JP7371453B2 - Test board, test board with cells, and test method - Google Patents

Description

The present disclosure relates to a test board that performs tests by measuring electrical signals emitted by cells, and particularly to a test board for testing cardiotoxicity of drugs, a test board with cells using the same, and a test method.

Currently, cell culture of various animals and plants is being carried out, and new cell culture methods are being developed. Cell culture technology is used for purposes such as elucidating the biochemical phenomena and properties of cells and producing useful substances. Furthermore, attempts are being made to examine the physiological activity and toxicity of artificially synthesized drugs using cultured cells.

For example, Patent Document 1 discloses a cardiomyocyte bioassay chip in which an adhesive/non-adhesive pattern is formed on a multi-point electrode, but since this adhesive/non-adhesive pattern has no directionality, Orientation culture cannot be performed.

Patent Document 2 describes a substrate portion on one surface of which at least one linear cell adhesive region is formed as a cell culture support for forming string-like myocardial cell aggregates having pulsatility. A cell culture support for forming a string-like myocardial cell aggregate is described, but there is no description of providing an electrode.

Patent Document 3 describes a bioassay system for observing the collective properties of cells while observing the state of myocardial beating cells with a voltage response or using a microscope. A cell culture substrate is described in which a cell adhesion region and a cell adhesion inhibiting region are adjacent to each other in the adhesive region.

Patent Document 4 includes a plurality of microelectrodes and a plurality of conductive cells, the microelectrode is in contact with the conductive cell, and the microelectrode is configured to transmit a signal input to the cell or A substrate is described that is connected to an electronic interface (eg, a signal generator and/or recorder) to record electrical signals from the conductive cells.

In the drug development process, toxicity tests are first conducted using non-human animals (rats, mice, monkeys, pigs, dogs, etc.). However, because there are large species differences between non-human animals and humans, even if no toxicity is observed in non-human animals, toxicity may be observed when administered to humans, which poses a major problem. In particular, because myocardial cell action potential differs between species, it may not be possible to evaluate human cardiotoxicity (toxicity that adversely affects the heart) in non-human animal experiments.As a result, drug-induced arrhythmia, etc. This was often the reason for cancellation. Therefore, evaluation of cardiotoxicity in humans is important, and development of a toxicity testing method using human cardiomyocytes is desired.

In recent years, advances in differentiation techniques for human ES cells (embryonic stem cells) and human iPS cells (induced pluripotent stem cells) have made it possible to stably induce differentiation into cardiomyocytes. In particular, studies are progressing on toxicity testing methods for testing the toxicity of drugs using cardiomyocytes derived from human iPS cells (see, for example, Non-Patent Document 1).

Patent No. 4812271 Patent No. 4822012 Patent No. 5278400 Special table 2016-518117 publication

Zhu R. , Blazeski A. , Poon E. , Costa KD., Tung L., Boheler KR., Stem Cell Res. And Ther. , 2014. Oct 20;5(5):117. Physical development cues for the maturation of human pluripotent stem cell-derived cardiomyocytes. Tatiana Trantidou et al. , Scientific Reports, 5 (11067), June 2015, Biorealistic cardiac cell culture platforms with integrated monitoring of extra cellular action potentials.

As a method for evaluating cardiotoxicity using iPS cell-derived cardiomyocytes as described above, for example, there is a method of using a multipoint electrode system and measuring extracellular potential from a myocardial sheet formed on the multipoint electrodes. . The cell membrane potential of cardiomyocytes changes as they beat, and when one cell beats, adjacent cells are induced to beat, and the change in membrane potential propagates. In the above method, it is analyzed how this electrical signal changes when a test substance such as a drug is added. However, this method has large variations depending on the cell lot, and there is a need for a testing method that can perform stable evaluations with little variation.

Furthermore, it is known that cardiomyocytes obtained by inducing differentiation from human ES/iPS cells are immature compared to cardiomyocytes in the body. Normally, as cells mature, proteins that generate electrical signals are generated in the cell membrane, but immature cells do not have enough of this protein. As a result, there are insufficient ion channels in the cell membrane, and the response to drugs differs from that of mature cells, making it impossible to accurately evaluate cardiotoxicity.

