An electric impedance tomography sensor 10 for cell imaging is provided, comprising a substrate 25 and a measurement unit 45 comprising four or more electrodes 20 provided on the substrate 25 and a cell placement section 28 provided on the substrate 25 and designed to have a cell or a group of cells placed thereon, where the electrodes 20 are arranged around the cell placement section 28.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an electrical impedance tomography sensor for cell imaging and a cell imaging device. [Background technology]

[0002] Ion channels, proteins on cell membranes, have attracted attention in fields such as drug discovery and cell identification. Ion channels are ion permeation pathways in the cell membrane, and are responsible for the formation of resting membrane potentials, the generation of action potentials, the control of chemical transmitter release, muscle contraction, and hormone secretion.

[0003] In the field of drug discovery, ion channels are involved in many diseases, so drug discovery targeting ion channels is progressing. For example, amlodipine, a dihydropyridine calcium ion channel antagonist, is one of the ion channel-targeting drugs and is taken worldwide.

[0004] Ion channel drugs are an important drug discovery target related to various diseases, but they account for only about 5% of all pharmaceuticals. One reason for this is the difficulty of evaluating drug efficacy in anisotropic cells in a minimally invasive, easy, and highly efficient manner. For example, the patch clamp method is a highly accurate method for electrically measuring ion flow through ion channels, but the evaluation speed is slow and requires advanced experimental techniques and experience. Therefore, there is a need for a minimally invasive, easy, and highly efficient method for evaluating ion flow through ion channels.

[0005] In the field of cell identification, cell identification techniques that distinguish target cells from heterologous cells in a minimally invasive manner are important in regenerative medicine. One example of a cell identification technique is flow cytometry. Flow cytometry is capable of highly efficient cell identification, but because it is an invasive method that uses fluorescent reagents, it is unsuitable for the regenerative medicine application of liver cells. From the perspective of quality and safety, there is a need for a minimally invasive method for measuring and evaluating the state of cells and tissues that does not require fluorescent reagents for the regenerative application of stem cells.

[0006] Non-Patent Document 1 discloses a technique for measuring the impedance of the fluid outside a cell group over time using an interdigital impedance sensor as a minimally invasive and efficient technique for identifying cells. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] D. Kawashima, S.Li, H.Obara, M. Takei, Low-frequency impedance-based cell discrimination considering ion transport model in cell suspension, IEEE Transactions on Biomedical Engineering, 68, 3, 1015-1023 (2021) Summary of the Invention [Problem to be solved by the invention]

[0008] However, the technique of Non-Patent Document 1 has a problem in that the measurement accuracy is poor when cells have anisotropy.

[0009] The present invention has been made in consideration of the above-mentioned problems, and aims to provide an electrical impedance tomography sensor for cell imaging and a cell imaging device that are capable of evaluating ion channels in a minimally invasive and highly efficient manner even when cells have anisotropy. [Means for solving the problem]

[0010] In order to solve the above problems, the present invention proposes the following means. <1> A cell imaging apparatus according to one aspect of the present invention comprises:The measuring unit comprises a substrate, four or more electrodes provided on the substrate, and a cell arrangement section provided on the substrate in which a cell or a group of cells is arranged, the electrodes being arranged around the cell arrangement section, and the electrodes being arranged on each of a plurality of concentric circles having different radii centered around the center of the cell arrangement section. A cell imaging device comprising an electrical impedance tomography sensor for cell imaging, The device comprises a current / voltage application measurement unit that applies a current or a potential difference between the electrodes, and measures the potential difference and phase based on a current application / voltage measurement pattern when the current is applied, and measures the current and phase based on a voltage application / current measurement pattern when the potential difference is applied between the electrodes; a Jacobian matrix calculation unit that calculates a Jacobian matrix based on the current application / voltage measurement pattern or the voltage application / current measurement pattern, mesh coordinates obtained by dividing the cell placement unit, and the coordinates of each of the electrodes; an electrical property distribution calculation unit that calculates the electrical property distribution of the cell or the cell group from the Jacobian matrix calculated by the Jacobian matrix calculation unit and the potential difference and phase or the current and phase measured by the current / voltage application measurement unit; and a cell identification unit that uses an ion transport model to evaluate the ion transport ability of the cell or the cell group from the electrical property distribution, thereby identifying the cell or the cell group.

[0016] <2> Above< 1 In the cell imaging device described above, the current / voltage application / measurement unit may apply the current or the potential difference at a frequency of 1 Hz or more and 1 THz or less.

[0017] < 3 > Above< 1 In the cell imaging device described above, the current / voltage application / measurement unit may apply the current or the potential difference at a frequency of 1 kHz or more and 1 MHz or less.

[0018] < 4 > Above< 1 In the cell imaging device described above, the current / voltage application and measurement unit may apply the current or the potential difference at a frequency exceeding 1 MHz.

