JP7798302B2 - Method for evaluating the effect of drugs on cardiomyocytes - Google Patents

Description

One aspect of the present invention relates to a method for evaluating the effect of a drug on cardiomyocytes.

New drug candidates are evaluated for the presence or absence of the expected effect (main effect) using in vitro test systems, and only those with high efficacy are advanced to the next stage of development. Therefore, it is important that the in vitro test system can detect the effect of the drug even at low concentrations and detect few false positives.
If serious side effects are found in drugs that have progressed to clinical development, their development as new drugs may be discontinued. Cardiotoxicity (especially fatal arrhythmias) is the most serious side effect, along with hepatotoxicity and neurotoxicity. Furthermore, among drugs that have been released onto the market, not only drugs in the cardiovascular field but also antiallergic drugs, digestive medicines, and antibiotics are often withdrawn from the market due to the occurrence of cardiotoxicity. Therefore, in the early stages of drug discovery, it is necessary not only to detect the main effects of drugs but also to evaluate their cardiotoxicity.

In recent years, techniques have been developed to evaluate the primary effects and cardiotoxicity of drugs using human cardiomyocytes induced to differentiate from induced pluripotent stem cells (iPS cells). For example, Non-Patent Document 1 discloses that cardiomyocytes induced to differentiate from iPS cells are treated with a trypsin solution, then seeded in a culture medium for cardiomyocytes and cultured to create a pulsating sheet of cardiomyocytes. The resulting sheet of cardiomyocytes is then exposed to a drug within a certain concentration range, and electrophysiological changes in the cardiomyocytes are measured by recording extracellular potentials. Patent Document 1 also discloses a method for testing the drug responsiveness of cardiomyocytes, in which the rate of oxygen supply to the cardiomyocytes is increased by using a configuration for increasing the amount of oxygen supplied to the cardiomyocytes.

International Publication No. 2019/131806

Pharmacol Toxicol Methods. 2017 Mar-Apr;84:111-127. doi:10.1016/j. vascn. 2016.12.003. Epub, 2016, Dec10.

The present inventors considered that in order to efficiently select highly safe compounds in the early stages of drug discovery, it is important to establish an in vitro test system that can not only detect the main effect of a drug but also accurately evaluate cardiotoxicity.
Conventional in vitro test systems have sometimes been unable to evaluate the effects of certain drugs on cardiomyocytes. For example, certain drugs cannot be detected at low drug concentrations, while high drug concentrations cause cardiomyocyte pulsation to cease, making detection difficult in in vitro test systems. The proarrhythmic effect of bepridil is an example of this. Furthermore, sensitivity was sometimes insufficient, such that the drug's effect on cardiomyocytes could not be detected unless the drug concentration was high. Furthermore, nonspecific effects were also detected, resulting in many false positives.
One aspect of the present invention provides a method that can accurately evaluate the effect of a drug on cardiomyocytes.

The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by providing the following configuration, which has led to the completion of the present invention.
Aspects of the present invention relate to, for example, the following [1] to [17].
[1] A method for evaluating the effect of a drug on cardiomyocytes, comprising:
(A) seeding cardiomyocytes on the culture surface of a culture vessel;
a step (B) of culturing the cardiomyocytes obtained in the step (A);
a step (D) of exposing the cultured cardiomyocytes to the drug; and a step (E) of analyzing and evaluating an indicator of cellular function of the cardiomyocytes obtained in the step (D),
The method, wherein at least a portion of the culture surface of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer.
[2] A step (C) of further culturing the cardiomyocytes obtained in the step (B),
The method according to [1], wherein steps (C) and (D) are carried out in a medium (β) containing 1 to 100 μg/mL of free fatty acids.
[3] The method according to [2], wherein the medium (β) further contains at least one selected from 1 to 100 μg/mL of lysophosphatidylcholine, 1 to 100 μg/mL of triacylglyceride, 1 to 100 μg/mL of phosphatidylcholine, 1 to 100 μg/mL of phosphatidic acid, 0.1 to 10 μg/mL of cholesterol, and 0.1 to 10 μg/mL of sphingomyelin.
[4] The method according to any one of [1] to [3], wherein steps (A) and (B) are carried out in a serum-free medium (α) containing a serum substitute.
[5] The method according to any one of [1] to [4], wherein the 4-methyl-1-pentene polymer is a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).
[6] The method according to any one of [1] to [5], wherein the entire culture surface of the culture vessel is formed from a base material containing a 4-methyl-1-pentene polymer.
[7] The method according to any one of [1] to [6], wherein the culture surface is coated with a coating agent containing laminin.
[8] The method according to any one of [1] to [7], wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells.
[9] The method according to any one of [1] to [8], wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells by protein-free cardiac differentiation (PFCD) method.
[10] The method according to any one of [1] to [9], wherein the effect is cardiotoxicity.
[11] The method according to [10], wherein the cardiotoxicity is proarrhythmic effect.
[12] The method according to [10], wherein the cardiotoxicity is myocardial damage.
[13] The method according to [12], wherein the indicator of cell function is an indicator of mitochondrial function.
[14] The method according to any one of [1] to [11], wherein the indicator of cell function is a calcium ion waveform.
[15] The method according to any one of [1] to [11] and [14], wherein early after-depolarizations (EADs) are detected in the step (E).
[16] The method according to any one of [1] to [9], wherein the effect is a tachycardia effect or a bradycardia effect.
[17] The method according to [16], wherein the indicator of cell function is a calcium ion waveform.

According to one aspect of the present invention, the effect of a drug on cardiomyocytes can be evaluated with high accuracy. More specifically, according to one aspect of the present invention, the effect of a drug can be evaluated even at low concentrations. Furthermore, according to one aspect of the present invention, false positives can be reduced. According to one aspect of the present invention, the effect of a drug that was considered difficult to detect in conventional in vitro test systems using iPS cell-derived cardiomyocytes, etc., can be detected.

FIG. 1 shows the results of comparing the expression levels of cTnT, MYL2, Kir2.1, and PGC1α when C plates and T plates were used. Figure 2 shows calcium ion waveforms in cardiomyocytes when 0.1% DMSO was added. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. Figure 3 shows calcium ion waveforms in cardiomyocytes after the addition of 1 μM bepridil. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. The arrows in the figure indicate EAD-like waveforms. FIG. 4 shows an enlarged view of the EAD-like waveform in the calcium ion waveform of FIG. Figure 5 shows calcium ion waveforms in cardiomyocytes when 2 μM bepridil was added. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. Figure 6 shows calcium ion waveforms in cardiomyocytes when 4 μM bepridil was added. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. Figure 7A shows calcium ion waveforms in cardiomyocytes after the addition of 11.1 μM pentamidine to the T-plate. The imaging time was 20 seconds, with a frame rate of 14.23 frames/second. The results are shown before addition (pre), 10 minutes after addition (10 min), 24 hours after addition (24 h), and 48 hours after addition (48 h). Figure 7B shows calcium ion waveforms in cardiomyocytes when 11.1 μM pentamidine was added to the C-plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. The results are shown before addition (pre), 10 minutes after addition (10 min), 24 hours after addition (24 h), and 48 hours after addition (48 h). Figure 8A shows calcium ion waveforms in cardiomyocytes after the addition of 33.3 μM pentamidine to a T-plate. The imaging time was 20 seconds, with a frame rate of 14.23 frames/second. The results are shown before addition (pre), 10 minutes after addition (10 min), 24 hours after addition (24 h), and 48 hours after addition (48 h). Figure 8B shows calcium ion waveforms in cardiomyocytes after the addition of 33.3 μM pentamidine to a C-plate. The imaging time was 20 seconds, with a frame rate of 14.23 frames/second. The results are shown before addition (pre), 10 minutes after addition (10 min), 24 hours after addition (24 h), and 48 hours after addition (48 h). Figure 9A shows calcium ion waveforms in cardiomyocytes after the addition of 100 μM pentamidine to a T-plate. The imaging time was 20 seconds, with a frame rate of 14.23 frames/second. The results are shown before addition (pre), 10 minutes after addition (10 min), 24 hours after addition (24 h), and 48 hours after addition (48 h). Figure 9B shows calcium ion waveforms in cardiomyocytes when 100 μM pentamidine was added to the C-plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. The results are shown before addition (pre), 10 minutes after addition (10 min), 24 hours after addition (24 h), and 48 hours after addition (48 h). 10 shows calcium ion waveforms in cardiomyocytes when 20 nM isoproterenol was added. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 11 shows calcium ion waveforms in cardiomyocytes when 100 nM isoproterenol was added. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 12 shows calcium ion waveforms in cardiomyocytes when 500 nM isoproterenol was added. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. Figure 13 shows photographs of cardiomyocytes observed under a microscope using G, T, and V plates. The upper and lower images are bright-field images, while the lower images are fluorescent images of GCaMP observed under a fluorescence microscope. The objective magnification for both the upper and lower images is 4x. 14 shows calcium ion waveforms in cardiomyocytes when 0.1% DMSO was added using a T-plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 15 shows calcium ion waveforms in cardiomyocytes when 0.1% DMSO was added using a G plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 16 shows calcium ion waveforms in cardiomyocytes when 0.06 μM bepridil was added using a T-plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 17 shows calcium ion waveforms in cardiomyocytes when 0.06 μM bepridil was added using a G plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 18 shows calcium ion waveforms in cardiomyocytes when 0.25 μM bepridil was added using a T-plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 19 shows calcium ion waveforms in cardiomyocytes when 0.25 μM bepridil was added using a G plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 20 shows calcium ion waveforms in cardiomyocytes when 1 μM bepridil was added using a T-plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 21 shows calcium ion waveforms in cardiomyocytes when 1 μM bepridil was added using a G plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 22 shows calcium ion waveforms obtained when 4 μM bepridil was added using a T-plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 23 shows calcium ion waveforms in cardiomyocytes when 4 μM bepridil was added using a G plate. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. 24 shows calcium ion waveforms in cardiomyocytes when verapamil was added at a final concentration of 10 nM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. Figure 25 shows calcium ion waveforms in cardiomyocytes when verapamil was added at a final concentration of 100 nM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 26 is a conceptual diagram for explaining CAD30 and CAD80. 27 shows calcium ion waveforms in cardiomyocytes when verapamil was added at concentrations of 60 nM to 4 μM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 28A is a graph showing CAD30 when verapamil was added at 60 nM to 4 μM, and FIG. 28B is a graph showing CAD80 when verapamil was added. 29 shows calcium ion waveforms in cardiomyocytes when E-4031 was added at concentrations of 1 nM to 1000 nM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 30A is a graph showing CAD30 when E-4031 was added at 1 nM to 1000 nM, and FIG. 30B is a graph showing CAD80 when E-4031 was added. 31 shows calcium ion waveforms in cardiomyocytes when bepridil was added at concentrations of 60 nM to 4 μM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 32A is a graph showing CAD30 when bepridil was added at 60 nM to 4 μM, and FIG. 32B is a graph showing CAD80 when bepridil was added at 60 nM to 4 μM. 33 shows calcium ion waveforms in cardiomyocytes when bepridil was added at concentrations of 312 nM to 20 μM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 34A is a graph showing CAD30 when bepridil was added at 312 nM to 20 μM, and FIG. 34B is a graph showing CAD80 when bepridil was added at 312 nM to 20 μM. 35 shows calcium ion waveforms in cardiomyocytes when risperidone was added at concentrations of 1 nM to 1 μM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 36A is a graph showing CAD30 when risperidone was added at 1 nM to 1 μM, and FIG. 36B is a graph showing CAD80 when risperidone was added at 1 nM to 1 μM. 37 shows calcium ion waveforms in cardiomyocytes when risperidone was added at concentrations of 60 nM to 4 μM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 38A is a graph showing CAD30 when risperidone was added at 60 nM to 4 μM, and FIG. 38B is a graph showing CAD80 when risperidone was added at 60 nM to 4 μM. 39 shows calcium ion waveforms in cardiomyocytes when terfenadine was added at concentrations of 0.25 nM to 16 nM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 40A is a graph showing CAD30 when terfenadine was added at 0.25 nM to 16 nM, and FIG. 40B is a graph showing CAD80 when terfenadine was added. 41 shows calcium ion waveforms in cardiomyocytes when ranolazine was added at concentrations of 10 nM to 10,000 nM. The imaging time was 20 seconds, and the frame rate was 14.23 frames/second. FIG. 42A is a graph showing CAD30 when ranolazine was added at 10 nM to 10,000 nM, and FIG. 42B is a graph showing CAD80 when ranolazine was added.