The present disclosure has been made in view of the above-mentioned problems, and aims to obtain simple and stable evaluation results in tests (particularly drug cardiotoxicity tests) performed by measuring electrical signals emitted by cells. The main purpose is to provide a test board that can perform

In order to solve the above problem, the present inventors conducted extensive studies and found that a test substrate used in tests that measure electrical signals emitted by cells has a cell non-adhesive region and a cell sandwiched between the cell non-adhesive regions. By providing a linear region with cell adhesion and further providing an electrode for detecting electrical signals from the cells in the linear region, cells can be cultured in a linear orientation. They found that the response to drugs is similar to that of living heart muscle because it can promote the maturation of heart muscle cells in the body. Furthermore, the inventors have discovered that the test results are stable because the membrane potential propagates in one direction along the linear region, leading to the completion of the present invention.

In order to achieve the above object, the present disclosure provides at least two or more cell non-adhesive regions, a linear region having cell adhesive properties sandwiched between the two adjacent cell non-adhesive regions, and a cell adhesive region. A test board is provided, which includes at least one electrode provided in the linear region and a lead wire connected to the electrode, which detects an electrical signal from the test board.

Further, the present disclosure provides a test substrate with cells, in which cells are placed on at least one electrode of the above-described test substrate.

Furthermore, the present disclosure includes a step of placing a cell on at least one electrode of the above-mentioned test substrate and culturing it, a first measurement step of detecting and measuring an electrical signal from the cell with the electrode, The method includes a step of adding a test substance that acts on the cells onto the test substrate, and a second measurement step of detecting and measuring an electrical signal from the cells on which the test substance acts, using the electrode, and The measurement results obtained in the first measurement step and the measurement results obtained in the second measurement step are compared, and the change in the electrical signal exerted on the cells by the test substance is examined. Provides a test method for testing toxicity against.

According to the test substrate of the present disclosure, since cells can be oriented and cultured and electrical signals can be detected, stable test results can be obtained easily. In particular, human ES/iPS-derived cardiomyocytes can be cultured in a linear orientation, thereby promoting maturation. Therefore, even when human ES/iPS-derived cardiomyocytes are used, drug response results closer to biological reactions can be obtained.

(A) A partial top view showing an embodiment of the test board of the present disclosure, (B) A schematic cross-sectional view taken along line XX' in (A), (C) A perspective photograph showing an embodiment of the test board of the present disclosure It is. FIG. 7 is a partial top view showing another embodiment of the test board of the present disclosure. FIG. 1 is a partial schematic cross-sectional view showing an embodiment of the cell-attached test substrate of the present disclosure.

The present disclosure relates to a test substrate, a cell-attached test board, and a test method.
Each will be explained in detail below. Note that the present disclosure can be implemented in many different embodiments, and should not be interpreted as being limited to the contents of the embodiments exemplified below. In addition, in order to make the explanation clearer, the drawings may schematically represent the width, thickness, shape, etc. of each member compared to the embodiment, but this is only an example, and the interpretation of this disclosure is It is not limited to. In addition, in this specification and each figure, the same elements as those described above with respect to the previously shown figures are denoted by the same reference numerals, and detailed explanations may be omitted as appropriate.

In this specification, when expressing a mode in which another member is placed on top of a certain member, when it is simply expressed as "above" or "below", unless otherwise specified, it means that the member is in contact with a certain member. This includes both cases in which another member is placed directly above or below a certain member, and cases in which another member is placed above or below a certain member via another member. In addition, in this specification, when expressing the aspect of arranging another member on top of a certain member, when it is simply expressed as “on a surface”, unless otherwise specified, it means directly above or in contact with a certain member. This includes both a case where another member is placed directly below a certain member and a case where another member is placed above or below a certain member via another member.

As mentioned above, in tests that measure electrical signals emitted by cells, there is a need for a test method that is simple and can provide stable evaluation results, and in particular, a simple toxicity test method using mature cardiomyocytes is needed. is desired.

As methods for promoting the maturation of cardiomyocytes, electrical stimulation methods, methods of applying mechanical loads, oriented culture methods, etc. are known. The present inventors investigated these methods and found that special equipment and substrates are required to perform electrical stimulation and mechanical loading methods, and that there are problems when conducting toxicity tests using mature cells. It was discovered that it was necessary to re-seeding the seeds from the maturation substrate onto the toxicity test substrate, which required a complicated operation. On the other hand, with the oriented culture method, we thought that if cells could be oriented and cultured on a toxicity test substrate, it would be possible to perform everything from oriented culture to toxicity testing on the same substrate.