[0020] < 5 > Above< 1 >~< 4 The cell imaging device described in any one of the above items may further include a cell supply unit that supplies the cells or the cell group to the cell placement unit.

[0021] < 6 > Above< 1 >~< 5 The cell imaging device described in any one of the above items may be provided with a stimulation unit that stimulates the cells or the cell group. [Effects of the Invention]

[0022] According to the above aspects of the present invention, it is possible to provide an electrical impedance tomography sensor for cell imaging and a cell imaging device that are capable of evaluating ion channels in a minimally invasive and highly efficient manner even when the cells are anisotropic. [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a schematic diagram of a cell imaging device according to an embodiment of the present invention. [Figure 2] FIG. 2 is a schematic diagram of a current / voltage application and measurement unit. [Figure 3] FIG. 10 is a diagram for explaining a current application voltage measurement pattern. [Figure 4] 1 is a flowchart of cell identification. [Figure 5] FIG. 10 is a schematic diagram of a cell imaging apparatus according to a second embodiment. [Figure 6] FIG. 10 is a schematic diagram of a current / voltage application and measurement unit according to the second embodiment. [Figure 7] FIG. 10 is a diagram for explaining differences in image images due to differences in cell tumors. [Figure 8] FIG. 10 is a plan view of a modified example of an electrical impedance tomography sensor for cell imaging. [Figure 9] FIG. 10 is a plan view of a modified example of an electrical impedance tomography sensor for cell imaging. [Figure 10] FIG. 10 is a cross-sectional view of the electrical impedance tomography sensor for cell imaging taken along line AA in FIG. 9. [Figure 11] 1 is a photograph showing an example of a cell imaging device. [Figure 12] Photograph of the electrodes of an electrical impedance tomography sensor for cell imaging. [Figure 13] 1 is a micrograph of a spheroid. [Figure 14] FIG. 1 is a diagram showing the time change of two-dimensional imaging. [Figure 15]This is a predicted image of 3D imaging. DETAILED DESCRIPTION OF THE INVENTION

[0024] (Cell imaging device) A cell imaging device 100 according to one embodiment of the present invention will be described below with reference to the drawings. As shown in Fig. 1, the cell imaging device 100 includes a current / voltage application / measurement unit 1 and an imaging calculation unit 50. The imaging calculation unit 50 includes a Jacobian matrix calculation unit 3, an electrical property distribution calculation unit 4, a cell identification unit 5, and an output unit 6.

[0025] The imaging calculation unit 50 of the cell imaging device 100 includes, for example, a central processing unit (CPU), read only memory (ROM), random access memory (RAM), and hard disk drive (HDD) / solid state drive (SSD). The Jacobian matrix calculation unit 3, electrical property distribution calculation unit 4, cell identification unit 5, and output unit 6 are realized by the CPU executing a predetermined program. The program may be acquired via a recording medium or via a network. Alternatively, a dedicated hardware configuration may be used to realize the configuration of the cell imaging device 100. Each unit will be described below.

[0026] (Current and voltage application measurement section) The current / voltage application and measurement unit 1 will be described using Figure 2. The drawings used in the following description may show characteristic parts enlarged for the sake of clarity, and the dimensional ratios of each component may differ from the actual ones. The materials, dimensions, etc. exemplified in the following description are merely examples, and the present invention is not limited to them. Appropriate changes can be made within the scope of the effects of the present invention.

[0027] First, let us define the directions. The direction parallel to the surface of the substrate 25 is the x direction, and the direction perpendicular to the x direction along the surface of the substrate 25 is the y direction. The z direction is perpendicular to the surface of the substrate 25. The z direction is perpendicular to the x and y directions. Hereinafter, the +z direction may be expressed as "up" and the -z direction as "down". Up and down do not necessarily coincide with the direction in which gravity is applied.

[0028] As shown in FIG. 2, the current / voltage application measurement unit 1 includes a cell imaging electrical impedance tomography sensor 10 and a control unit 30. The cell imaging electrical impedance tomography sensor 10 includes a substrate 25, Q electrodes 20 provided on the substrate 25, and a cell placement unit 28 provided on the substrate 25 in which cells are placed. The measurement unit 45 includes the electrodes 20 and the cell placement unit 28. Here, the number Q of electrodes 20 is 4 or more. More preferably, the number of electrodes 20 is 8 or more. In the example shown in FIG. 2, the number Q of electrodes 20 is 8. Having four or more electrodes 20 enables evaluation of the distribution of electrical properties of cells. The placement positions of the electrodes 20 are not particularly limited as long as they are arranged around the cell placement unit 28. Preferably, the electrodes 20 are arranged circumferentially around the center of the cell placement unit 28. More preferably, the electrodes 20 are evenly arranged around the circumference of the cell placement unit 28. After placing a cell on the cell placement section 28 of the electrical impedance tomography sensor 10 for cell imaging, the current / voltage application / measurement section 1 applies a predetermined current or potential difference between the electrodes 20 and measures the potential difference or current.