In Figures 2 to 6, 10 to 12, 14 to 25, 27, 29, 31, 33, 35, 37, 39, and 41, the vertical axis on the left is Intensity, the vertical axis on the right is Movement, and the horizontal axis is Time (frame), and the graphs show Fluorescence. Also, in Figures 7 to 9, the vertical axis is Intensity, the horizontal axis is Time (frame), and the graphs show Fluorescence. Also, in Figures 28, 30, 32, 34, 36, 38, 40, and 42, the vertical axis of A shows CAD30, and the vertical axis of B shows CAD80.

Next, the present invention will be described in detail.
One aspect of the present invention is a method for evaluating the effect of a drug on cardiomyocytes, comprising the steps of: (A) seeding cardiomyocytes on a culture surface of a culture vessel; (B) culturing the cardiomyocytes obtained in step (A); (D) exposing the cultured cardiomyocytes to the drug; and (E) analyzing and evaluating an indicator of cellular function of the cardiomyocytes obtained in step (D), wherein at least a portion of the culture surface of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer.
The culture vessel in which at least a part of the culture surface is formed from a base material containing a 4-methyl-1-pentene polymer is referred to as a culture vessel (X).

[Method for evaluating the effect of a drug on cardiomyocytes]
The drug in one aspect of the present invention is not particularly limited, and may be a substance known to have an effect on cardiomyocytes, such as isoproterenol, pentamidine, bepridil, verapamil, E-4031, terfenadine, astemizole, chromanol 293b, mexiletine, nifedipine, propranolol, milrinone, or a drug described in Non-Patent Document 1. Of these, isoproterenol, pentamidine, bepridil, verapamil, and E-4031 are preferred because the method of one aspect of the present invention allows for accurate evaluation of the effect on cardiomyocytes.

Isoproterenol is a non-selective beta-agonist with tachycardiac and inotropic effects. Pentamidine is an antibiotic that can induce arrhythmias. Bepridil is a calcium channel blocker used to treat angina pectoris, but is known to induce a potentially fatal ventricular arrhythmia known as torsade de pointes (TdP) and QT prolongation as side effects. Milrinone is a phosphodiesterase III inhibitor that also has tachycardiac and inotropic effects. Verapamil is an L-type calcium channel blocker. E-4031 is a hERG-type potassium channel blocker. Terfenadine is an anti-allergy drug known to cause QT prolongation. Astemizole is an anti-allergy drug known to cause QT prolongation. Chromanol 293b is a voltage-gated potassium channel KCNQ1 inhibitor. Mexiletine is a voltage-gated sodium channel blocker. Nifedipine is an L-type calcium channel blocker. Propranolol is a beta-blocker with bradycardic effects.

Ksenia B. et al., International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment, Cell Reports 24, September 25, 2018, pp. 3582-3592 (hereinafter referred to as "Document A") reports the results of an international, multi-center validation study on a method for assessing the proarrhythmic effects of drugs using human cardiomyocytes differentiated from iPS cells. As the drug in one embodiment of the present invention, for example, the 28 drugs evaluated in Reference A, namely, ibutilide, dl-sotalol, azimilide, dofetilide, quinidine, disopyramide, vandetanib, bepridil, domperidone, ondansetron, astemizole, cisapride, pimozide, clarithromycin, risperidone, terfenadine, chlorpromazine, clozapine, ranolazine, metoprolol, mexiletine, loratadine, tamoxifen, nitrendipine, nifedipine, diltiazem, and verapamil can be used. Among these, drugs for which it is difficult to predict the risk level in conventional proarrhythmia models using cardiomyocytes differentiated from iPS cells are preferred, drugs for which the clinical risk level of TdP is high or medium but the risk level of TdP is predicted to be low in conventional proarrhythmia models using cardiomyocytes differentiated from iPS cells are more preferred, and drugs for which the clinical risk level of TdP is low but the risk level of TdP is predicted to be high in conventional proarrhythmia models using cardiomyocytes differentiated from iPS cells are even more preferred, with bepridil, risperidone, terfenadine, and ranolazine being even more preferred.

The drug may be a substance whose effect on cardiomyocytes is unknown, such as a new drug candidate, a substance suspected of having cardiotoxicity, or a candidate substance expected to have a major effect on cardiomyocytes.
The drug may be a low molecular weight compound included in a low molecular weight drug, or a high molecular weight compound included in a high molecular weight drug, such as a protein, antibody, nucleic acid, or polysaccharide. The drug may be a novel substance or a known substance.

The effects of a drug include the main effects and side effects of the drug.
The main action of a drug on cardiomyocytes is an action that is originally expected among the pharmacological actions of the drug, such as bradycardic action (negative chronotropic action), tachycardic action (positive chronotropic action), cardiotonic action (positive inotropic action), and anaphylactic action (negative inotropic action).The main action of a drug on cardiomyocytes is preferably a tachycardic action or a bradycardic action, because the method of the present invention can detect the drug even at a low concentration.

A drug's side effects on cardiomyocytes refer to pharmacological effects of the drug that are unrelated to or interfere with treatment. Side effects on cardiomyocytes include, for example, cardiotoxicity, which includes proarrhythmic effects and myocardial injury. Proarrhythmic effects are effects that worsen existing arrhythmias or cause new arrhythmias, such as QT prolongation, early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), torsades de pointes (TdP), triggered activity arrhythmias, and reentrant arrhythmias. Myocardial injury may result in irreversible or reversible myocardial damage.

The side effect can be detected with high accuracy by the method of one aspect of the present invention, and is preferably a proarrhythmic effect, more preferably an effect that causes QT prolongation or early afterdepolarization (EAD).
The side effect is preferably myocardial injury, more preferably mitochondrial toxicity, since it can be detected with high accuracy by the method of one aspect of the present invention.

A method according to one aspect of the present invention can be used to evaluate the safety and efficacy of pharmaceuticals in the drug discovery process. Specifically, using a substance with unknown effects on cardiomyocytes as a drug allows the evaluation of the substance's main effects or side effects even at low concentrations, and false positives are unlikely to be detected. Furthermore, substances that may pose cardiotoxicity problems in subsequent in vivo testing can be detected as substances with predicted cardiotoxicity in an in vitro test system at an earlier stage. Furthermore, using a substance with known effects on cardiomyocytes as a drug allows the confirmation of known main effects or side effects of the substance, even at low concentrations, or the evaluation of new main effects or side effects of the substance, and false positives are unlikely to be detected. In particular, using a substance with reported or suspected cardiotoxicity in vivo as a drug allows for easier detection of cardiotoxicity than in vivo testing, and allows for analysis of the mechanism of cardiotoxicity.

[Cardiomyocytes]
The cardiomyocytes may be cardiomyocytes differentiated from pluripotent stem cells, or primary cultured cardiomyocytes isolated from the heart of an organism. Alternatively, commercially available cardiomyocytes differentiated from pluripotent stem cells, such as iCell Cardiomyocytes from FUJIFILM Cellular Dynamics, MiraCell Cardiomyocytes v2 from Takara Bio, Cor. 4U from Axogenesis, CarmyA from Myoridge, and ReproCardio2 from REPROCELL, may also be used.

Pluripotent stem cells are a general term for stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), and trophoblast stem cells. cell: TS cell), epiblast stem cell (EpiS cell), embryonic germ cell (EG cell), multipotent germline stem cell: mGS cell), induced pluripotent stem cell (iPS cell), Muse cell (Multi-lineage differentiating The pluripotent stem cells are preferably ES cells or iPS cells.

The cardiomyocytes are preferably cardiomyocytes differentiated from pluripotent stem cells, more preferably cardiomyocytes differentiated from induced pluripotent stem cells, and even more preferably mature cardiomyocytes differentiated from induced pluripotent stem cells. Cardiomyocytes differentiated from induced pluripotent stem cells can be produced by known cardiomyocyte differentiation induction methods, such as the protein-free cardiac differentiation (PFCD) method (see International Publication No. WO 2015/182765).
The cardiomyocytes are preferably cardiomyocytes differentiated from induced pluripotent stem cells by protein-free cardiac differentiation (PFCD) method.

Mature cardiomyocytes differentiated from induced pluripotent stem cells are a term used in the art in contrast to immature cardiomyocytes differentiated from induced pluripotent stem cells, and have higher ion channel function than immature cardiomyocytes differentiated from induced pluripotent stem cells.
Mature cardiomyocytes differentiated from induced pluripotent stem cells are, for example, cells that have been present for at least 14 days, preferably at least 20 days, and more preferably at least 30 days since the initiation of differentiation induction of induced pluripotent stem cells. Here, the day on which differentiation induction of induced pluripotent stem cells is initiated is the day on which induced pluripotent stem cells maintained in an undifferentiated state are exposed to a treatment to transition to a differentiated state, and this day is designated as day 0. Furthermore, since mature cardiomyocytes can be cultured for long periods while maintaining their differentiated state, there is no particular upper limit on the number of days from the initiation of differentiation induction, and mature cardiomyocytes may be cultured or stored for long periods while maintaining their differentiated state. For example, mature cardiomyocytes may be cells that have been present for at least 365 days since the initiation of differentiation induction of induced pluripotent stem cells. Mature cardiomyocytes differentiated from induced pluripotent stem cells preferably refer to cells that have been present for at least 30 days since the initiation of differentiation induction of induced pluripotent stem cells using the protein-free cardiac differentiation (PFCD) method.
Mature cardiomyocytes differentiated from induced pluripotent stem cells are preferable because their properties are closer to those of cardiomyocytes isolated from the heart of an organism than immature cardiomyocytes differentiated from induced pluripotent stem cells.

The origin of the cardiomyocytes is not particularly limited and may be mammalian, avian, amphibian, reptile, fish, etc., but is preferably mammalian, more preferably human, monkey, mouse, rat, pig, dog, sheep, cat, or goat, and even more preferably human.

The cardiomyocytes may be normal cardiomyocytes, cardiomyocytes containing a gene mutation, or disease model cardiomyocytes.
Various genes may be introduced into the cardiomyocytes so that they transiently or constitutively express a sensor protein, etc. It is preferable that the cardiomyocytes transiently or constitutively express a sensor protein, since this allows an indicator of cell function to be detected by fluorescence or chemiluminescence.
Examples of sensor proteins include calcium sensors such as GCaMP, cameleon, pericam, G-GECO, B-GECO, R-GECO, GEX-GECO, GEM-GECO, and CEPIA; ATP sensors such as mito-MaLion, MaLionB, MaLionG, MaLionR, and ATeam; magnesium sensors such as MARIO; glucose sensors such as Green Glifon and Red Glifon; lactate sensors such as Green Lindoblum; and pyruvate sensors such as Green Pegassos. The sensor protein is preferably a calcium sensor or an ATP sensor, more preferably a calcium sensor, and even more preferably GCaMP.

Cardiomyocytes may be used in the form of single cells, in the form of cardiomyocyte clusters, or in the form of cardiomyocyte sheets.

Since the culture vessel (X) can efficiently supply oxygen to the cardiomyocytes, the cardiomyocytes may be either adherent or floating, but adherent cardiomyocytes are preferred.

[Culture container (X)]
At least a part of the culture surface of the culture vessel (X) is formed from a base material containing a 4-methyl-1-pentene polymer.

In one aspect of the present invention, the term "culture vessel" refers to any vessel used for culturing cells. Various known culture vessels can be used as the culture vessel, and the shape and size are not particularly limited. Examples of the culture vessel include dishes, flasks, plates, bottles, bags, and tubes. The culture vessel is typically used in an apparatus such as an incubator, a mass culture apparatus, or a perfusion culture apparatus.
Here, the culture surface means a surface that comes into contact with the medium and/or cells when culturing the cells, or a surface that will come into contact with the medium and/or cells.

In the culture vessel (X), "at least a portion of the culture surface of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer" means that at least a portion of the culture surface is formed from a substrate containing a 4-methyl-1-pentene polymer, and the entire culture surface may be formed from a substrate containing a 4-methyl-1-pentene polymer.

The culture vessel (X) is preferably a culture vessel whose bottom surface includes a culture surface in order to hold or store the culture medium. When the culture vessel (X) is a dish, flask, or plate, the bottom surface includes the culture surface, and therefore at least a portion or all of the bottom surface, side surfaces, and top surfaces thereof are formed from a substrate containing a 4-methyl-1-pentene polymer. When at least a portion or all of the bottom surface is formed from a substrate containing a 4-methyl-1-pentene polymer, oxygen can be efficiently supplied to the culture medium via the 4-methyl-1-pentene polymer, facilitating the efficient proliferation and differentiation of cells in the culture medium. Furthermore, it becomes easier to culture cells at high density while maintaining their functions.

The entire culture surface of the culture vessel (X) is preferably formed from a substrate containing a 4-methyl-1-pentene polymer. That is, when the culture vessel (X) is a dish, flask, or plate, the inside of the bottom surface is the culture surface, and therefore, of the bottom, side, and top surfaces, it is preferable that the entire bottom surface be formed from a substrate containing a 4-methyl-1-pentene polymer.