Further, as methods for orienting cells, there are a method of forming a concavo-convex pattern and a method of forming an adhesive/non-adhesive pattern. The present inventors also studied these methods and found that the method using a concavo-convex pattern (for example, Non-Patent Document 2) produces a certain degree of orientation, but has a problem with maturation. This is presumed to be because intercellular adhesion is formed not only in the long axis direction but also in the short axis direction, but in vivo, intercellular adhesion is mainly formed in the long axis direction. Incidentally, Non-Patent Document 2 discloses a technique in which a concavo-convex pattern is formed on a multi-point electrode system substrate, and this concavo-convex pattern is used to culture and test cells.

Therefore, the present inventors developed a test substrate that is provided with a plurality of cell non-adhesive regions and a linear region having cell adhesive properties sandwiched between the cell non-adhesive regions. It is possible to culture cells in an oriented manner in the linear region, and in particular, it is possible to mature myocardial cells.Furthermore, because the membrane potential propagates in one direction, the electrodes installed in the linear region provide stable test results. The present invention was completed based on the discovery that the following can be obtained.

A. Test Substrate The test board of the present disclosure includes at least two or more cell non-adhesive regions, a linear region having cell adhesive properties sandwiched between the two adjacent cell non-adhesive regions, and an electrically The device is characterized in that it includes at least one electrode provided in the linear region that detects a signal, and a lead wire connected to the electrode.

The inspection board of the present disclosure will be described with reference to the drawings. FIG. 1(A) is a partial top view showing an embodiment of the test board of the present disclosure, FIG. 1(B) is a schematic cross-sectional view taken along line XX' in FIG. 1(A), and FIG. 1(C) is a , is a perspective photograph showing an embodiment of the test board of the present disclosure. The test substrate 10 of the present disclosure includes a plurality of cell non-adhesive regions 1, a linear region 2 having cell adhesive properties sandwiched between adjacent cell non-adhesive regions 1, and a linear region 2 provided in the linear region 2. It is characterized by comprising at least one electrode 3 and a lead wire 4 connected to the electrode 3. In FIG. 1, a cell non-adhesive layer 11 whose surface is a cell non-adhesive region is a multi-point electrode substrate 6 comprising an insulating layer 22 constituting a linear region 2, an electrode 3, an extraction wiring 4, and a support portion 5. formed on top. Hereinafter, the configuration of the test board of the present disclosure will be described in detail.

1. Cell Non-Adhesive Area The cell non-adhesive area in the present disclosure is an area where cells do not substantially adhere. "Substantially no cells adhere" refers to an area where cells do not adhere at all during cell culture, or where a small number of cells adhere but the adherent cells do not or hardly spread, and as a result, those cells do not adhere at all or do not spread. A state in which there is almost no growth. By sandwiching a linear region having cell adhesive properties, which will be described later, between such cell non-adhesive regions, cells can be oriented and cultured in the linear direction with good directionality in the linear region. Therefore, in particular, when iPS-derived cardiomyocytes are cultured, their maturation progresses, and the drug response becomes similar to that of living myocardium. Furthermore, during evaluation, since the membrane potential propagates in one direction, the evaluation results are stable.
The test substrate of the present disclosure has at least two or more cell non-adhesive regions.

(1) Cell non-adhesion property Cell non-adhesion property is determined by whether or not cell adhesion or spreading is difficult to occur due to the chemical and physical properties of the surface. As an index for determining cell non-adhesion, the cell adhesion extension rate when cells are actually cultured can be used. The cell non-adhesive surface preferably has a cell adhesion extension rate of less than 20%, more preferably less than 10%, even more preferably 5% or less, and still more preferably 1%. Most preferably the following surfaces:

The cell adhesion extension rate in the present invention is determined by seeding cells to be cultured on the surface to be measured at a seeding density of 4,000 cells/cm ² or more and less than 30,000 cells/cm ² in an incubator at 37°C and a CO ₂ concentration of 5%. It is defined as the percentage of cells that have adhered and spread after 14.5 hours of culture ({(number of adherent cells)/(number of seeded cells)}×100(%)).

Furthermore, the cell adhesion extension rate is measured after exchanging the medium and removing non-adherent cells immediately before the measurement. In addition, the cell adhesion extension rate should be measured at locations other than locations where cell density tends to be specific (for example, the center of a given area where cell density tends to be high, or the periphery of a given area where cell density tends to be low). The measurement is carried out using the following methods.