[0029] After placing a cell on the cell placement section 28 of the electrical impedance tomography sensor 10 for cell imaging, the current / voltage application / measurement section 1 applies a predetermined current or potential difference between the electrodes 20 and measures the potential difference or current. When applying a current, the potential difference is measured based on a predetermined current application / voltage measurement pattern (a pattern in which two electrodes are selected in sequence from a large number of electrodes, a current is applied, and the potential difference is measured sequentially). At this time, it is desirable to also measure the phase (the time lag between the applied current and the measured potential difference). When applying a potential difference, the current is measured based on a predetermined voltage application / current measurement pattern (a pattern in which two electrodes are selected in sequence from a large number of electrodes, a potential difference is applied, and the current is measured sequentially). At this time, it is desirable to also measure the phase (the time lag between the applied potential difference and the measured current). Hereinafter, the case of applying a current will be mainly described, and detailed description of the case of applying a potential difference may be omitted.

[0030] (substrate) There are no particular limitations on the substrate 25. For example, a resin substrate, a glass substrate, or the like can be used as the substrate 25.

[0031] (electrode) The electrode 20 is provided on a substrate 25. It is electrically connected to the control unit 30. In this embodiment, the electrode 20 and the wiring connection unit 35 are connected by substrate wiring 32, and the wiring connection unit 35 and the control unit 30 are connected by external wiring 36. The material and shape of the electrode 20 are not particularly limited as long as it can apply a current or a potential difference to the cells. Examples of materials for the electrode 20 include metals such as Au, Ag, and Cu, and conductive polymers.

[0032] The width and length of the electrode 20 are not particularly limited as long as they can accurately apply a current or potential difference to a cell or a group of cells. The width of the electrode 20 is, for example, 0.15 mm. The length of the electrode 20 is, for example, 0.40 mm.

[0033] (Cell arrangement part) The cell placement section 28 is provided on the substrate 25. The size of the cell placement section 28 is not particularly limited as long as it is large enough to place a cell or a group of cells. For example, the cell placement section 28 may be a circular area with a diameter of 1 mm. Examples of the cell group include spheroids and organoids.

[0034] A single cell or a group of cells such as a spheroid or an organoid may be statically placed by a known method on cell placement section 28. Alternatively, the cell or group of cells may be caused to flow continuously or intermittently on cell placement section 28 by a known method.

[0035] (Control unit) The control unit 30 includes, for example, a multiplexer for switching between current application electrodes (or voltage application electrodes for applying a potential difference) that apply a current and voltage measurement electrodes (or current measurement electrodes for measuring a current) that measure a potential difference, and an impedance analyzer for measuring voltage (or current) and phase. The impedance analyzer is a component that measures impedance, i.e., the ratio of the measured potential difference (applied potential difference) to the applied current (measured current), and its phase, by changing the applied frequency and amplitude. The control unit 30 executes a predetermined program in, for example, a CPU and controls the multiplexer and impedance analyzer to perform impedance measurement (measurement of the ratio of the potential difference to the current and its phase). The control unit 30 may perform impedance measurement by controlling only the current / voltage application measurement unit 1, or may perform impedance measurement by controlling the control unit 30 according to a program executed in the imaging calculation unit 50. The results of the impedance measurement are sent to the electrical property distribution calculation unit 4. The method of transmitting information to the electrical property distribution calculation unit 4 is not particularly limited. The data may be sent from the control unit 30 to the electrical property distribution calculation unit 4 of the imaging calculation unit 50 via a wired connection, or may be sent to the electrical property distribution calculation unit 4 of the imaging calculation unit 50 wirelessly.

[0036] The control unit 30 applies a current between the electrodes 20 and measures the potential difference based on a predetermined current application / voltage measurement pattern (a pattern that determines between which electrodes the current is applied and between which electrodes the potential difference is measured). Alternatively, the control unit 30 applies a potential difference between the electrodes 20 and measures the current based on a predetermined voltage application / current measurement pattern. When applying a current or a potential difference, there are no particular limitations on which electrodes 20 the current (potential difference) is applied between and which electrodes the potential difference (current) is measured between. However, it is preferable to "uniformly" apply the current (potential difference) to the electrodes 20 arranged two-dimensionally or three-dimensionally and measure the potential difference (current). "Uniformly apply the current (potential difference) and measure the potential difference (current)" means that the current / potential difference is applied and measured so that all of the electrodes 20 are used at least once to apply or measure the current / potential difference.