The thickness of the substrate containing a 4-methyl-1-pentene polymer is not particularly limited, but is preferably 20 μm to 400 μm, more preferably 20 μm to 300 μm, and even more preferably 20 μm to 200 μm. The thickness of the substrate containing a 4-methyl-1-pentene polymer is selected appropriately depending on the shape of the culture vessel, but adjusting it to the above range makes it easier to obtain an appropriate oxygen concentration in the culture medium necessary for cell proliferation and differentiation, and also makes it easier to obtain sufficient strength as a culture vessel.

The culture vessel (X) is preferably a culture vessel having at least one well, more preferably a plate having at least one well, and even more preferably a plate having 6, 12, 24, 48, 96, 384, 1536, or other wells. Culture vessels with well-like depressions on the bottom generally require a thick bottom to stabilize the complex shape of the bottom, making it difficult to adequately supply oxygen to cells. However, when the bottom is formed from a substrate containing 4-methyl-1-pentene polymer, the shape is stable and sufficient oxygen is supplied to cells, even in plates with 1, 6, 12, 24, 48, 96, 384, 1536, or other wells.

The shape of the bottom of the culture vessel (X) is not particularly limited, and examples include a flat bottom, a round bottom (U-bottom), a flat bottom (F-bottom), a conical bottom (V-bottom), a flat bottom with a curved edge, etc. When processing into a round bottom (U-bottom), a flat bottom (F-bottom), a conical bottom (V-bottom), a flat bottom with a curved edge, etc., it can be processed in one step using general injection molding or press molding, or it can be produced by first creating a film or sheet and then performing secondary processing such as vacuum forming or pressure forming. The shape of the bottom is selected depending on the purpose of the culture, but a flat bottom is usually desirable when culturing cells in two dimensions, and a round bottom (U-bottom) or conical bottom (V-bottom) is usually desirable when culturing cells in three dimensions.

The portions of the culture vessel (X) other than the culture surface may be made of a material other than the base material containing 4-methyl-1-pentene polymer. The material is not particularly limited, and known materials can be used. Examples of such materials include polystyrene (PS), polydimethylsiloxane (PDMS), thermosetting resin, cyclic olefin polymer, cyclic olefin copolymer, and glass.

At least the culture surface of the culture vessel (X) may be coated with a coating agent containing a natural polymer material, a synthetic polymer material, or an inorganic material. Coating can be carried out by a known method.
The coated culture vessel (X) exhibits superior adhesion and proliferation of cardiomyocytes. This is thought to be because the components coated on the culture surface serve as a scaffold for the cells. Therefore, when attaching and culturing cardiomyocytes, it is preferable that the culture surface of the culture vessel (X) be coated with a coating agent containing a natural polymer material, a synthetic polymer material, or an inorganic material.

The natural polymer materials, synthetic polymer materials, and inorganic materials are not particularly limited. Examples of natural polymer materials include collagen, gelatin, alginic acid, hyaluronic acid, glycosaminoglycans such as chondroitin sulfate, fibronectin, laminin, fibrinogen, osteopontin, tenascin, vitronectin, thrombospondin, agarose, elastin, keratin, chitosan, fibrin, fibroin, and sugars; synthetic polymer materials include polyglycolic acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, synthetic proteins, polyhydroxyethyl methacrylate, and polyethyleneimine; and inorganic materials include β-tricalcium phosphate and calcium carbonate.

The natural polymer materials, synthetic polymer materials, or inorganic materials may be processed, such as by vitrification, before use. Examples of such processing include vitrigel, which is obtained by vitrifying a hydrogel such as a conventional extracellular matrix component and then rehydrating it, and collagen vitrigel, which is composed of a high-density collagen fiber network made from collagen.

From the viewpoint of improving the adhesiveness and proliferation of cardiomyocytes and maintaining the function of cardiomyocytes for a longer period of time, the coating agent preferably contains a protein or peptide such as collagen, gelatin, laminin, or polylysine, more preferably contains laminin, collagen, or polylysine, and even more preferably contains laminin. That is, it is preferable that the culture surface of the culture vessel (X) is coated with a coating agent containing laminin.
The coating agent may contain one of the above components alone or two or more of them in combination.

The coating agent may be used directly for coating, or may be diluted with a solvent such as water, PBS, or culture medium before use. From the perspective of cardiomyocyte adhesion, the coating agent is preferably diluted with a solvent such as water, PBS, or culture medium before use, and more preferably diluted with culture medium before use. The culture medium used to dilute the coating agent is not particularly limited, and may be the same as the culture medium used in step (A), or a different culture medium. The culture medium used to dilute the coating agent is preferably a culture medium that does not contain animal serum or serum substitute, more preferably DMEM, and particularly preferably the special basal culture medium described below in the Examples.

After being used for coating, the coating agent may be removed from the culture vessel (X) or left in the culture vessel (X), but from the standpoint of adhesion of cardiomyocytes, it is preferable to leave it in the culture vessel (X).

At least the culture surface of the culture vessel (X) may be processed. Examples of surface processing include surface modification such as forming a concave-convex structure, hydrophilization, and hydrophobicity.

The method used for surface modification is not particularly limited, but examples include hydrophilization treatments such as corona treatment, plasma treatment, ozone treatment, and ultraviolet treatment; hydrophobic treatments such as esterification, silylation, and fluorination; surface graft polymerization, chemical vapor deposition, etching, or addition of specific functional groups such as hydroxyl groups, amino groups, sulfonic groups, thiol groups, and carboxyl groups; treatments using specific functional groups such as silane coupling, titanium coupling, and zirconium coupling; surface roughening using oxidizing agents; and physical treatments such as rubbing and sandblasting. These surface modification treatments may be performed alone or in combination with two or more. When performing surface modification treatments, it is preferable to perform them on at least the culture surface.

At least the culture surface of the culture vessel (X) is preferably subjected to a hydrophilization treatment, more preferably a corona treatment or a plasma treatment. By subjecting the surface of the culture vessel (X) to a hydrophilization treatment, the wettability of the surface of the culture vessel (X) is increased, improving adhesion between the culture vessel (X) and cardiomyocytes, allowing cardiomyocytes to grow uniformly on the surface of the culture vessel (X). Furthermore, by subjecting the surface of the culture vessel (X) to a hydrophilization treatment, it becomes easier to coat a coating agent on the culture surface of the culture vessel (X). In particular, it becomes easier to uniformly load and adhere the coating agent to the culture surface of the culture vessel (X). Furthermore, even after the loading treatment, the components of the coating agent do not peel off when washed with physiological saline or in a cell culture environment, allowing the culture vessel (X) to maintain a stable initial state and be used for cell culture.
When plasma treatment is performed, nitrogen, hydrogen, helium, oxygen, argon, etc. are used as the entraining gas, and preferably at least one gas selected from nitrogen, helium, and argon is selected.

The culture vessel (X) may be disinfected or sterilized to prevent contamination. The disinfection or sterilization method is not particularly limited and includes physical disinfection methods such as steam circulation, boiling, intermittent irradiation, and ultraviolet light; chemical disinfection using gases such as ozone or disinfectants such as ethanol; heat sterilization methods such as high-pressure steam and dry heat; irradiation sterilization methods such as gamma ray sterilization, electron beam sterilization, and high-frequency sterilization; and gas sterilization methods such as ethylene oxide gas sterilization and hydrogen peroxide gas plasma sterilization. Among these, ethanol disinfection, high-pressure steam sterilization, gamma ray sterilization, electron beam sterilization, and ethylene oxide gas sterilization are preferred because of their simple operation and ability to achieve sufficient sterilization. These disinfection or sterilization methods may be performed alone or in combination of two or more.

The culture surface of the culture vessel (X) preferably has a water contact angle of 50° or more, more preferably 55° or more, and even more preferably 60° or more. Furthermore, the culture surface of the culture vessel (X) preferably has a water contact angle of 100° or less, more preferably 90° or less, and even more preferably 84° or less.

By adjusting the water contact angle of the culture surface of the culture vessel (X) to be below the upper limit or above the lower limit, for example, cardiomyocytes can more easily adhere to the culture surface and grow uniformly on the culture surface. Furthermore, it is easier to coat a natural polymer material, synthetic polymer material, or inorganic material uniformly on the culture surface of the culture vessel (X) and ensure adhesion. Even after coating, the natural polymer material, synthetic polymer material, or inorganic material does not peel off when washed with saline or in a cell culture environment, maintaining a stable initial state and allowing it to be used for cell culture.

The method for measuring the water contact angle is not particularly limited, and known methods can be used, but the sessile drop method is preferred. For example, the water contact angle can be measured in accordance with Japanese Industrial Standard JIS-R3257 (Test method for wettability of glass substrate surfaces) by dropping a water droplet of 4 μL or less, which can be considered spherical, onto the surface of a measurement sample made from the same material as the culture vessel (X) under constant temperature and humidity conditions of 25±5°C and 50±10%. The sessile drop method measures the angle of the contact interface between the measurement sample and the water droplet within one minute after the water droplet makes contact with the surface of the measurement sample.

The method for manufacturing the culture vessel (X) is not particularly limited, and neither is the equipment used for manufacturing. When the entire culture vessel (X) is formed from a substrate containing a 4-methyl-1-pentene polymer, for example, a film or sheet containing the 4-methyl-1-pentene polymer can be formed, and the film or sheet can be molded as needed to produce the culture vessel (X) in the desired shape. The culture vessel (X) can also be obtained by direct molding using methods such as extrusion molding, solution casting, injection molding, and blow molding. When only a portion of the culture vessel (X) is formed from a substrate containing a 4-methyl-1-pentene polymer, for example, a film or sheet containing the 4-methyl-1-pentene polymer can be formed, and the film or sheet can be appropriately bonded to another substrate to obtain the culture vessel (X). The bonding method is not particularly limited, and the substrate containing the 4-methyl-1-pentene polymer and the other substrate may be formed integrally, or they may be bonded together using an adhesive or pressure-sensitive adhesive.

Specific methods for forming the film or sheet include, for example, the usual inflation method and T-die extrusion method. Production is usually carried out under heating. When using T-die extrusion method, the extrusion temperature is preferably 100°C to 400°C, and more preferably 200°C to 300°C. The roll temperature is also preferably 45°C to 75°C, and more preferably 55°C to 65°C.

The film or sheet may also be produced by a solution casting method in which the 4-methyl-1-pentene polymer is dissolved in a solvent, poured onto a resin or metal, and slowly dried while leveling to form a film (sheet). There are no particular restrictions on the solvent used, but hydrocarbon solvents such as cyclohexane, hexane, decane, and toluene may be used. Two or more solvents may also be mixed, taking into consideration the solubility of the 4-methyl-1-pentene polymer and drying efficiency. The polymer solution can be applied by a method such as table coating, spin coating, dip coating, die coating, spray coating, bar coating, roll coating, or curtain flow coating, followed by drying and peeling to form a film or sheet.

[4-methyl-1-pentene polymer]
In one embodiment of the present invention, 4-methyl-1-pentene homopolymers and copolymers of 4-methyl-1-pentene with other monomers are collectively referred to as "4-methyl-1-pentene polymers."

As an example of a 4-methyl-1-pentene polymer, a copolymer of 4-methyl-1-pentene and another monomer may be any of a random copolymer, an alternating copolymer, a block copolymer, and a graft copolymer. As a copolymer of 4-methyl-1-pentene and another monomer, a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is preferred because it has high strength, is resistant to tearing and cracking, and exhibits little deflection when used as a substrate.

The 4-methyl-1-pentene polymer is preferably at least one polymer selected from 4-methyl-1-pentene homopolymers and copolymers of 4-methyl-1-pentene with at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), and more preferably a copolymer of 4-methyl-1-pentene with at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

Examples of the olefin include ethylene, propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and 1-eicosene. The olefin can be selected appropriately depending on the physical properties required for the substrate. For example, from the standpoint of appropriate oxygen permeability and excellent rigidity, the olefin is preferably an α-olefin having 8 to 18 carbon atoms, and more preferably at least one selected from 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, and 1-octadecene. When the carbon number of the olefin is within the above range, the polymer has better processability and tends to be less susceptible to poor appearance of the substrate due to cracks or edge breakage. Furthermore, the rate of defective substrates is reduced.

The olefins can be used alone or in combination of two or more. When combining two or more different olefins, from the viewpoint of material strength, the olefins preferably have two or more carbon atoms, and more preferably have ten or more carbon atoms. When combining two or more different α-olefins, it is particularly preferable to combine at least one selected from 1-tetradecene and 1-hexadecene with at least one selected from 1-heptadecene and 1-octadecene.