(2) Width Although the cell non-adhesive region is not particularly limited, it is preferable that the cell non-adhesive region is provided in a linear shape parallel to the linear region. The width of the cell non-adhesive region when provided in a linear manner is preferably 100 μm or more, particularly preferably 125 μm or more, particularly preferably 150 μm or more. This is because if it is above the above range, it is easy to form cell non-adhesive regions in a stripe shape, and oriented culture can be reliably performed only in the linear regions. Note that, in consideration of inspection efficiency, the width of the above region is usually 800 μm or less.

Here, "width of the cell non-adhesive region" means the width of the cell non-adhesive region sandwiched between two adjacent linear regions in the direction perpendicular to the line direction of the linear region. Specifically, it means the width W1 in FIG.

(3) Length The length of the cell non-adhesive region is not particularly limited as long as it can sandwich the linear region described below, and is a multi-point electrode substrate that can be used for manufacturing test substrates. It can be determined by the size of

(4) Cell non-adhesive layer The cell non-adhesive region is formed by forming a pattern of cell non-adhesive regions on the substrate surface that has a cell adhesive region (for example, a region exhibiting normal wettability). Preferably, this is provided by forming a certain cell non-adhesive layer. Further, as the substrate having cell adhesive properties, it is preferable to use a multi-point electrode substrate.

The material for the cell non-adhesive layer is not particularly limited as long as it is capable of forming a fine pattern and exhibits cell non-adhesive properties on its surface. A resist material is preferably used. Specific examples include biosurfine AWP-MPH (manufactured by Toyo Gosei Kogyo Co., Ltd.), SPRA-101 (manufactured by Tokyo Ohka Kogyo Co., Ltd.), Lipidure-CR3001 (manufactured by NOF Corporation), and the like.

The thickness of the cell non-adhesive layer is not particularly limited, but can be 1 μm or less, particularly 200 nm or less.
Further, there may be a step between the cell non-adhesive region and the linear region, or the region may be flat without a step.

2. Linear Region A linear region is a cell adhesive region that is sandwiched between two adjacent cell non-adhesive regions, to which cells adhere or are likely to adhere. The linear region is not particularly limited as long as it is linear, and is usually linear, but may be curved, broken, or a combination of these.

(1) Cell adhesion Cell adhesion is determined by whether or not cells tend to adhere or spread depending on the chemical and physical properties of the surface. The cell adhesive surface preferably has a cell adhesion extension ratio defined above of 60% or more, and more preferably a cell adhesion extension ratio of 80% or more.

It is preferable that the linear region exhibiting cell adhesiveness as described above exhibits wettability, and the water contact angle of the linear region is preferably, for example, 40° or more and 70° or less, particularly 45°. It is preferable that the angle is greater than or equal to 65°.

As a method for measuring the contact angle of pure water, the θ/2 method shown below can be used.
Place 0.5 to 1 uL of water on the substrate and observe the droplet with a camera from the side of the substrate. Then, the coordinates of the left and right end points of the droplet are extracted by image analysis, and the left and right end points are determined. Let this distance be 2r. Next, the straight line passing through the left and right end points is defined as the boundary surface, and the intersection of the perpendicular bisector between the left and right end points and the droplet contour is defined as the vertex. Let this distance be h. At this time, the contact angle θ is
θ=2 arctan(h/r)
It can be found as

(2) Width The width of the linear region (width in the direction perpendicular to the linear direction) is preferably within the range of 20 μm to 70 μm, particularly preferably within the range of 30 μm to 60 μm. This is because within the above range, maturation of human ES/iPS-derived cardiomyocytes progresses more reliably.

(3) Length The length of the linear region is not particularly limited and varies depending on the size of the multipoint electrode substrate that can be used for manufacturing the test substrate, but is preferably 100 μm or more, particularly preferably 200 μm or more.

(4) Others The linear region is usually an area on the surface of the multi-point electrode substrate used for manufacturing the test substrate where the cell non-adhesive layer is not formed, so the material constituting the linear region is usually , is the material of the substrate part in the multi-point electrode substrate, and includes insulating resins such as polyimide resin and acrylic resin.

In the present disclosure, at least one electrode is provided in one linear region, and preferably two or more electrodes are provided in one linear region. For example, when using a multi-point electrode substrate with 64 (8×8) electrodes, it is preferable that eight electrodes be provided in one linear region.