[0037] The current application voltage measurement pattern for electrode 20 will be described using the electrode arrangement in FIG. 3 as an example. Numbers representing the positions of electrode 20 are assigned counterclockwise from the first electrode, which serves as the reference, for example. The number M of current application voltage measurement patterns differs for each current application voltage measurement pattern. Each current application voltage measurement pattern will be described below. Note that the current application voltage measurement patterns described below can also be applied to voltage application current measurement patterns. Examples of current application voltage measurement patterns will be described below, but the present invention is not limited to the following current application voltage measurement patterns.

[0038] First, we will explain the current application voltage measurement pattern using the counter electrode method. In this case, a current is applied between a pair of opposing electrodes. Using Figure 3(a) as an example, current is applied between opposing electrodes, such as electrodes 1 and 9, and electrodes 2 and 10. In the case of Figure 3(a), the number of electrodes Q is 16, so there are a total of eight possible patterns. The potential difference is measured between pairs of electrodes, such as electrodes 2 and 3, and electrodes 3 and 4, excluding the electrode to which the current is applied. Since measurements are performed on pairs of electrodes 2 and 3 through electrodes 15 and 16, there are 13 possible voltage measurement patterns for one current application pattern. Therefore, with the counter electrode method, the total number of measurements (measurement patterns) M is 104. Here, when a current is applied and a potential difference is measured, the measurement pattern is a voltage measurement pattern. When a potential difference is applied and a current is measured, the measurement pattern is a current measurement pattern.

[0039] Next, we will explain the current application voltage measurement pattern using the adjacent electrode method. In this case, current is applied between adjacent electrodes. For example, using Figure 3(b), current is applied to adjacent electrodes, such as electrodes 1 and 2, and electrodes 2 and 3. In the case of Figure 3(b), the number of electrodes Q is 16, so there are 16 possible patterns in total. The potential difference is measured in electrode pairs, such as electrodes 3 and 4, excluding the electrode to which the current is applied. Measurements are then made from electrodes 3 and 4 to electrodes 15 and 16, so there are 13 possible voltage measurement patterns for one current application pattern. Therefore, in the case of the adjacent electrode method, the number of measurements (measurement patterns) M is 208 in total.

[0040] This section explains the current application and voltage measurement patterns for the reference method. In this case, the potential difference is measured for all combinations between a reference electrode and electrodes other than the reference electrode. For example, using Figure 3(c), current is applied between the reference electrode and electrodes other than the reference electrode, such as electrodes 1 and 2, or electrodes 1 and 3. In the case of Figure 3(c), the number of electrodes Q is 16, so there are 16 possible combinations in total. The potential difference is measured for electrode pairs such as electrodes 3 and 4, excluding the electrode to which the current is applied. Measurements are performed for electrode pairs from electrodes 3 and 4 to electrodes 15 and 16. Therefore, there are 13 possible voltage measurement patterns for one current application pattern. Therefore, the total number of measurements (measurement patterns) M for the reference method is 208.

[0041] We will explain the current application voltage pattern using the two-terminal method. In this case, the electrode that applies the current and the electrode that measures the potential difference are the same, and the voltage is measured for all combinations of electrodes. If the number of electrodes Q is 16, the number of measurements (measurement patterns) M will be 120 in total.

[0042] The applied current value (or potential difference) and its application frequency are preferably, for example, 1.0 mA or less and an application frequency of 1 Hz to 1 THz, taking into consideration the effect on cells and the simplicity of the device. When imaging the ion concentration in the fluid outside the cell group, the application frequency is preferably 1 kHz to 1 MHz. When imaging the ion concentration inside the cell group, the application frequency is preferably greater than 1 MHz.

[0043] Hereinafter, an example will be described in which the potential difference is measured using the neighboring method in the cell imaging device 100 of this embodiment. Note that, although the following description will be given using an example in which the electrodes 20 are arranged two-dimensionally, the present invention can also be applied to a case in which the electrodes 20 are arranged three-dimensionally.

[0044] (Jacobian matrix calculation section 3) The Jacobian matrix is a sensitivity matrix that indicates how much the measured potential difference when a current is applied (or the measured current when a voltage is applied) changes with respect to changes in the reference of the electrical properties (conductivity, permittivity) distributed in space. The Jacobian matrix (sensitivity matrix) of a cell or cell group differs depending on the spatial distribution of the electrical properties of the cell or cell group, etc. If the Jacobian matrix of the cell or cell group is known, the electrical property distribution can be calculated. The Jacobian matrix calculation unit 3 calculates the Jacobian matrix for the region of the cell or cell group arranged in the cell arrangement unit 28 based on a predetermined current application voltage measurement pattern (or voltage application current measurement pattern), mesh coordinates, and the coordinates of each electrode 20. The Jacobian matrix calculation unit 3 calculates the Jacobian matrix J g using, for example, the following formula (3) based on the image data in which the cell or cell group is arranged, ion concentration, etc. Hereinafter, the Jacobian matrix J g will be described.