The content of structural units derived from 4-methyl-1-pentene in the 4-methyl-1-pentene polymer is preferably 60 to 100 mol %, more preferably 80 to 99.5 mol %, and even more preferably 85 to 98 mol %.
Furthermore, when the 4-methyl-1-pentene polymer is a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), the content of structural units derived from at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer is preferably 0.1 to 40 mol%, more preferably 0.5 to 20 mol%, and even more preferably 2 to 15 mol%. The content of these structural units is calculated based on the total amount of repeating structural units in the 4-methyl-1-pentene polymer being 100 mol%. When the content of the structural units is within the above range, a homogeneous culture surface with excellent processability can be obtained, and the substrate has a good balance between toughness and strength, resulting in less deflection.

The 4-methyl-1-pentene polymer may contain structural units other than structural units derived from 4-methyl-1-pentene and structural units derived from ethylene and α-olefins having 3 to 20 carbon atoms (hereinafter also referred to as "other structural units"), as long as the effects of the present invention are not impaired. The content of other structural units is, for example, 0 to 10.0 mol%. When the 4-methyl-1-pentene polymer contains other structural units, the other structural units may be one type or two or more types.

Monomers from which other structural units are derived include, for example, cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins. Examples of cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins include the compounds described in paragraphs [0035] to [0041] of JP 2013-169685 A.

The 4-methyl-1-pentene polymer may be used alone or in combination of two or more types.

Commercially available 4-methyl-1-pentene polymers can also be used. Specific examples include TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31, and DX845 (all registered trademarks) manufactured by Mitsui Chemicals, Inc. Also, 4-methyl-1-pentene polymers manufactured by other manufacturers that meet the above requirements can be preferably used. These commercially available products can be used alone or in combination of two or more.

4-methyl-1-pentene polymers typically have a melting point of 200°C to 240°C and are highly heat-resistant. Furthermore, because they do not hydrolyze and have excellent water resistance, boiling water resistance, and steam resistance, substrates containing 4-methyl-1-pentene polymers can be sterilized by high-pressure steam. 4-methyl-1-pentene polymers also have high visible light transmittance (typically over 90%) and do not emit autofluorescence, making it easy to observe cardiomyocytes in culture vessels formed from substrates containing 4-methyl-1-pentene polymers. Furthermore, they exhibit excellent chemical resistance to most chemicals and are resistant to drug sorption, so they do not interfere with the effects of substances that maintain cardiomyocytes and are also resistant to changes in the concentration of drugs used to evaluate their effects. 4-methyl-1-pentene polymers are heat-sealable, allowing for easy thermal fusion not only between themselves but also with other materials. Furthermore, because they are thermoformable, they can be easily molded into culture vessels of any shape, including by imprinting or insert molding.

The weight-average molecular weight (Mw) of the 4-methyl-1-pentene polymer, measured by gel permeation chromatography (GPC) using standard polystyrene as the reference material, is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,000,000, and even more preferably 30,000 to 500,000. The sample concentration during GPC measurement can be, for example, 1.0 to 5.0 mg/ml. The molecular weight distribution (Mw/Mn) of the 4-methyl-1-pentene polymer is preferably 1.0 to 30, more preferably 1.1 to 25, and even more preferably 1.1 to 20. The solvent used for GPC is preferably orthodichlorobenzene. Measurement conditions include, but are not limited to, those shown in the examples below.

By setting the weight-average molecular weight (Mw) to the above upper limit or less, in the molding method of 4-methyl-1-pentene polymer described below, the occurrence of defects such as gels in the film produced by melt molding is more likely to be suppressed, making it easier to produce a film with a uniform surface. Furthermore, when producing by solution casting, the solubility in solvents is improved, making it easier to suppress defects such as gels in the film, making it easier to produce a film with a uniform surface.

In addition, by setting the weight-average molecular weight (Mw) to the above-mentioned lower limit or higher, the culture vessel (X) tends to have sufficient strength. Furthermore, by setting the molecular weight distribution within the above-mentioned range, stickiness on the surface of the culture vessel produced tends to be reduced, and the toughness of the culture vessel tends to be sufficient, making it easier to suppress cracks during bending and cutting during molding.

When two or more types of 4-methyl-1-pentene polymers are used, the weight average molecular weight (Mw) and molecular weight distribution (Mw/Mn) of each of the 4-methyl-1-pentene polymers may be within the above ranges.

The 4-methyl-1-pentene polymer preferably has an oxygen permeability coefficient of 100 to 2500 cm ³ × mm/(m ² × 24h × atm), more preferably 1000 to 2500 cm ³ × mm/(m ² × 24h × atm). When the oxygen permeability coefficient is within this range, the polymer has excellent oxygen permeability, allowing cardiomyocytes to maintain good morphology, to proliferate efficiently depending on the culture period, and to detect the effects of drugs.

Specifically, the oxygen permeability coefficient can be measured by the following method. A measurement sample is prepared from a substrate composed of a 4-methyl-1-pentene polymer, and the oxygen permeability coefficient [cm ³ × mm/(m ² × 24h × atm)] at a temperature of 23°C and humidity of 0% is measured using a differential pressure gas permeability measurement method. The equipment used for the measurement is not particularly limited as long as it uses a differential pressure gas permeability measurement method, but an example is the MT-C3 differential pressure gas permeability measurement device manufactured by Toyo Seiki Seisaku-sho. The measurement sample is prepared by cutting a 90 × 90 mm test piece from a 50 μm thick substrate composed of a 4-methyl-1-pentene polymer, and the measurement section diameter is preferably 70 mm (permeation area: 38.46 cm ² ). Due to the high oxygen permeability, it is more preferable to apply an aluminum mask to the sample in advance to set the actual permeation area to 5.0 cm ² . The measurement sample may or may not have undergone microfabrication or surface modification treatment, but no treatment is preferred. The oxygen permeability [cm ³ /(m ² ×24h×atm)] is calculated by dividing the oxygen permeability coefficient by the thickness (μm) of the substrate.

Because 4-methyl-1-pentene polymer has the excellent properties described above, culture vessels in which at least the culture surface is formed from a substrate containing 4-methyl-1-pentene polymer do not adversely affect culture, and have good shape stability, light transparency, moldability, and oxygen permeability. They can also be sterilized, making them extremely excellent culture vessels for culturing cardiomyocytes and evaluating the effects of drugs.

The method for producing the 4-methyl-1-pentene polymer may be any method capable of polymerizing 4-methyl-1-pentene, an olefin, or other monomers. Furthermore, a chain transfer agent, such as hydrogen, may be used in the presence of the polymer to control the molecular weight or molecular weight distribution. There are no limitations on the equipment used for production. The polymerization method may be a known method, such as a gas-phase method, a slurry method, a solution method, or a bulk method. The slurry method or the solution method is preferred. The polymerization method may be a single-stage polymerization method or a multi-stage polymerization method, such as a two-stage polymerization method, in which multiple polymers with different molecular weights are blended into the polymerization system. Whether single-stage or multi-stage polymerization is used, when hydrogen is used as a chain transfer agent, it may be added all at once or in portions, for example, at the initial, middle, or final stages of polymerization. The polymerization may be carried out at room temperature or, if necessary, heated. From the standpoint of polymerization efficiency, however, it is preferably carried out at 20°C to 80°C, and particularly preferably at 40°C to 60°C. The catalyst used in the production is not limited, but from the viewpoint of polymerization efficiency, it is preferable to use, for example, a solid titanium catalyst component (I) described in WO 2006/054613 or an olefin polymerization catalyst (metallocene catalyst) containing a transition metal compound (A) described in WO 2014/050817.

When the substrate containing a 4-methyl-1-pentene polymer is a composition containing a 4-methyl-1-pentene polymer, the content of the 4-methyl-1-pentene polymer is preferably 90% by mass or more but less than 100% by mass, more preferably 95% by mass or more but less than 100% by mass, and particularly preferably 99% by mass or more but less than 100% by mass, relative to 100% by mass of the composition. When the content of the 4-methyl-1-pentene polymer in the composition is 90% by mass or more, the oxygen permeability, transparency, strength, etc. of the substrate are further improved.
In this case, examples of components other than the 4-methyl-1-pentene polymer include additives such as heat resistance stabilizers, light resistance stabilizers, processing aids, plasticizers, antioxidants, lubricants, antifoaming agents, antiblocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents.

[Step (A)]
Step (A) is a step of seeding cardiomyocytes onto the culture surface of the culture vessel (X). The method for seeding the cardiomyocytes onto the culture surface of the culture vessel (X) is not particularly limited, and for example, cardiomyocytes suspended in a medium are added to the culture vessel (X) using a pipette or the like, and the culture vessel (X) is shaken as necessary to distribute the cardiomyocytes evenly within the culture vessel (X), followed by allowing the culture vessel (X) to stand in an incubator.

The density at which cardiomyocytes are seeded is not particularly limited as long as it is possible to maintain or proliferate the cardiomyocytes, but is preferably 0.1 x 10 ⁵ cells/cm ² to 10.0 x 10 ⁵ cells/cm ² , more preferably 0.3 x 10 ⁵ cells/cm ² to 5.0 x 10 ⁵ cells/cm ² , and even more preferably 0.5 x 10 ⁵ cells/cm ² to 3.0 x 10 ⁵ cells/cm ² .
When the seeding density of the cardiomyocytes is within the above range, the cardiomyocytes are more likely to adhere to the culture vessel (X) and proliferate more efficiently, which is preferable.

The medium used in step (A) is not particularly limited as long as it is a medium in which cardiomyocytes can survive, and may be appropriately selected depending on the cell type used. Examples of the medium used for seeding include any basal cell culture medium, differentiation medium, medium dedicated to primary culture, etc., specifically, serum-free medium (α) described below, medium (β) described below, Eagle's Mini Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI 1640, Ham's F-12, MCDB medium, Williams' Medium E, special maintenance medium described below in the Examples, special basal medium, iCell cardiomyocyte maintenance medium (manufactured by FUJIFILM Cellular Dynamics), iCell cardiomyocyte thawing medium (manufactured by FUJIFILM Cellular Dynamics), and mixed media thereof. These media may also be supplemented with serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, etc.
Since cardiomyocytes tend to adhere to the culture vessel (X), the medium used in step (A) is preferably an iCell cardiomyocyte thawing medium or a serum-free medium (α) described later in the Examples, more preferably a serum-free medium (α).

The amount of medium used in step (A) is not particularly limited and can be determined appropriately depending on the cell species and the type of medium.
The culture conditions are not particularly limited and can be carried out by known methods, but the culture is usually carried out at a temperature of about 25 to 40°C and a carbon dioxide concentration of about 5%.

[Serum-free medium (α)]
Serum-free medium (α) is a medium that does not contain animal serum but contains a serum substitute. Serum-free medium (α) can be prepared by adding a serum substitute to a basal medium.
The alternative serum refers to a serum substitute that replaces animal serum added as a nutrient necessary for cell survival during cell culture, and may also be referred to as serum protein substitute (SPS), serum substitute supplement (SSS), etc. The alternative serum is not particularly limited, and any known alternative serum may be used. Commercially available replacement serum includes Knockout Serum Replacement (ThermoFisher SCIENTIFIC, No. 10828028), StemSure ^™ Serum Replacement (SSR, Fujifilm Wako Pure Chemical Industries, Ltd.), PL SOLUTION (PL Bioscience), PL MATRIX (PL Bioscience), and Artificial Serum (Cell Science Institute, Inc.). Among these, Knockout Serum Replacement is preferred.
The content of the serum substitute in the serum-free medium (α) is not particularly limited, but is preferably 1 to 20% (v/v), more preferably 5 to 15% (v/v), and even more preferably 8 to 12% (v/v).

The basal medium is not particularly limited as long as it is a medium for cell culture that does not contain animal serum, and examples of the basal medium include Grace's medium, IPL-41 medium, Schneider's medium, Opti-PRO ^™ SFM medium, Opti-MEM ^™ I medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle ^™ 293 medium, CD-CHO, and AGT. Examples of the medium include ^TM medium, RPMI medium, DMEM medium, MEM medium, GMEM medium, Eagle's MEM medium, BME medium, DME medium, αMEM medium, IMEM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, ASF103 medium, ASF104 medium, ASF301 medium, TC-100 medium, Sf-900II medium, Ex-cell405 medium, Express-Five medium, Drosophila medium, Ham's F-12K medium, and mixed media thereof.
Since cardiomyocytes tend to adhere to the culture vessel (X), the basal medium is preferably a DMEM medium, an RPMI medium, or an MEM medium, more preferably a DMEM medium.

The serum-free medium (α) is preferably DMEM medium, RPMI medium, or MEM medium containing Knockout Serum Replacement, and more preferably DMEM medium containing Knockout Serum Replacement.

In addition to serum substitutes, serum-free medium (α) may also contain various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, etc.

[Process (B)]
Step (B) is a step of culturing the cardiomyocytes seeded in step (A). The method for culturing cardiomyocytes is not particularly limited, and may be performed according to a known protocol.

Details regarding the culture medium used for culture in step (B) are the same as those used in step (A). The medium used in step (B) may be the same as or different from the medium used in step (A), but it is preferable that it be the same as the medium used in step (A) because this facilitates proliferation of cardiomyocytes.