Although the number of linear regions in the test substrate of the present disclosure varies depending on the number of electrodes on the multi-point electrode substrate used for manufacturing the test substrate, for example, a multi-point electrode substrate with 64 (8×8) electrodes is used. In some cases, there may be eight lines (eight rows).
The distance between adjacent linear regions is preferably 100 μm or more. This is because the cells can be reliably oriented and cultured in the linear region. Note that the distance between the adjacent linear regions is usually the same as the width W1 of the cell non-adhesive region described above.

3. Electrode At least one, preferably two or more electrodes in each linear region are provided in the present disclosure. It is provided to detect electrical signals from cultured cells, and the detected electrical signals are measured by a separately installed measuring means via lead-out wiring, which will be described later. In the measuring means, the cell potential can be measured, for example, between electrodes provided in the linear region, or between an electrode provided in the linear region and a separately installed standard electrode. When evaluating the cardiotoxicity of a drug, it is possible to perform the cardiotoxicity evaluation by observing how the potential changes when the drug is added.

The electrodes in the present disclosure are those of a system commercially available as a multi-point electrode substrate (for example, a multi-point electrode substrate (MED-P545A-NR) manufactured by AlphaMed Scientific). This is preferable in terms of the manufacturing process. Usually, the electrodes on a multipoint electrode substrate are arranged in, for example, 4×4 or 8×8. The electrode size is not particularly limited, but is preferably within the range of 10 μm to 50 μm×10 μm to 50 μm.

Further, the shape of the upper surface of the electrode is not particularly limited, and may be any of a circle, an ellipse, a square, a rectangle, and a polygon, but a square is preferred.

The electrode width may be smaller or larger than the line width of the linear region, or may be the same. However, as shown in FIG. 2, if the width W3 of the electrode 3 is larger than the line width W2 of the linear region 2, depending on the accuracy of patterning the cell non-adhesive layer, the cell non-adhesive layer may be placed on the top surface of the electrode. A sexual layer may be formed. Therefore, as shown in FIG. 2, it is preferable to form the cell non-adhesive layer in a pattern with an interval of about 5 μm to 10 μm around the electrode 3. This is because it is preferable for cells to be in contact with or in close proximity to the entire upper surface of the electrode. Usually, this region is provided with the material forming the linear region.

The material of the electrode is not particularly limited, but examples include platinum black, carbon nanotubes (CNT), gold, ITO (indium tin oxide), and titanium.

The distance between the electrodes (L in FIG. 2) is usually within the range of 100 μm to 500 μm, and preferably within the range of 200 μm to 450 μm.

4. Lead Out Wiring The test board of the present disclosure includes lead wires connected to electrodes. The lead-out wiring is not particularly limited as long as it is made of a conductive material, but it is preferably transparent, and specific examples thereof include ITO (indium tin oxide) and the like.

5. Supporting Part In the present disclosure, as shown in FIG. may be formed on the top. Examples of the material for the support portion 5 include glasses and plastics commonly used for cell culture, but are not particularly limited thereto. The shape of the support part may be any shape as long as it is suitable for the purpose of cell culture, and examples thereof include a sheet shape and a petri dish shape.

6. Others (1) Adhesive layer There may be an adhesive layer below the cell non-adhesive layer, that is, between the multi-point electrode substrate and the cell non-adhesive layer. The adhesion layer is not particularly limited as long as it can increase the adhesion between the multipoint electrode substrate and the cell non-adhesive layer, and examples thereof include a coupling layer, particularly a silane coupling layer.

(2) Method for manufacturing a test substrate As a method for manufacturing a test substrate according to the present disclosure, for example, a cell non-adhesive layer may be formed on a multi-point electrode substrate in areas other than the linear areas. is possible. Specifically, it can be manufactured by forming a resist for forming a cell non-adhesive layer on a multi-point electrode substrate, and performing exposure through a photomask, development, etc. An example of the method for manufacturing a test substrate according to the present disclosure will be described in detail below.

First, after cleaning the multi-point electrode substrate, a silane coupling treatment is performed. As the multi-point electrode substrate, a commercially available system (for example, a multi-point electrode substrate (MED-P545A-NR) manufactured by AlphaMed Scientific) can be used. By performing the silane coupling treatment, it is possible to improve the adhesion between the cell non-adhesive layer and the multi-point electrode substrate. The coupling agent used in the silane coupling treatment can be appropriately selected depending on the type of cell non-adhesive layer material, and specifically includes methacryloxysilane, vinylsilane, aminosilane, epoxysilane, etc. be able to. In particular, aminosilane is preferred.