[0045] First, the region including the cell arrangement unit 28 is divided into a two-dimensional mesh (in the case of three dimensions, it is divided into a three-dimensional mesh) so that an appropriate resolution can be obtained according to the Q electrodes 20. For example, when the number of electrodes 20 is Q = 8, the cell arrangement unit 28 may be divided into a total of 4096 points by dividing it 64 times in the x direction and approximately 64 times in the y direction to create a mesh n (1 ≤ n ≤ N). In this case, the number of meshes N is 4096. The number of meshes and the shape can be appropriately set according to the number of electrodes 20 and the required resolution. If the electrode arrangement is three-dimensional, it is divided into a three-dimensional mesh.

[0046] The Jacobian matrix J g is represented by the following formula (1). M in formula (1) indicates the number of current application voltage measurement patterns, and N indicates the number of meshes. The Jacobian matrix J g n at mesh n (1 ≤ n ≤ N) is represented by formula (2). The Jacobian matrix element J g nm at the current application voltage measurement pattern m (1 ≤ m ≤ M) of mesh n (1 ≤ n < N) is calculated using the following formula (3). Here, σ nindicates the conductivity in mesh n as an example of an electrical property distribution, but other electrical property distributions (conductivity difference distribution Δσ, permittivity distribution, permittivity difference distribution, phase distribution, phase difference distribution) may also be used. n indicates the area of ​​the nth mesh (volume in the case of three dimensions). Zm(e,d) indicates the measured potential difference V in the current application voltage measurement pattern m. e indicates the current application electrode pair in the current application voltage measurement pattern m, and d means the voltage measurement electrode pair in the current application voltage measurement pattern m. v(i e ) denotes the potential difference between the voltage measurement electrode pair d induced by applying a current to the current application electrode pair e. d ) is the potential difference between the current application electrode pair e induced by the current application to the voltage measurement electrode pair d. ∇ is the nabla symbol, which is the differential operator.

[0047]

number

[0048] (Electrical Property Distribution Calculation Unit) The electrical property distribution calculation unit 4 calculates the Jacobian matrix J of the cell or cell group sent from the Jacobian matrix calculation unit 3. g The electrical property distribution of the cell or cell group is calculated from the potential difference and phase (or current and phase) measured by the current / voltage application measurement unit 1. Here, the electrical property distribution is, for example, the conductivity distribution σ, the conductivity difference distribution Δσ, the permittivity distribution, the permittivity difference distribution, the phase distribution, and the phase difference distribution. Hereinafter, the conductivity and the conductivity difference (conductivity at time t relative to the reference at time t0) may be described separately, and the symbol Δ is used to represent the difference. The following explanation focuses on the conductivity distribution σ. The Jacobian matrix J of the cell and cell group g The problem of finding the conductivity distribution σ from the measured impedance Z is called an ill-posed inverse problem, and can be found, for example, by using iterative calculations. The number of iterations is represented by the number on the right. The initial conductivity distribution σ at the 0th iteration is 0 is the Jacobian matrix J of a cell or a group of cells (the number in the upper right corner is the number of iterations). gis used to calculate from the following equation (4). T indicates a transposed matrix. Z in equation (4) is a column vector with M elements of a predetermined current application voltage measurement pattern (or voltage application current measurement pattern), as shown in the following equation (5). Zm in equation (5) is expressed by the following equation (6). m in equation (6) is the current application voltage measurement pattern. The conductivity distribution σ of a cell or cell group is the initial conductivity distribution σ 0 is used as the start of the iteration count and is calculated using the following formula (7). In formula (7), i represents the number of iterations. In formula (7), R represents a regularization matrix, and λ represents an arbitrary parameter for converging the calculation, for example, 0.01. R is expressed, for example, by the following formula (8), and is the Jacobian matrix J of the cell or cell group. g The calculated electrical property distribution of the cell or cell group is sent to the cell identification unit 5 or the output unit 6. The electrical property distribution calculation unit 4 may convert the electrical property distribution (for example, conductivity distribution) into another electrical property distribution and send it to the cell identification unit 5 or the output unit 6. For example, the electrical property distribution calculation unit 4 may convert the conductivity distribution σ into an ion concentration distribution by using the following equation (9) and send it to the cell identification unit 5. In the following equation (9), C exp (x, y, t) denotes the measured ion concentration at the coordinate (x, y) at time t, γ is a constant, and σ(x, y, t) denotes the conductivity at the coordinate (x, y) at time t.