The amount of medium used in step (B) is not particularly limited and can be determined appropriately depending on the type of cell and medium.
The frequency of medium replacement is not particularly limited, but it is preferable not to replace the medium during the culture period in step (B) except for the day after seeding.
The culture conditions are not particularly limited and can be carried out by known methods, but the culture is usually carried out at a temperature of about 25 to 40°C and a carbon dioxide concentration of about 5%.

The culture period in step (B) is not particularly limited, but is preferably 6 hours to 3 days, more preferably 10 hours to 2 days, and even more preferably 12 to 36 hours.
The end of the culture period in step (B) can be determined, for example, by whether the cardiomyocytes have sufficiently adhered to the culture vessel (X). Whether the cardiomyocytes have sufficiently adhered to the culture vessel (X) can be evaluated, for example, by microscopic observation.

The culture in step (B) can be either adherent culture or suspension culture, as oxygen can be efficiently supplied in the culture vessel (X), but adherent culture is preferred.

Steps (A) and (B) are preferably performed in a serum-free medium (α). When steps (A) and (B) are performed in a serum-free medium (α), cardiomyocytes tend to adhere to the culture vessel (X).

[Step (C)]
Step (C) is a step of further culturing the cardiomyocytes obtained in step (B).
The method according to one aspect of the present invention preferably includes a step (C) of further culturing the cardiomyocytes cultured in the step (B). By including the step (C), it becomes easier to evaluate the action of a drug with high accuracy.
The method for culturing cardiomyocytes is not particularly limited, and may be carried out according to known protocols.

The medium used for the culture in step (C) is not particularly limited as long as it is a medium in which cardiomyocytes can survive, and may be appropriately selected depending on the cell type used. Examples of the medium used for the culture include any basal cell culture medium, differentiation medium, medium dedicated to primary culture, etc., specifically, serum-free medium (α), medium (β) described below, Eagle's Mini-Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), α-MEM, Glasgow MEM (GMEM), IMDM, RPMI 1640, Ham's F-12, MCDB medium, Williams' Medium E, CardioGro (manufactured by Funakoshi Co., Ltd.), iCell Cardiomyocyte Maintenance Medium (manufactured by FUJIFILM Cellular Dynamics), iCell Cardiomyocyte Thawing Medium (manufactured by FUJIFILM Cellular Dynamics), and mixed media thereof. These media may also be supplemented with serum, various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, and the like.
The medium used in step (C) is preferably an iCell cardiomyocyte maintenance medium or medium (β) described below, more preferably medium (β) described below, because it allows cardiomyocytes to proliferate more efficiently and the action of the drug to be detected with high accuracy.

The medium used for the culture in step (C) may be changed during the culture period in step (C), and two or more types may be used in combination.
It is preferable to use medium (β) for at least part of the culture period in step (C) because this allows cardiomyocytes to proliferate more efficiently and the action of the drug to be detected with high accuracy. Among the two or more media used for culture in step (C), the order and period in which medium (β) is used are not limited, but medium (β) is preferably used as the second or subsequent medium. The period for culture using medium (β) is preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, and particularly preferably 60% or more of the culture period in step (C).

The amount of medium used in step (C) is not particularly limited and can be determined appropriately depending on the cell species and the type of medium.
The frequency of medium change is not particularly limited, but it is preferable to change the medium every day or every 2 or 3 days.
The culture conditions are not particularly limited and can be carried out by known methods, but the culture is usually carried out at a temperature of about 25 to 40°C and a carbon dioxide concentration of about 5%.

The culture period in step (C) is not particularly limited, but is preferably 3 to 30 days, more preferably 7 to 20 days, and even more preferably 10 to 16 days.
The end of the culture period in step (C) can be determined, for example, by whether the cardiomyocytes are sufficiently synchronized, more specifically, whether the cardiomyocytes exhibit a stable beating pattern. Whether the cardiomyocytes exhibit a stable beating pattern can be evaluated, for example, by calcium ion waveforms.

[Medium (β)]
The medium (β) is a medium containing 1 to 100 μg/mL of free fatty acids. There are no particular limitations on other components of the medium (β), as long as the medium contains 1 to 100 μg/mL of free fatty acids.

The term "free fatty acid" refers to a non-esterified fatty acid, and the number of carbon atoms in the fatty acid is not limited.
The type of free fatty acid is not limited, and examples thereof include short-chain fatty acids having 2 to 6 carbon atoms, such as acetic acid, butyric acid, and caproic acid; medium-chain fatty acids having 8 to 10 carbon atoms, such as caprylic acid and capric acid; and long-chain fatty acids having 12 to 22 carbon atoms, such as lauric acid, myristic acid, myristoleic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. The fatty acids may be saturated or unsaturated.
The medium (β) may contain one kind of the above-mentioned free fatty acids alone or two or more kinds of them in combination.

The free fatty acid content of the medium (β) is preferably 1 to 70 μg/mL, more preferably 2 to 50 μg/mL, and even more preferably 10 to 50 μg/mL.
When the medium (β) contains free fatty acids in an amount within the above range, the action of a drug can be evaluated more accurately.

The method for measuring the free fatty acid content of the culture medium (β) is not particularly limited, and the free fatty acid concentration can be measured, for example, by a method utilizing a coupled enzyme reaction.

Preferably, the medium (β) further contains at least one selected from 1 to 100 μg/mL of lysophosphatidylcholine, 1 to 100 μg/mL of triacylglyceride, 1 to 100 μg/mL of phosphatidylcholine, 1 to 100 μg/mL of phosphatidic acid, 0.1 to 10 μg/mL of cholesterol, and 0.1 to 10 μg/mL of sphingomyelin.
The lysophosphatidylcholine content is more preferably 1 to 50 μg/mL, even more preferably 1 to 20 μg/mL. The triacylglyceride content is more preferably 1 to 50 μg/mL, even more preferably 1 to 20 μg/mL. The phosphatidylcholine content is more preferably 1 to 50 μg/mL, even more preferably 1 to 25 μg/mL. The phosphatidic acid content is more preferably 1 to 50 μg/mL, even more preferably 1 to 10 μg/mL. The cholesterol content is more preferably 0.1 to 8 μg/mL, even more preferably 0.1 to 5 μg/mL. The sphingomyelin content is more preferably 0.1 to 8 μg/mL, even more preferably 0.1 to 5 μg/mL.
When the medium (β) contains the lipids within the above ranges, the action of the drug can be evaluated more accurately.

The method for measuring the content of the above lipids in the culture medium (β) is not particularly limited, and can be measured using known methods.

[Step (D)]
Step (D) is a step of exposing the cultured cardiomyocytes to the drug. The cultured cardiomyocytes are the cardiomyocytes cultured in step (B) when step (C) is not performed, and are the cardiomyocytes cultured in step (C) when step (C) is performed.
The method for exposing cardiomyocytes to a drug is not particularly limited. For example, the drug may be added directly to the medium, or the drug may be dispersed or dissolved in an appropriate solvent and then added to the medium.
The solvent is not particularly limited, but examples thereof include water, ethanol, methanol, and DMSO.
The concentration of the drug to be added may be determined depending on the type of drug. In the method of one embodiment of the present invention, the effect of the drug can be easily detected even at a low concentration, so the concentration of the drug to be added can be set lower than the known concentrations described in, for example, Non-Patent Document 1. The drug can be added to the medium to a final concentration in the range of, for example, 0.001 nM to 10 mM.

The period for exposing cardiomyocytes to a drug is not particularly limited as long as the drug can exert its effect on cardiomyocytes, and can be determined appropriately depending on the type of cardiomyocytes or drug.
The exposure period is preferably 1 second to 5 days, more preferably 1 minute to 3 days, and even more preferably 5 minutes to 3 days.
The culture conditions are not particularly limited and can be carried out by known methods, but the culture is usually carried out at a temperature of about 25 to 40°C and a carbon dioxide concentration of about 5%.

Details regarding the medium used in step (D) are the same as those used in step (B) if step (C) is not present, and the same as those used in step (C) if step (C) is present. The medium used in step (D) may be the same as or different from the medium used in step (C) or step (B), but it is preferable that it be the same, as this allows for accurate evaluation of the drug's effects.

The method according to one aspect of the present invention preferably includes step (C), and steps (C) and (D) are carried out in a medium (β). When steps (C) and (D) are carried out in a medium (β), the action of a drug, particularly cardiotoxicity, can be evaluated more accurately, and proarrhythmic properties, particularly EAD-like waveforms, can be easily detected.
It is more preferable that steps (A) and (B) are performed in a serum-free medium (α), and steps (C) and (D) are performed in a medium (β). When performed under such medium conditions, cardiomyocytes are more likely to adhere to the culture vessel (X), and the drug action, particularly cardiotoxicity, can be evaluated more stably and accurately, and arrhythmogenicity, particularly EAD-like waveforms, can be more easily detected.

[Step (E)]
Step (E) is a step of analyzing and evaluating an indicator of the cellular function of the cardiomyocytes exposed to the drug in step (D).
The indicator of cardiomyocyte cellular function is not particularly limited as long as it indicates cardiomyocyte function, and may be an indicator of general cellular function or an indicator of cardiomyocyte-specific function. Examples of indicators of cardiomyocyte cellular function include action potential, gene expression, contractility, sarcomere length, intracellular concentrations of sodium ions, potassium ions, or calcium ions or changes therein, pulsation, cell viability, an indicator of mitochondrial function, and concentrations or changes therein of energy-related substances such as glucose, pyruvate, lactate, and ATP.
The indicator of cellular function of cardiomyocytes is preferably calcium ion concentration or its changes, because it is easy to measure and allows analysis of the movement of ions that cause pulsation of cardiomyocytes. Changes in the intracellular calcium ion concentration can be detected as a waveform indicating changes in calcium ion concentration. That is, the indicator of cellular function of cardiomyocytes is preferably calcium ion waveform.

Methods for analyzing indicators of cardiomyocyte cellular function are not particularly limited. Action potentials can be analyzed, for example, by extracellular potential analysis using a multi-electrode system, MEA (multi-electrode array) analysis, or intracellular potential analysis using whole-cell patch clamp. Gene expression can be analyzed, for example, by RT-PCR analysis of the expression of cardiomyocyte-specific genes. Contractility can be analyzed, for example, by measuring the deflection velocity from the movement of the cardiomyocyte's periphery using a motion analyzer. Sarcomere length can be analyzed, for example, by visualizing α-actinin present in sarcomeres with GFP labeling. Intracellular calcium ion concentration or its changes can be analyzed, for example, by calcium imaging. Pulsation can be analyzed, for example, using a motion analyzer. Cell viability can be analyzed, for example, by cell viability assays using tetrazolium compounds or mitochondrial membrane potential-dependent dyes, or by measuring ATP levels. Indicators of mitochondrial function can be analyzed, for example, by a swelling assay, which is an indicator of mitochondrial toxicity, e.g., mitochondrial permeability transition (MPT), or activity measurement of electron transport chain enzyme complexes. Concentrations or changes in concentrations of energy-related substances such as glucose, pyruvate, lactate, and ATP can be analyzed, for example, by imaging methods for glucose, pyruvate, lactate, ATP, etc.

By analyzing calcium ion waveforms, for example, QT prolongation, bradycardia (negative chronotropy), tachycardia (positive chronotropy), inotropy (positive inotropy), weakness (negative inotropy), early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), torsades de pointes (TdP), triggered activity arrhythmia, or reentrant arrhythmia can be detected.
In step (E), early after-depolarizations (EADs) are preferably detected by analyzing calcium ion waveforms. Also, in step (E), tachycardia or bradycardia is preferably detected by analyzing calcium ion waveforms.

[Other Step: Step (F)]
The method according to one aspect of the present invention may include, after step (E), step (F) of comparing the analytical results obtained in step (E) with analytical results obtained in the absence of the drug. In other words, step (F) is a step of comparing the indicators of cellular function obtained in step (E) in the presence and absence of the drug to determine the level of the effect of the drug.
The criterion for judgment is whether an index of cell function in the presence of a drug is higher or lower than, or has changed from, an index of cell function in the absence of the drug.
"In the absence of a drug" generally refers to the case before the drug is added, or when only a solvent for dispersing or dissolving the drug is added.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these.