Next, after performing heat treatment as necessary, a resist composition capable of forming a cell non-adhesive layer is applied onto the multipoint electrode substrate, and heated as necessary to obtain a cell non-adhesive layer. . Thereafter, exposure and development are performed through a photomask designed so that the region from which the resist layer is removed is the linear region containing the electrode. The resist composition is the same as the photosensitive resist material explained above in "A. Inspection Substrate (4) Cell Non-Adhesive Layer", so the explanation here will be omitted.

Next, as necessary, heating is performed to suppress swelling of the cell non-adhesive layer, residue removal treatment (corona treatment, etc.), cleaning, and sterilization are performed to produce a test substrate.

B. Test Substrate with Cells The present disclosure provides a test substrate with cells, in which cells are placed on at least one electrode of the above-described test substrate. FIG. 3 is a partial schematic sectional view showing an embodiment of the cell-attached test substrate 30 of the present disclosure, in which cells 7 are placed on the electrodes 3 of the test substrate of the present disclosure.

The cell-attached test board of the present disclosure may be a cell-attached test board on which cells to be cultured are placed and on which a test using the cultured cells can be performed after culturing, or a cell-attached test board on which cells that have already been cultured are placed. It may also be a cell-attached test board that can perform tests using these cultured cells.

1. Cells The cells in the cell-attached test substrate of the present disclosure are not particularly limited as long as they can be tested by transmitting and receiving electrical signals, and cells that require linear orientation and culture are preferably used. Such cells include cardiomyocytes and nerve cells, with cardiomyocytes derived from human ES/iPS cells being particularly preferred. Particularly when testing for cardiotoxicity of drugs, the maturity of myocardial cells is important in order to obtain stable test results. With the cell-attached test substrate of the present disclosure, it is possible to achieve the maturation of cardiomyocytes, particularly iPS-derived cardiomyocytes, and the response to drugs is similar to that of living myocardium, so that stable test results can be obtained.

2. Test Board The test board used in the present disclosure is the same as that described in "A. Test Board" above, so the description here will be omitted.

C. Inspection Method The present disclosure includes a step of placing cells on at least one electrode of the above-described test substrate and culturing them, a first measurement step of detecting and measuring an electrical signal from the cells with the electrode, and The method includes a step of adding a test substance that acts on the cells onto the test substrate, and a second measurement step of detecting and measuring an electrical signal from the cells on which the test substance acts, using the electrode, and The measurement results obtained in the first measurement step and the measurement results obtained in the second measurement step are compared, and the change in the electrical signal exerted on the cells by the test substance is examined. Provides a test method for testing toxicity against.
In addition, in this indication, the method using the test board with cells shown in the above-mentioned "B. Test board with cells" may be used, and in this case, the following "first measurement step" is performed.

(1) Cell culture step Cells are placed on at least one electrode and cultured under predetermined conditions. The cells are preferably placed on or close to the top surface of the electrode, and it is preferable from the viewpoint of accurately detecting electrical signals that the cells are placed on the entire top surface of the electrode. The time for culturing cells depends on the presence or absence of cell manipulation during culturing, but is usually 1 week to 4 weeks, preferably 1 week to 2 weeks. The culture temperature is usually 37°C. It is preferable to culture in an atmosphere with a CO ₂ concentration of about 5%.
With the test substrate of the present disclosure, cells can be oriented and cultured in a linear direction, and in particular, human ES/iPS-derived cardiomyocytes can be oriented and cultured to mature.

(2) First measurement step This is a step of detecting and measuring electrical signals from cells using the electrodes. For example, between two or more electrodes in the linear region of the test substrate of the present disclosure, between at least one electrode in the linear region and a separately provided standard electrode, or between two or more electrodes in the linear region, or between at least one electrode in the linear region and a standard electrode provided separately, It is possible to measure changes in cell potential between the electrodes.

(3) Test substance addition process The test substance is added by adding the test substance to the culture medium. The test substance can be an artificially synthesized chemical substance, particularly a drug to be tested for cardiotoxicity.

(4) Second measurement step Electrical signals from cells affected by the test substance are detected and measured by the same method as the first measurement step above.