[0049]

number

[0050]

number

[0051] (Cell Identification Department) The cell identification unit 5 uses an ion transport model to evaluate the ion transport capacity of a cell or cell group from the electrical property distribution sent from the electrical property distribution calculation unit 4, thereby identifying the cell or cell group. The evaluation of ion transport capacity can be performed, for example, by the following method. Figure 4 shows a flowchart of cell identification. First, an initial value of the ion transport capacity of the cell is set (S1). Here, examples of the ion transport capacity of the cell include a diffusion coefficient and membrane permeability. Next, a numerical analysis is performed using the set ion transport value of the cell and the ion transport model, and a model ion concentration C num (x, y, t)(M) is calculated (S2). Here, the ion transport model is, for example, Fick's diffusion equation expressed by the following equation (10). In the following equation (10), D(x, y) is the diffusion coefficient (m 2 / s), t is time (s), and ∇ is the nabla symbol, which is the differential operator. Next, the obtained model ion concentration C num (x,y,t) and measured ion concentration C exp The error between (x, y, t) is calculated (S3). It is determined whether the error value is within the specified range (S4). If the error value is outside the range, the ion transport capacity value is set again (S5), and the model ion concentration C num If the error value is within the specified range, the cell identification unit 5 identifies the cell or cell group based on the value of the ion transport ability of the cell (S6). Information on the identified cell or cell group is sent to the output unit 6.

[0052]

number

[0053] (output section) The output unit 6 outputs the electrical property distribution of the cell or cell group sent from the electrical property distribution calculation unit 4 and the identification information of the cell or cell group sent from the cell identification unit 5. The output destination of the electrical property distribution and identification information of the cell or cell group is not particularly limited. The output destination may be a display unit such as a liquid crystal display or a storage device such as a hard disk drive.

[0054] Second Embodiment Next, a cell imaging device 100A according to a second embodiment will be described with reference to Figures 5 and 6. In this second embodiment, the same components as those in the first embodiment are denoted by the same reference numerals, and their description will be omitted, with only the differences being described. As shown in Figure 6, the cell imaging device 100A includes a current / voltage application / measurement unit 1A and an imaging calculation unit 50. The imaging calculation unit 50 includes a Jacobian matrix calculation unit 3, an electrical property distribution calculation unit 4, a cell identification unit 5, and an output unit 6.

[0055] (Current and voltage application measurement section) The current / voltage application measurement unit 1A will be described with reference to FIG. 6. As shown in FIG. 6, the current / voltage application measurement unit 1A includes a cell imaging electrical impedance tomography sensor 10A, a control unit 30, stimulators 60A and 60B that apply stimuli to cells or cell groups, and a cell supply unit 70 that supplies cells or cell groups to a cell placement unit 28. The cell imaging electrical impedance tomography sensor 10A includes a substrate 25, Q electrodes 20 provided on the substrate 25, and a cell placement unit 28 provided on the substrate 25 and in which cells are placed. The cell imaging electrical impedance tomography sensor 10A includes two or more measurement units, each consisting of an electrode 20 and a cell placement unit 28. In the second embodiment, the cell imaging electrical impedance tomography sensor 10A has three measurement units: measurement units 45A, 45B, and 45C. Here, the number Q of electrodes 20 in each measurement unit is four or more. The number of electrodes 20 may be the same or different for each measurement unit. By providing multiple measurement units, the electrical impedance tomography sensor for cell imaging 10A can simultaneously measure multiple cells or cell groups. It is more preferable that the electrodes 20 of each measurement unit are evenly arranged around the circumference of the cell placement unit 28 of each measurement unit. After placing a cell on the electrical impedance tomography sensor for cell imaging 10, the current / voltage application measurement unit 1A applies a predetermined current or voltage between the electrodes 20 and measures the potential difference or current. Measurements at each measurement unit may be performed simultaneously, or the measurement time may be staggered for each measurement unit.

[0056] Stimulation units 60A and 60B have the function of providing stimulation to cells or cell groups. The type of stimulation is not particularly limited as long as it can provide stimulation to cells or cell groups, and examples include mechanical stimulation, electrical stimulation, and chemical stimulation. Stimulation unit 60A is a chemical solution supply means that supplies a chemical solution. Stimulation unit 60B is an electrode that electrically stimulates cells or cell groups.

[0057] By applying stimulation to cells or cell groups using stimulation units 60A and 60B and imaging the ion concentrations inside and / or outside the cell group, it is possible to confirm the operation of ion channels distributed three-dimensionally in the cell membrane of the cell group. For example, amlodipine, a hydropyridine calcium ion channel antagonist, inhibits L-type Ca ion channels but not other types such as N-type and T-type Ca ion channels. Therefore, as shown in Figure 7, a significant anisotropy is evident in the ion concentration imaging images inside and / or outside the cell group. By providing stimulation unit 60A, it is possible to visualize the behavior of ion channels due to chemical stimulation and to investigate the effects of chemical stimulation in detail.