[Measurement of weight average molecular weight (Mw) and molecular weight distribution (Mw/Mn)]
The weight average molecular weight (Mw) and molecular weight distribution (Mw/Mn) of the 4-methyl-1-pentene polymer used in the examples were measured by gel permeation chromatography (GPC).
Specifically, the weight average molecular weight (Mw) and number average molecular weight (Mn) of a polymer dissolved in orthodichlorobenzene were measured under the following conditions, with the molecular weight calibrated using standard polystyrene.
Apparatus: Gel permeation chromatograph HLC-8321GPC/HT type (manufactured by Tosoh Corporation)
Data analysis software: Empower3 (Waters)
Detector: differential refractometer Serially connected columns: TSKgel GMH6-HT (2 columns) and TSKgel GMH6-HTL (2 columns)
Column temperature: 140°C
Flow rate: 1.0 mL/min Sample concentration: 1.5 mg/mL

[Production Example 1] Production of substrate A 4-methyl-1-pentene polymer, TPX (registered trademark) (manufactured by Mitsui Chemicals, Inc.: molecular weight (Mw) = 428,000, molecular weight distribution (Mw/Mn) = 4.1, content of structural units derived from 4-methyl-1-pentene is 97.2 mol%, and the total content of structural units derived from an α-olefin having 16 carbon atoms and the content of structural units derived from an α-olefin having 18 carbon atoms is 2.8 mol%), was used and fed into a T-die extruder equipped with a full-flight screw that extrudes a substrate layer. The extrusion temperature was set to 270°C, the roll temperature was set to 60°C, and the roll rotation speed conditions were changed to obtain a film 1 having a thickness of 50 μm.

Using the film 1 as a measurement sample, the oxygen permeability coefficient [cm ³ × mm/(m ² × 24h × atm)] was measured at a temperature of 23°C and humidity of 0% using a differential pressure gas permeability measuring device MT-C3 manufactured by Toyo Seiki Seisakusho. The measurement diameter was 70 mm (permeation area: 38.46 cm ² ). Because a high oxygen permeability coefficient was expected, an aluminum mask was applied to the sample in advance, and the actual permeation area was set to 5.0 cm ² . The oxygen permeability coefficient was 1912 cm ³ × mm/(m ² × 24h × atm).

The film 1 was plasma treated using an atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.) by filling the chamber with a nitrogen gas flow (treatment speed 2 m/min, output 4.5 kW, 2 round trips).

The water contact angle was measured using plasma-treated Film 1 as the measurement sample. The water contact angle was measured in accordance with Japanese Industrial Standard JIS-R3257 (Test method for wettability of substrate glass surfaces). A water droplet of 4 μL or less, which can be considered spherical under constant temperature and humidity conditions of 25±5°C and 50±10%, was dropped onto the surface of the measurement sample, and the angle of the contact interface between the measurement sample and the water droplet was measured using the sessile drop method within one minute after the water droplet made contact with the measurement sample surface. The water contact angle of Plasma-treated Film 1 was 60.3°.

[Manufacturing Example 2] Preparation of Culture Vessel The plasma-treated film 1 was cut to a size of 8 cm x 12 cm and attached to the bottom of a 96-well polystyrene (PS) container frame via a medical adhesive (manufactured by 3M) to prepare a 96-well culture plate. The plate was then packed in a gamma-ray-resistant bag and sterilized by irradiating with 10 kGy of gamma rays. This was used as a T-plate. The culture area per well was approximately 0.32 ^cm2 .

As controls for the T plate, a 96-well TCPS (Tissue Culture Polystyrene) plate (CellBIND, Corning, product number 3300, also referred to as the "C plate"), a 96-well PDMS (Polydimethylsiloxane) plate (VECELL, product number V96WGPB, also referred to as the "V plate", oxygen permeability coefficient 19121 [cm ³ × mm/(m ² × 24h × atm)], bottom thickness 350 μm), and a 96-well TCPS plate (Greiner, product number 655090, also referred to as the "G plate") were used.

[Preparation of culture medium and reagents]
We used a human iPS cell-derived cardiomyocyte GCaMP-transfected line (Myoridge, Inc., product number G-011106) that had been cryopreserved in liquid nitrogen. These cells were cardiomyocytes 37 days after the start of differentiation induction of human iPS cells by protein-free cardiac differentiation (PFCD) and were transfected with a gene to constitutively express GCaMP as a calcium sensor.

The reagents used were as follows. All were stored refrigerated.
CDI maintenance medium (iCell cardiomyocyte maintenance medium, FUJIFILM Cellular Dynamics, 557-33591)
CDI thawing/seeding medium (iCell cardiomyocyte thawing medium, FUJIFILM Cellular Dynamics, 550-33581)

Special maintenance medium (CarmyA maintenance medium UG, manufactured by Myoridge, ME-01A00241, supplied as a liquid medium and an accompanying supplement, which are mixed before use. Corresponds to medium (β)).
It contains 11.3 μg/mL of lysophosphatidylcholine, 9.79 μg/mL of triacylglycerides, 5.29 μg/mL of phosphatidylcholine, 2.07 μg/mL of phosphatidic acid, 0.88 μg/mL of cholesterol, and 0.80 μg/mL of sphingomyelin. It also contains 35.29 μg/mL of free fatty acids.
Carmy A special seeding medium (Carmy A seeding medium kit UG, manufactured by Myoridge, ME-02A00211. Corresponding to serum-free medium (α))
Special basal medium (serum-free medium included in CarmyA Seeding Medium Kit UG, manufactured by Myoridge, ME-02A00211)

iMatrix-511silk (product number 387-10131, manufactured by Nippi)
CultureSure ^™ Y-27632 (product number 034-24024, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., lot number KCG7025)

Isoproterenol (Tokyo Chemical Industry Co., Ltd.)
Bepridil (Kissei Pharmaceutical Co., Ltd.)
Pentamidine (Kissei Pharmaceutical Co., Ltd.)
E-4031 manufactured by Kissei Pharmaceutical Co., Ltd. Verapamil manufactured by Kissei Pharmaceutical Co., Ltd. Risperidone manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Terfenadine manufactured by Sigma-Aldrich Ranolazine manufactured by Tokyo Chemical Industry Co., Ltd. DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[Calcium transient analysis method]
Using a fluorescence microscope system IX83 (Olympus), changes in GCaMP fluorescence intensity were measured as calcium transients, and analysis was performed using Carmy-Analyzer software (Myoridge).

[Reference Examples 1 and 2] Changes in cardiomyocyte-specific gene expression levels due to differences in test plate materials (method)
As test plates, C plate (Reference Example 1) and T plate (Reference Example 2) were used to compare the expression levels of cardiomyocyte-specific genes.
<Cultivation schedule>
Cell culture was carried out according to the following schedule: The day the cells woke up is designated as Day 0, and subsequent days are indicated.
Day 1: Reagent preparation and setup Day 0: Frozen cardiomyocytes awakening, pre-coating of plates, seeding onto 96-well test plates, and culturing Days 1-5: Culture with 150 μL of CDI maintenance medium every other day Days 7-13: Culture with 200 μL of CDI maintenance medium every other day Day 14: Gene expression analysis

<Method for coating test plates with iMatrix-511silk>
70 μL of iMatrix-511silk was added to 9 mL of PBS(-) and diluted to prepare a coating solution. 100 μL of the coating solution was added to a 96-well test plate at a concentration of 1.21 μg/cm ² and allowed to stand in an incubator at 37°C for 1 hour. The solution was removed by aspiration before seeding the cells.

<Awakening and seeding of frozen cardiomyocytes>
Y-27632 was added to the CDI thawing/seeding medium to a final concentration of 10 μM, and used as the thawing medium. The medium was heated to 37° C. before use.
Frozen cardiomyocytes were thawed in a 37°C water bath and suspended in thawing medium. The cells were centrifuged at 300 x g for 5 minutes and the supernatant was removed. The cells were suspended in thawing medium and adjusted to 2 x ¹⁰ - 5 x ¹⁰ cells/mL. The cell number was counted and the cells were seeded at 8 x ¹⁰ cells/well into an iMatrix-511silk-coated 96-well test plate and placed in a 37°C, 5% _CO2 incubator. The experiment was performed in triplicate.

<RNA extraction and RT-qPCR>
cTnT, MYL2, Kir2.1, PGC1α (PPARGC1A), and GAPDH were analyzed.

(1) Reagents and Equipment RNA extraction: miRNeasy Mini Kit (product number 217004, manufactured by Qiagen)
cDNA synthesis: ReverTraAce® qPCR RT MasterMix with gDNA Remover (product number FSQ-301, manufactured by TOYOBO)
qPCR reaction: PowerUp SYBR Green Master Mix (product number A25776, ThermoFisher)
QuantStudio6 Flex Real-time PCR system (ThermoFisher)
Nanophotometer spectrophotometer C40 (manufactured by Wakembie Tech Co., Ltd.)

(2) RNA Extraction After removing the culture supernatant of cardiomyocytes, QIAZOL (Qiagen) was added and suspended to lyse the cells, and the lysate was collected in a tube. RNA was extracted according to the protocol attached to the miRNeasy MiniKit, and the RNA concentration was measured using a Nanophotometer C40 spectrophotometer.

(3) RT-qPCR
Using 253 ng of the extracted RNA, reverse transcription was performed according to the protocol provided with the PowerUp SYBR Green Master Mix to synthesize cDNA. Subsequently, 6 ng of cDNA was used to perform qPCR using the standard method. A five-point calibration curve was prepared by collecting 10 μL of each 10 ng/μL cDNA sample and diluting them 1/10.

<Evaluation of gene expression>
RNA extracted from the cells was used to analyze gene expression levels using the QuantStudio6Flex Real-time PCR system. The primer sequences used are shown in Table 1, and the PCR conditions are shown in Table 2.

(result)
The results of analyzing gene expression levels are shown in Figure 1. Gene expression levels were expressed as relative values when the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was set at 1, and are shown as ±SD (n=3).
When the T plate was used (Reference Example 2), the expression levels of cTnT, MYL2, Kir2.1, and PGC1α (PPARGC1A) were higher than when the C plate was used (Reference Example 1). These results demonstrate that culturing cardiomyocytes using the T plate increases the expression of cardiomyocyte-specific genes and can improve cardiomyocyte function.

[Examples 1 to 9, Comparative Examples 1 to 9] Changes in drug responsiveness due to differences in test plate materials (method)
As test plates, C plates (Comparative Examples 1 to 9) and T plates (Examples 1 to 9) were used, and drugs were added to analyze calcium transients.
<Cultivation schedule>
Cell culture was carried out according to the following schedule: The day the cells woke up is designated as Day 0, and subsequent days are indicated.
Day 1: Preparation and setup of reagents Day 0: Awakening frozen cardiomyocytes, precoating of plates, seeding onto 96-well test plates (step (A)), and culturing (step (B))
Day 1: Culture medium was replaced with CDI maintenance medium (step (C))
Day 4 to 13: Culture with medium change every 2 days with special maintenance medium (step (C))
Day 14: Drug addition and calcium transient analysis using special maintenance medium (step (D) and step (E))

<Method for coating test plates with iMatrix-511silk>
19.2 μL of iMatrix-511silk was added to 3 mL of special basal medium and diluted to prepare a coating solution. The coating solution was added to a 96-well test plate at 100 μL/well (working concentration 1 μg/cm ² ) and allowed to stand in a 37°C incubator for 1 hour. The coating solution was not removed, and cardiomyocytes were seeded while the coating solution was still in place.

<Awakening and seeding of frozen cardiomyocytes>
Y-27632 was added to Carmy A special seeding medium to a final concentration of 10 μM, and used as a special thawed seeding medium. The medium was allowed to cool to room temperature before use.
Frozen cardiomyocytes were thawed in a 37°C water bath and suspended in a special thawing and seeding medium. The cells were centrifuged at 300 x g for 5 minutes and the supernatant was removed. The cells were suspended in the special thawing and seeding medium and adjusted to 2 x ¹⁰ - 5 x ¹⁰ cells/mL. The cell number was counted and the cells were seeded at 6 x ¹⁰ cells/well (medium volume 200 μL) into an iMatrix-511silk-coated 96-well test plate and placed in a 37°C, 5% _CO2 incubator. The experiment was performed in triplicate.

<Addition of drugs>
The drugs were added to a final concentration of 0.1% DMSO, 1 μM, 2 μM, and 4 μM bepridil, 11.1 μM, 33.3 μM, and 100 μM pentamidine, and 20 nM, 100 nM, and 500 nM isoproterenol.
In the T plate, bepridil was added to final concentrations of 1 μM, 2 μM, and 4 μM, pentamidine to final concentrations of 11.1 μM, 33.3 μM, and 100 μM, and isoproterenol to final concentrations of 20 nM, 100 nM, and 500 nM, respectively, to give Examples 1 to 9.
Comparative Examples 1 to 9 were prepared by adding bepridil to C plates at final concentrations of 1 μM, 2 μM, and 4 μM, pentamidine to final concentrations of 11.1 μM, 33.3 μM, and 100 μM, and isoproterenol to final concentrations of 20 nM, 100 nM, and 500 nM, respectively.