Next, the measurement results obtained in the first measurement step and the measurement results obtained in the second measurement step are compared, and the changes in the electrical signal that the test substance exerts on the cells are examined to determine the toxicity of the test substance to the cells. can be inspected. If the electrical signal is disrupted, it can be determined that the test substance is toxic. In the test method of the present disclosure, cells are cultured in an oriented manner and the membrane potential propagates in one direction, so stable test results can be obtained. In particular, when human iPS cell-derived cardiomyocytes are used, the cells can be matured and their response to drugs will be similar to that of living myocardium, so they can be suitably used for drug discovery screening.

Note that mature cardiomyocytes are suitably used not only for drug discovery screening but also for regenerative medicine. Therefore, the test substrate of the present disclosure can be used for culturing cells in regenerative medicine and for testing cultured cells. It can also be used as a substrate.

Note that the present disclosure is not limited to the above embodiments. The above-mentioned embodiments are illustrative, and any embodiment that has substantially the same configuration as the technical idea stated in the claims of the present disclosure and provides similar effects is the present invention. within the technical scope of the disclosure.

Hereinafter, the present disclosure will be specifically described using Examples.
(Example)
A multi-point electrode substrate (MED-P545A-NR) manufactured by AlphaMed Scientific was subjected to VUV cleaning for 6 minutes. After spin coating 0.1% 3-aminopropyltrimethoxysilane on the cleaned substrate (200 rpm 3s → slope 3s → 1000 rpm 60s → slope 3s → end), spin coat 3% Biosurfine AWP-MPH (200 rpm 3s → slope 3s→1000rpm 60s→slope 3s→end). It was heated at 60° C. for 15 minutes and exposed to UV light (300 mJ/cm ² ) through a photomask. At this time, after aligning the pattern on the multipoint electrode substrate and the pattern on the photomask, UV exposure was performed to form a pattern aligned with the base. After processing, development was performed by rinsing with water for 120 seconds. The developed substrate was heated on a hot plate under the conditions of 70° C. for 5 minutes → 170° C. for 10 minutes. Finally, corona treatment (output: 0.2 kW) was performed three times to produce a test substrate. The width of the linear region on the test substrate was 50 μm, and the width of the cell non-adhesive region was 400 μm.

(evaluation)
The patterned substrate was coated with FN (fibronectin), and human iPS-derived cardiomyocytes (iCell2, 1.5×10 ⁵ cells) were seeded. When observed after 10 days of culture, striations of myocardial cells were observed in Examples. Immunostaining for cardiac troponin T and α-actinin, which are proteins that make up sarcomeres, showed that sarcomeres were maturing.

1... Cell non-adhesive area 2... Linear area 3... Electrode 4... Output wiring 5... Support part 6... Multi-point electrode substrate 7... Cell 10... Test substrate 11... Cell non-adhesive layer 22... Insulating layer 30... Cell Inspection board with

Claims (5)

at least two or more cell non-adhesive regions;
a linear region having cell adhesive properties sandwiched between the two adjacent cell non-adhesive regions;
two or more electrodes provided in the linear region that detect electrical signals from cells;
a lead wire connected to the electrode;
A test substrate comprising: the cell non-adhesive region is a region in which a cell non-adhesive layer is formed, and the cell non-adhesive layer has a film thickness of 1 μm or less. The test substrate according to claim 1, wherein the distance between the electrodes in the linear region is within a range of 100 μm to 500 μm. A test board with cells, wherein cells are placed on two or more electrodes of the test board according to claim 1 or 2 . A step of placing cells on two or more electrodes of the test substrate according to claim 1 or claim 2 and culturing them;
a first measurement step of detecting and measuring electrical signals from the cells with the electrodes;
adding a test substance that acts on the cells onto the test substrate;
a second measurement step of detecting and measuring an electrical signal from cells on which the test substance acted, using the electrode;
By comparing the measurement results obtained in the first measurement step and the measurement results obtained in the second measurement step, and examining the change in the electrical signal of the test substance on the cells, the test substance can be measured. A test method for testing toxicity to cells. A first measurement step of detecting and measuring electrical signals from the cells using the electrodes of the cell-attached test board according to claim 3 ;
adding a test substance that acts on the cells onto the test substrate;
a second measurement step of detecting and measuring an electrical signal from cells on which the test substance acted, using the electrode;
By comparing the measurement results obtained in the first measurement step and the measurement results obtained in the second measurement step, and examining the change in the electrical signal of the test substance on the cells, the test substance can be measured. A test method for testing toxicity to cells.