[0058] The cell supply unit 70 supplies cells or cell groups to the cell placement unit 28. The cell supply unit is not particularly limited as long as it can supply cells or cell groups to the cell placement unit 28. The cell supply unit 70 may be stationary in the cell placement unit 28 to place cells, or may flow the cells or cell groups so as to pass through the cell placement unit 28 continuously or intermittently.

[0059] <Modification 1 of Electrical Impedance Tomography Sensor for Cell Imaging> Next, we will explain an electrical impedance tomography sensor for cell imaging 10B, which is a modified example of the electrical impedance tomography sensor for cell imaging. As shown in FIG. 8 , the electrical impedance tomography sensor for cell imaging 10B includes a substrate 25, Q electrodes 20A and 20B provided on the substrate 25, and a cell placement section 28 provided on the substrate 25 in which cells are placed. The electrical impedance tomography sensor for cell imaging 10B includes a measurement section 45D consisting of the electrodes 20A and 20B and the cell placement section 28. The number of electrodes 20A is four or more, and the number of electrodes 20B is four or more. The number of electrodes 20A and the number of electrodes 20B may be the same or different. The electrodes 20A and 20B of the electrical impedance tomography sensor for cell imaging 10B are arranged concentrically around the center of the cell placement section 28. Specifically, the electrodes 20A and 20B are arranged on the circumferences of concentric circles C1 and C2 of different radii. Electrode 20A is arranged on an inner circle C1 with a smaller radius, and electrode 20B is arranged on an outer circle C2 with a larger radius. Electrodes 20A and 20B are preferably arranged evenly on the circumference of each of circles C1 and C2. Furthermore, electrodes 20A and 20B are preferably arranged so that they do not overlap in the radial direction. By arranging electrodes 20A and 20B in this manner, three-dimensional cell imaging can be performed even when the electrodes are arranged on the same plane.

[0060] <Modification 2 of Electrical Impedance Tomography Sensor for Cell Imaging> Next, an electrical impedance tomography sensor for cell imaging 10C, which is a modified example of the electrical impedance tomography sensor for cell imaging, will be described with reference to FIGS. 9 and 10. FIG. 9 is a plan view of the electrical impedance tomography sensor for cell imaging 10C. FIG. 10 is a cross-sectional view taken along line AA in FIG. 9. As shown in FIGS. 9 and 10, the electrical impedance tomography sensor for cell imaging 10C includes a substrate 25, Q electrodes 20C and 20D provided on the substrate 25, and a cell placement section 28 provided on the substrate 25 and in which cells are placed. The electrical impedance tomography sensor for cell imaging 10C also includes a measurement section 45D consisting of the electrodes 20C and 20D and the cell placement section 28. An electrode layer 80A is formed by the electrode 20C and an insulator 81A surrounding the electrode 20C. Similarly, an electrode layer 80B is formed by the electrode 20D and an insulator 81B surrounding the electrode 20D. In this embodiment, an electrode layer 80B is provided on an electrode layer 80A. The electrical impedance tomography sensor 10C for cell imaging includes an electrode layer 80B including an electrode 20C, an insulator 81A covering the periphery of the electrode 20C, and the electrode layer 80A, and an electrode 20D and an insulator 81B covering the periphery of the electrode 20D. The electrical impedance tomography sensor 10C for cell imaging may include two or more stacked electrode layers. The electrodes 20C and 20D are each arranged in a circle in the XY plane. The number of electrodes 20C is four or more, and the number of electrodes 20D is four or more. The number of electrodes 20C and electrodes 20D may be the same or different. By arranging the electrodes 20C and 20D three-dimensionally in this manner, three-dimensional cell imaging can be performed.

[0061] The electrical impedance tomography sensor 10 for cell imaging and the cell imaging device of the present disclosure have been described in detail above. Note that the technical scope of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention. Furthermore, within the spirit of the present invention, the components in the above-described embodiment can be replaced with well-known components as appropriate, and the above-described modifications can be combined as appropriate.

[0062] (Example) Next, an example of an experiment conducted to verify the effectiveness of the electrical impedance tomography sensor for cell imaging and the cell imaging device of the present disclosure will be described.