(result)
The results for 0.1% DMSO are shown in Figure 2, the results for 1 μM bepridil in Figure 3, an enlarged EAD-like waveform of the results for 1 μM bepridil in Figure 4, the results for 2 μM bepridil in Figure 5, the results for 4 μM bepridil in Figure 6, the results for 11.1 μM pentamidine in Figures 7A and 7B, the results for 33.3 μM pentamidine in Figures 8A and 8B, the results for 100 μM pentamidine in Figures 9A and 9B, the results for 20 nM isoproterenol in Figure 10, the results for 100 nM isoproterenol in Figure 11, and the results for 500 nM isoproterenol in Figure 12. "Pre" indicates before drug addition, and "Post" indicates after drug addition.
When DMSO, bepridil, or pentamidine was added, the results are shown 10 minutes after addition. When isoproterenol was added, the results are shown 10 minutes, 24 hours, and 48 hours after addition.

Addition of 0.1% DMSO slightly increased the fluorescence intensity in both the T plate and the C plate, but did not affect the calcium ion waveform (Figure 2).

When 1 μM bepridil was added to the T plate (Example 1), EAD (early afterdepolarization)-like waveforms were detected, albeit weakly, in two of three wells (Figures 3 and 4). Even when the bepridil concentration was increased to 2 μM in the T plate (Example 2), EAD-like waveforms were not detected more prominently (Figure 5), and when the concentration was increased to 4 μM (Example 3), pulsatile waveforms were no longer detected (Figure 6). On the other hand, when the bepridil concentration was changed to 1 μM, 2 μM, or 4 μM in the C plate (Comparative Examples 1 to 3), EAD-like waveforms were not detected (Figures 3, 4, 5, and 6).

Bepridil is a calcium channel blocker used to treat angina pectoris, but is known to induce a potentially fatal ventricular arrhythmia called torsade depointes (TdP) as a side effect. However, its proarrhythmic effect is difficult to detect in in vitro test systems using cardiomyocytes differentiated from iPS cells. However, in Example 1, it was possible to detect EAD-like waveforms that trigger torsade depointes (TdP), demonstrating that the proarrhythmic effect of bepridil can be detected.

When 11.1 μM pentamidine was added, arrhythmia was detected 24 hours after addition on the T plate (Example 4) (Figure 7A), but no arrhythmia was detected on the C plate (Comparative Example 4) (Figure 7B). When the pentamidine concentration was increased to 33.3 and 100 μM, arrhythmia was not detected even on the T plate (Examples 5 and 6, Figures 8A and 9A). When 100 μM pentamidine was added to the C plate (Comparative Example 6), acute arrhythmia was detected 10 minutes later (Figure 9B).

Pentamidine is an antibacterial drug known to induce arrhythmias, and its mechanism of action is believed to be inhibition of membrane trafficking of hERG (human ether-a-go-go related gene) protein. Therefore, it is known that prolonged exposure to pentamidine is required for the development of its proarrhythmic effect.
The fact that arrhythmia was detected 24 hours after the addition of pentamidine in Example 4 is reasonable considering the mechanism of action of pentamidine, and therefore it is believed that the T plate was able to detect pentamidine's inhibitory effect on membrane translocation (trafficking) of hERG protein. On the other hand, the acute arrhythmia detected on the C plate (Comparative Example 6) occurred 10 minutes after the addition of pentamidine, which is likely a nonspecific effect other than the inhibitory effect on membrane translocation (trafficking) of hERG protein. The absence of such a nonspecific effect on the T plate suggests that the use of the T plate can reduce false positives when evaluating the effects of drugs.

When 20 nM isoproterenol was added, tachycardia was stably confirmed in the T plate (Example 7) (Figure 10). On the other hand, although tachycardia was confirmed in the C plate (Comparative Example 7), the calcium ion waveform was disturbed and unstable. When the isoproterenol concentration was increased to 100 and 500 nM, tachycardia was stably confirmed in the T plate (Examples 8 and 9), and the tachycardia pattern was stably confirmed in the C plate (Comparative Examples 8 and 9) (Figures 11 and 12).

Isoproterenol is a β1 and β2 receptor agonist that increases heart rate (tachycardia-inducing effect). Compared to C-plates, T-plates were able to stably detect the increase in heart rate (tachycardia-inducing effect) even at lower concentrations of isoproterenol. It is believed that T-plates can evaluate the effects of drugs with higher sensitivity than C-plates.

[Reference Examples 3, 4, and 5] Study of test plates with high oxygen permeability (method)
The influence on cardiomyocyte culture was investigated using test plates made of PDMS with high oxygen permeability, including G plates (made of TCPS, Reference Example 3), T plates (made of PDMS, Reference Example 4), and V plates (made of PDMS, Reference Example 5).

<Cultivation schedule>
Cell culture was carried out according to the following schedule: The day the cells woke up is designated as Day 0, and subsequent days are indicated.
Day 1: Reagent preparation and setup Day 0: Using a special thawing seeding medium, frozen cardiomyocytes were awakened, pre-coated on a plate, seeded on a 96-well test plate, and cultured Day 1: Culture medium was replaced with a special maintenance medium Days 3-13: Culture medium was replaced with a special maintenance medium every two days Day 6: Microscopic observation

Coating of the test plate with iMatrix-511silk, waking and seeding of the frozen cardiomyocytes were carried out in the same manner as in Examples 1 to 9, except that a specially prepared medium for thawing and seeding was used.
Six days after seeding, the cells were observed under a microscope in a bright field and photographed. They were also observed under a fluorescence microscope and video and still images were taken.

(result)
Figure 13 shows photographs of cells observed 6 days after seeding. The photographs on the left are from Reference Example 3, which used a G plate; the photographs in the center are from Reference Example 4, which used a T plate; and the photographs on the right are from Reference Example 5, which used a V plate. In Reference Example 3, which used a G plate, and Reference Example 4, which used a T plate, the cardiomyocytes adhered sufficiently to the test plate. Furthermore, fluorescent observation revealed that synchronized pulsation-like waveforms were detected. On the other hand, in Reference Example 5, which used a V plate, the cardiomyocytes did not adhere sufficiently to the test plate, and cell clumps were observed. Furthermore, fluorescent observation revealed that synchronized pulsation-like waveforms were not detected.

These results revealed that even if the test plate was made of PDMS, which has high oxygen permeability, it was impossible to culture cardiomyocytes, especially to detect pulsatile waveforms.

[Examples 10 to 17, Comparative Examples 10 to 17] Culture medium examination (method)
T plates or G plates were used as test plates, and special maintenance medium or CDI maintenance medium was used as the medium from day 1 onwards after seeding, and drugs were added, and calcium transients were analyzed.
<Cultivation schedule>
Cell culture was carried out according to the following schedule: The day the cells woke up is designated as Day 0, and subsequent days are indicated.
Day 1: Reagent preparation and setup Day 0: Pre-coating plates with a special basal medium, activating frozen cardiomyocytes using a special thawing and seeding medium, seeding them onto a 96-well plate for testing (step (A)), and culturing them (step (B))
Day 1: Culture with special maintenance medium or CDI maintenance medium (step (C))
Day 3 to 13: Culture with medium change every 2 days with special maintenance medium or CDI maintenance medium (step (C))
Day 14: Medium replacement, drug addition, calcium transient analysis (step (D), step (E))

<Culture medium>
The test plates were coated with iMatrix-511silk using a special basal medium. The frozen cardiomyocytes were thawed and seeded using a special thawing and seeding medium.
The medium used for medium replacement, drug addition, and calcium transient analysis on day 1 after seeding was a special maintenance medium or a CDI maintenance medium.

<Addition of drugs>
The drugs were added so that DMSO had a final concentration of 0.1% and bepridil had final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively.
In the T plates, special maintenance medium was used as the medium from day 1 onwards, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively, as Examples 10 to 13, and CDI maintenance medium was used as the medium from day 1 onwards, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively, as Examples 14 to 17.
On the G plate, a special maintenance medium was used as the medium from day 1 onwards, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively. Comparative Examples 10 to 13 were used, and CDI maintenance medium was used as the medium from day 1 onwards, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively. Comparative Examples 14 to 17 were used, and CDI maintenance medium was used as the medium from day 1 onwards, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively.

(result)
The results are shown in Figures 14 to 23. "Special maintenance medium" refers to the case where the special maintenance medium was used as the medium from day 1 onwards after seeding, and "CDI" refers to the case where the CDI maintenance medium was used as the medium from day 1 onwards after seeding. Pre refers to before the addition of the drug, and Post refers to after the addition of the drug.

Addition of 0.1% DMSO slightly increased the fluorescence intensity on the T plate but did not affect the calcium ion waveform (Figure 14). However, on the G plate, a disturbance in the calcium ion waveform was observed (Figure 15, bottom of the special maintenance medium Post).
The pulsation-like waveform was more stable and the signal was stronger on the T plate than on the G plate. Furthermore, when the special maintenance medium was used as the medium on the T plate from day 1 onwards, the pulsation-like waveform was detected more stably and the signal was stronger than when the CDI maintenance medium was used.

No disturbances in the calcium ion waveform were detected when bepridil was added at 0.06 μM or 0.25 μM on the T plate (Examples 10, 11, 14, 15) or when bepridil was added at 0.06 μM or 0.25 μM on the G plate (Comparative Examples 10, 11, 14, 15) (Figures 16, 17, 18, 19).

When a special maintenance medium was used as the medium for T-plate seeding on day 1 or later, a weak EAD (early after depolarization)-like waveform was detected when 1 μM bepridil was added (Example 12) (FIG. 20). When a CDI maintenance medium was used as the medium for T-plate seeding on day 1 or later, a weak EAD-like waveform was detected when 1 μM bepridil was added (Example 16) (FIG. 20).
When special maintenance medium was used as the medium on the first day after seeding on G plates, the addition of 1 μM bepridil (Comparative Example 12) resulted in a disturbance in the calcium ion waveform, and the pulsatile waveform was no longer detected, and neither EAD-like waveforms nor QT prolongation were detected, although this varied depending on the well. When CDI maintenance medium was used as the medium on the first day after seeding on G plates, the addition of 1 μM bepridil (Comparative Example 16) resulted in a stable calcium ion waveform, but neither EAD-like waveforms nor QT prolongation were detected ( FIG. 21 ).

When the bepridil concentration was increased to 4 μM on T plates (Examples 14 and 17), pulsatile waveforms were no longer detected, regardless of the type of medium (Figure 22). Similarly, when the bepridil concentration was increased to 4 μM on G plates (Comparative Examples 14 and 17), pulsatile waveforms were no longer detected, regardless of the type of medium (Figure 23).

[Examples 18 to 21] Examination of medium change immediately before drug addition and verapamil. Using T plates as test plates, the influence of medium change immediately before drug addition was examined. In addition, using T plates as test plates, it was examined whether the effect of verapamil on cardiomyocytes could be evaluated.
(method)
<Exam Schedule>
Day 1: Reagent preparation and preparation Day 0: Pre-coating a T-plate with a special basal medium, activating frozen cardiomyocytes using a special thawing and seeding medium, seeding them onto a 96-well plate for testing (step (A)), and culturing them (step (B))
Day 1: Culture medium was replaced with special maintenance medium (step (C))
Day 4 to 13: Culture with medium change every 2 days with special maintenance medium (step (C))
Day 14: Medium replacement, drug addition, calcium transient analysis (step (D), step (E))

<Details of Day 14>
<Culture medium exchange>
Medium change A: The special maintenance medium in the plate was removed and new special maintenance medium was added.
Medium change B: The special maintenance medium was removed from the plate, new special maintenance medium was added, and then immediately removed, and new special maintenance medium was added again.

<Addition of drugs>
A drug (verapamil) was added to plates that underwent medium exchange A or B approximately 30 minutes after the medium exchange. The drug was dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with special maintenance medium to a concentration 10 times the target final concentration to prepare a drug solution for addition. One-tenth the volume of the measurement medium was used as the drug solution for addition. For example, when adding verapamil to a final concentration of 10 nM, the 10 mM stock solution was diluted with special maintenance medium to prepare a 100 nM drug solution for addition, and 15 μL of the drug solution for addition was added to 150 μL of measurement medium. DMSO was added to the control group at a final concentration of 0.1%.

The medium was replaced with medium A or B, and verapamil was added to a final concentration of 10 nM, as Examples 18 and 19, respectively.
The medium was replaced with medium A or B, and verapamil was added to a final concentration of 100 nM, as Examples 20 and 21, respectively.

<Calcium transient analysis>
Calcium transient analysis was performed 10 to 30 minutes after drug addition.

(result)
The results of adding verapamil at a final concentration of 10 nM are shown in Figure 24, and the results of adding verapamil at a final concentration of 100 nM are shown in Figure 25. "Pre" indicates before drug addition, and "Post" indicates after drug addition. Figure 24A shows the results before medium exchange A, Figure 24B shows the results before medium exchange B, Figure 24C shows the results after medium exchange A, and Figure 24D shows the results after medium exchange B. Figure 25A shows the results before medium exchange A, Figure 25B shows the results before medium exchange B, Figure 25C shows the results after medium exchange A, and Figure 25D shows the results after medium exchange B.