[0063] Using an electrical impedance tomography sensor for cell imaging and a cell imaging device, spheroids were placed stationary in the cell placement area, and the ion concentration of the extracellular fluid was imaged two-dimensionally from the conductivity distribution. Figure 11 is a photograph of an example of the cell imaging device disclosed herein. The cell imaging device used in this example comprises an electrical impedance tomography sensor for cell imaging, a multiplexer, an impedance analyzer (IM3570, HIOKI, Japan), and a PC for control and data collection. Figure 12 is an enlarged view of the electrodes of the electrical impedance tomography sensor for cell imaging 10. The electrodes of the electrical impedance tomography sensor for cell imaging consist of two layers of eight electrodes per layer within the xy plane of the imaging area, for a total of 16 electrodes. Each electrode has a length le = 0.40 mm and a width we = 0.15 mm. The number of electrode pairs for which impedance was measured between single-layer and multi-layer electrodes was 40 and 208, respectively. The electrode pairs were switched using a multiplexer, and a constant voltage was applied to each electrode pair using an impedance analyzer to measure the current. A cell spheroid (317 μm in diameter) (Figure 13 shows a micrograph of the spheroid) was fixed to the center of the sensor as the cellular tissue to be imaged, and 5 μL of sucrose solution was added. Impedance measurement was initiated using inverse problem analysis to determine the conductivity distribution using the impedance Zn normalized to the impedance at time t = 0 s. The frequency of the applied voltage was swept from 1 kHz to 1 MHz.

[0064] Figure 14 shows the results of 2D+time imaging of the ion concentration distribution in the extracellular fluid of a spheroid according to the present invention at t = 20 s, t = 27 s, and t = 34 s when using eight electrodes (40 electrode pair patterns) in the first layer. As shown in Figure 14, after the sucrose solution was dripped, as time passed, many ions were emitted from the upper left side of the image in Figure 14. Generally, when spheroids are formed, the cell distribution and properties become anisotropic. From the above, it was found that the cell imaging device disclosed herein can be used to evaluate ion channels in cells or cell groups.

[0065] Next, we present an example of 3D imaging using the two-layer, 16-electrode system (208 electrode pair patterns) shown in Figure 12. Figure 15(a) shows the three-dimensional potential distribution when using two layers of 16 electrodes. Figure 15(b) is a predicted 3D image of the ion concentration distribution in the extracellular fluid at a certain time, obtained using the present invention. The darker the color, the higher the ion concentration, and it can be seen that the concentration is high around the cell spheroid. The center is filled in because it is the spheroid. It is expected that the conductivity is high near the spheroid and decreases toward the outer edge. As shown in Figure 14, the conductivity distribution also changes over time t, and an increase in conductivity is observed near the spheroid. Therefore, it is expected that the present invention will enable three-dimensional imaging of ion outflow from the spheroid and diffusion into the surrounding solution. [Explanation of symbols]

[0066] 1 Current and voltage application measurement unit, 3 Jacobian matrix calculation unit, 4 Electrical property distribution calculation unit, 5 Cell identification unit, 6 Output unit, 10 Electrical impedance tomography sensor for cell imaging, 20 Electrode, 25 Substrate, 28 Cell placement unit, 100 Cell imaging device

Claims

1. A substrate, a measuring unit including four or more electrodes provided on the substrate and a cell placement unit provided on the substrate, on which a cell or a group of cells is placed; Equipped with the electrodes are arranged around the cell placement section, a cell imaging device including an electrical impedance tomography sensor for cell imaging, wherein the electrodes are arranged on the circumferences of a plurality of concentric circles having different radii centered on the center of the cell placement section, a current / voltage application / measurement unit that applies a current or a potential difference between the electrodes, measures the potential difference and phase based on a current application / voltage measurement pattern when the current is applied, and measures the current and phase based on a voltage application / current measurement pattern when the potential difference is applied between the electrodes; a Jacobian matrix calculation unit that calculates a Jacobian matrix based on the current application voltage measurement pattern or the voltage application current measurement pattern, mesh coordinates obtained by dividing the cell placement unit, and coordinates of each of the electrodes; an electrical property distribution calculation unit that calculates the electrical property distribution of the cell or the cell group from the Jacobian matrix calculated by the Jacobian matrix calculation unit and the potential difference and phase or the current and phase measured by the current / voltage application measurement unit; a cell identification unit that identifies the cell or the cell group by evaluating the ion transport ability of the cell or the cell group from the electrical property distribution using an ion transport model; A cell imaging device comprising:

2. The cell imaging device according to claim 1 , wherein the current or potential difference applied by the current / voltage application / measurement unit has an application frequency of 1 Hz or more and 1 THz or less.

3. 2. The cell imaging device according to claim 1, wherein the current or potential difference applied by the current / voltage application / measurement unit has an application frequency of 1 kHz or more and 1 MHz or less.

4. The cell imaging device according to claim 1 , wherein the current or potential difference applied by the current / voltage application / measurement unit has an application frequency of more than 1 MHz.

5. 5. The cell imaging device according to claim 1, further comprising a cell supplying section that supplies the cells or the cell groups to the cell placement section.

6. The cell imaging device according to any one of claims 1 to 5, further comprising a stimulating unit that applies a stimulus to the cell or the group of cells.

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