When 10 nM verapamil was added, no change in the waveform was observed in either medium change A or B (Figure 24). When 100 nM verapamil was added, in either medium change A or B, the change in intensity was smaller at tPost compared to Pre. In other words, the contractility of cardiomyocytes was weakened, and the attenuated effect (negative inotropic effect) of verapamil was detected (Figure 25).

Verapamil is a calcium ion channel blocker. Verapamil is known to have a cardio-weakening effect (negative inotropic effect) that reduces the calcium ion concentration in cardiomyocytes and attenuates the contractility of cardiomyocytes, as well as a QT shortening effect. Verapamil is a drug that has a low risk level for clinical TdP, and is predicted to have a low risk level for TdP even in a conventional proarrhythmia model using cardiomyocytes differentiated from iPS cells.
The method according to one embodiment of the present invention was able to detect the cardioinhibitory effect (negative inotropic effect) of verapamil.

No difference was observed between medium changes A and B.

[Examples 22 to 53] Examination of other drugs Next, using T plates as test plates and special maintenance medium as the medium from day 1 onwards from seeding, it was examined whether the effects of other drugs on cardiomyocytes could be evaluated.
In literature A, the concentration dependence and variable factors of the electrophysiological response of 28 drugs with known clinical TdP risk levels (high risk, medium risk, low risk, or no risk) are evaluated, and the usefulness of cardiomyocytes differentiated from human iPS cells as an in vitro proarrhythmic model is evaluated.
For many drugs, the clinical risk level of TdP correlates with the risk level estimated in a proarrhythmia model using cardiomyocytes differentiated from iPS cells, but there are exceptional drugs, i.e., drugs for which it is difficult to predict the risk level in a conventional proarrhythmia model using cardiomyocytes differentiated from iPS cells (Reference A, Figure 6). Therefore, the effects of drugs for which it is difficult to predict the risk level in a conventional proarrhythmia model using cardiomyocytes differentiated from iPS cells were evaluated using a method that is one aspect of the present invention.
Specifically, bepridil, risperidone, and terfenadine were used as representative examples of drugs that predict a low risk of TdP in conventional proarrhythmia models using cardiomyocytes differentiated from iPS cells, even though the clinical risk of TdP is high or medium. Furthermore, ranolazine was used as a representative example of drugs that predict a high risk of TdP in conventional proarrhythmia models using cardiomyocytes differentiated from iPS cells, even though the clinical risk of TdP is low.

(method)
<Exam Schedule>
Day 1: Reagent preparation and preparation Day 0: Pre-coating a T-plate with a special basal medium, activating frozen cardiomyocytes using a special thawing and seeding medium, seeding them onto a 96-well plate for testing (step (A)), and culturing them (step (B))
Day 1: Culture medium was replaced with special maintenance medium (step (C))
Day 4 to 13: Culture with medium change every 2 days with special maintenance medium (step (C))
Day 14: Medium replacement, drug addition, calcium transient analysis (step (D), step (E))

<Addition of drugs>
The drugs were added in the same manner as in Examples 18-21.
Verapamil was added to final concentrations of 60 nM, 250 nM, 1000 nM, and 4000 nM, respectively, to give Examples 22, 23, 24, and 25.
Examples 26, 27, 28, and 29 were prepared by adding E-4031 to final concentrations of 1 nM, 10 nM, 100 nM, and 1000 nM, respectively.
Bepridil was added to final concentrations of 60 nM, 250 nM, 1 μM, and 40 μM, respectively, to give Examples 30, 31, 32, and 33.
Bepridil was added to final concentrations of 312 nM, 1.25 μM, 5 μM, and 20 μM, respectively, to give Examples 34, 35, 36, and 37.
Examples 38, 39, 40 and 41 were prepared by adding risperidone to final concentrations of 1 nM, 10 nM, 0.1 μM and 1 μM, respectively.
Examples 42, 43, 44 and 45 were prepared by adding risperidone to final concentrations of 60 nM, 250 nM, 1 μM and 4 μM, respectively.
Examples 46, 47, 48 and 49 were prepared by adding terfenadine to final concentrations of 0.25 nM, 1 nM, 4 nM and 16 nM, respectively.
Examples 50, 51, 52, and 53 were prepared by adding ranolazine to final concentrations of 10 nM, 100 nM, 1000 nM, and 10,000 nM, respectively.

<Calcium transient analysis>
Calcium transient analysis was carried out in the same manner as in Examples 18-21.

<CAD30 and CAD80>
CAD30 (CaD30, calcium transient duration at 30%) and CAD80 (CaD80, calcium transient duration at 80%) were calculated from the waveform obtained by calcium transient analysis. The time at which the minimum peak value of the intensity was measured was defined as t0. When the minimum peak value was defined as 0 and the maximum peak value as 1, CAD30 was defined as the time elapsed from t0 to the time at which a 30% decrease (0.7) from the maximum peak value was measured, and CAD80 was defined as the time elapsed from t0 to the time at which an 80% decrease (0.2) from the maximum peak value was measured. A conceptual diagram is shown in Figure 26.
CAD30 and CAD80 are values that serve as indicators of QT interval, as conceptually shown in Figure 26. When both or either one of CAD30 and CAD80 are prolonged compared to the control, it can be said that QT is prolonged, and when both or either one of CAD30 and CAD80 are shortened, it can be said that QT is shortened.

(result)
The results are shown in Figures 27 to 42. Pre indicates before drug addition, and Post indicates after drug addition.
(Verapamil)
As shown in Figure 27, there was almost no change in intensity between pre- and post-treatment with 60 nM and 250 nM verapamil. Considering that the control group (DMSO-treated) showed an increase in intensity at post-treatment compared to pre-treatment, although data are not shown, it can be said that the increase in intensity was suppressed with 60 nM and 250 nM verapamil. In other words, verapamil reduced intensity, or in other words, attenuated the contractility of cardiomyocytes, and the anabolic effect (negative inotropy) of verapamil was detected. At 1000 nM, the anabolic effect (negative inotropy) of verapamil was clearly detected (Figure 27). As shown in Figure 28, 1000 nM and 4000 nM verapamil reduced CAD30 and CAD80 at post-treatment compared to pre-treatment. In other words, verapamil shortened the QT interval. Furthermore, these effects were verapamil concentration-dependent.
The method according to one embodiment of the present invention was able to detect the cardio-weakening effect (negative inotropic effect) and QT shortening effect of verapamil.

(Regarding E-4031)
E-4031 is a potassium ion channel blocker known to induce EAD and TdP. E-4031 is a drug that is associated with a high risk of clinical TdP and is also predicted to have a high risk of TdP in a conventional proarrhythmia model using cardiomyocytes differentiated from iPS cells.
29 and 30, QT prolongation was observed from 10 nM, and at 100 nM, the QT prolongation was more pronounced, and the proarrhythmic effect of E-4031 was detected in a concentration-dependent manner.
The method according to one embodiment of the present invention was able to detect the proarrhythmic effect of E-4031.

(About Bepridil)
Bepridil is a calcium channel blocker originally approved as an antianginal drug, but is also classified as a group IV antiarrhythmic drug (according to the Vaughan Williams classification). Bepridil inhibits not only calcium ion channels but also sodium and potassium ion channels, a so-called multi-ion channel blocking effect. Bepridil is known to induce QT prolongation and torsades de pointes (TdP) due to its potassium ion channel blocking effect.
As shown in Figures 31 and 32, QT prolongation was detected with the addition of 60 nM to 4000 nM bepridil, but no concentration dependency was observed. At 1 μM, an EAD-like waveform was detected. Next, experiments were conducted using a narrower concentration range of bepridil. As shown in Figures 33 and 34, QT prolongation was detected with the addition of 312 nM and 1250 nM bepridil.
The method according to one embodiment of the present invention was able to detect the proarrhythmic effect of bepridil.

(About risperidone)
Risperidone is an atypical antipsychotic drug that has antagonistic effects on dopamine 2 receptors and serotonin 2A receptors, but is known to induce QT prolongation.

As shown in Figures 35 and 36, concentration-dependent QT prolongation was detected by adding risperidone at concentrations of 1 nM to 1 μM. Therefore, experiments were conducted by varying the concentration range of risperidone. As shown in Figures 37 and 39, concentration-dependent QT prolongation was detected by adding risperidone at concentrations of 60 nM, 250 nM, and 1000 nM, and EAD-like waveforms were also detected, especially at 250 nM (arrow in Figure 37).
The method according to one embodiment of the present invention was able to detect the proarrhythmic effect of risperidone.

(About terfenadine)
Terfenadine is a histamine H1 receptor antagonist, but was withdrawn from the market due to a series of reports of QT prolongation, TdP, and cardiac arrest. Currently, fexofenadine, an active metabolite of terfenadine that does not have proarrhythmic effects, is widely used as a successor antiallergic drug.

39 and 40, no concentration-dependent QT prolongation was detected with the addition of 0.25 nM to 16 nM terfenadine, but abnormal waveforms were detected at 4 nM (arrow in FIG. 39).
The method according to one embodiment of the present invention was able to detect the proarrhythmic effect of terfenadine.

(About ranolazine)
Ranolazine is a sodium ion channel blocker. Ranolazine has a low risk of causing arrhythmia in vivo, but it is known that QT prolongation is detected in cardiomyocytes differentiated from iPS cells, leading to the determination that ranolazine has proarrhythmic effects. In other words, ranolazine is known to be determined as a false positive in conventional proarrhythmic models using cardiomyocytes differentiated from iPS cells.

41 and 42, even when ranolazine was added over a wide range of concentrations from 10 nM to 10,000 nM, no change in the waveform was observed and no QT prolongation was detected.
According to the method of one aspect of the present invention, ranolazine was not evaluated as having a proarrhythmic effect. In other words, according to the method of one aspect of the present invention, even drugs that would be judged as false positives in a conventional proarrhythmia model using cardiomyocytes differentiated from iPS cells can be correctly judged as negative, thereby reducing false positives.

Examples 30 to 53 revealed that, according to one aspect of the present invention, it is possible to detect the effects of drugs that were difficult to detect using conventional in vitro test systems using cardiomyocytes differentiated from iPS cells.

INCORPORATION BY REFERENCE
This application claims priority based on Japanese Patent Application No. 2022-002556 filed with the Japan Patent Office on January 11, 2022, and Japanese Patent Application No. 2022-104613 filed with the Japan Patent Office on June 29, 2022, the disclosures of which are incorporated herein in their entirety.

Claims (15)

A method for evaluating the effect of a drug on cardiomyocytes, comprising:
(A) seeding cardiomyocytes on the culture surface of a culture vessel;
a step (B) of culturing the cardiomyocytes obtained in the step (A);
a step (C) of culturing the cardiomyocytes obtained in the step (B) for an additional 3 to 30 days;
a step (D) of exposing the cultured cardiomyocytes to the drug; and a step (E) of analyzing and evaluating an indicator of cellular function of the cardiomyocytes obtained in the step (D),
the steps (C) and (D) are carried out in a medium (β) containing 1 to 100 μg/mL of free fatty acids and further containing at least one selected from 1 to 100 μg/mL of lysophosphatidylcholine, 1 to 100 μg/mL of triacylglyceride, 1 to 100 μg/mL of phosphatidylcholine, 1 to 100 μg/mL of phosphatidic acid, 0.1 to 10 μg/mL of cholesterol, and 0.1 to 10 μg/mL of sphingomyelin;
The method, wherein at least a portion of the culture surface of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer. 2. The method of claim 1, wherein steps (A) and (B) are carried out in a serum-free medium (α) containing a serum replacement. The method according to claim 1 or 2, wherein the 4-methyl-1-pentene polymer is a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). The method of claim 1 or 2, wherein the entire culture surface of the culture vessel is formed from a substrate containing a 4-methyl-1-pentene polymer. The method of claim 1 or 2, wherein the culture surface is coated with a coating agent containing laminin. The method of claim 1 or 2, wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells. The method according to claim 1 or 2, wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells by protein-free cardiac differentiation (PFCD) method. The method of claim 1 or 2, wherein the effect is cardiotoxicity. 9. The method of claim 8 , wherein the cardiotoxicity is proarrhythmic. The method of claim 8 , wherein the cardiotoxicity is myocardial damage. The method of claim 10 , wherein the indicator of cellular function is an indicator of mitochondrial function. The method of claim 1 or 2, wherein the indicator of cell function is a calcium ion waveform. The method of claim 1 or 2, wherein early after-depolarizations (EADs) are detected in step (E). The method of claim 1 or 2, wherein the effect is a tachycardial effect or a bradycardial effect. 15. The method of claim 14 , wherein the indicator of cellular function is a calcium ion waveform.