Method for evaluating the effect of drugs on cardiomyocytes

Abstract

One aspect of the present invention relates to a method for evaluating an action on cardiomyocytes, the method for evaluating the action of a drug on cardiomyocytes comprising a step (A) for seeding cardiomyocytes on the culture surface of a culture vessel, a step (B) for culturing the cardiomyocytes obtained in step (A), a step (D) for exposing the cultured cardiomyocytes to the drug, and a step (E) for analyzing and evaluating indicators of cell function of the cardiomyocytes obtained in step (D), at least part of the culture surface of the culture vessel being formed from a base material containing a 4-methyl-1-pentene polymer.

Description

[Technical Field]

[0001] One aspect of the present invention relates to a method for evaluating the effect of a drug on cardiomyocytes. [Background technology]

[0002] Candidate new drugs are evaluated for the presence or absence of the expected action (main action) using in vitro test systems, and only those with high efficacy proceed to the next stage of development. Therefore, it is important that the in vitro test system can detect the action of the drug even at low concentrations and detect few false positives. If a drug that has progressed to clinical development is found to have serious side effects, its development as a new drug 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not only drugs in the cardiovascular field but also anti-allergy drugs, digestive medicines, and antibiotics are often withdrawn from the market due to the occurrence of cardiotoxicity.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 effect of the drug but also to evaluate its cardiotoxicity.

[0003] 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, and the resulting sheet of cardiomyocytes is 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 a configuration for increasing the amount of oxygen supplied to the cardiomyocytes. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2019 / 131806 [Non-patent literature]

[0005] [Non-Patent Document 1] Pharmacol Toxicol Methods.2017 Mar-Apr;84:111-127.doi:10.1016 / j.vascn.2016.12.003.Epub, 2016, Dec10. Summary of the Invention [Problem to be solved by the invention]

[0006] 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 effects of drugs but also accurately evaluate their cardiotoxicity. Conventional in vitro test systems sometimes fail 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; 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. [Means for solving the problem]

[0007] 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. The embodiments 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); (D) exposing the cultured cardiomyocytes to the agent; and and a step (E) of analyzing and evaluating an indicator of the 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 the 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 cellular 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. [Effects of the Invention]

[0008] 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 has been considered difficult to detect in conventional in vitro test systems using iPS cell-derived cardiomyocytes or the like can be detected. [Brief explanation of the drawings]

[0009] [Figure 1] 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]Figure 2 shows calcium ion waveforms in cardiomyocytes with 0.1% DMSO added. The acquisition time was 20 seconds, and the frame rate was 14.23 frames / second. [Figure 3] Figure 3 shows calcium ion waveforms in cardiomyocytes after the addition of 1 μM bepridil. The acquisition time was 20 seconds, and the frame rate was 14.23 frames / second. The arrows in the figure indicate EAD-like waveforms. [Figure 4] FIG. 4 shows an enlarged view of the EAD-like waveform in the calcium ion waveform of FIG. [Figure 5] Figure 5 shows calcium ion waveforms in cardiomyocytes after the addition of 2 μM bepridil. The acquisition time was 20 seconds, and the frame rate was 14.23 frames / second. [Figure 6] Figure 6 shows calcium ion waveforms in cardiomyocytes after the addition of 4 μM bepridil. The acquisition time was 20 seconds, and the frame rate was 14.23 frames / second. [Figure 7A] Figure 7A shows calcium ion waveforms in cardiomyocytes after the addition of 11.1 μM pentamidine to the T-plate. The acquisition time was 20 seconds, with a frame rate of 14.23 frames / second. Results are shown for the time points before (pre), 10 minutes after (10 min), 24 hours after (24 h), and 48 hours after (48 h). [Figure 7B] Figure 7B shows calcium ion waveforms in cardiomyocytes after the addition of 11.1 μM pentamidine to the 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 8A] Figure 8A shows calcium ion waveforms in cardiomyocytes after the addition of 33.3 μM pentamidine to the T-plate. The acquisition time was 20 seconds, with a frame rate of 14.23 frames / second. Results are shown for the time points before (pre), 10 minutes after (10 min), 24 hours after (24 h), and 48 hours after (48 h). [Figure 8B]Figure 8B shows calcium ion waveforms in cardiomyocytes after the addition of 33.3 μM pentamidine to the 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] Figure 9A shows calcium ion waveforms in cardiomyocytes after the addition of 100 μM pentamidine to the T-plate. The imaging time was 20 seconds, with a frame rate of 14.23 frames / second. Results are shown for the time points before (pre), 10 minutes after (10 min), 24 hours after (24 h), and 48 hours after (48 h). [Figure 9B] Figure 9B shows calcium ion waveforms in cardiomyocytes after the addition of 100 μM pentamidine to the C-plate. The imaging time was 20 seconds, with a frame rate of 14.23 frames / second. Results are shown for the time points before (pre), 10 minutes after (10 min), 24 hours after (24 h), and 48 hours after (48 h). [Figure 10] Figure 10 shows calcium ion waveforms in cardiomyocytes after the addition of 20 nM isoproterenol. The acquisition time was 20 seconds, and the frame rate was 14.23 frames / second. [Figure 11] Figure 11 shows calcium ion waveforms in cardiomyocytes after the addition of 100 nM isoproterenol. The acquisition time was 20 seconds, and the frame rate was 14.23 frames / second. [Figure 12] Figure 12 shows calcium ion waveforms in cardiomyocytes after the addition of 500 nM isoproterenol. The acquisition time was 20 seconds, and the frame rate was 14.23 frames / second. [Figure 13] Figure 13 shows photographs of cardiomyocytes observed under a microscope using G-plates, T-plates, and V-plates. The upper row shows photographs observed in bright field, and the lower row shows photographs of GCaMP fluorescence observed under a fluorescence microscope. The objective magnification for both the upper and lower rows is 4x. [Figure 14]Figure 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. [Figure 15] Figure 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. [Figure 16] Figure 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. [Figure 17] Figure 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. [Figure 18] Figure 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. [Figure 19] Figure 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. [Figure 20] Figure 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. [Figure 21] Figure 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. [Figure 22] Figure 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. [Figure 23]Figure 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. [Figure 24] Figure 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] 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. [Figure 26] FIG. 26 is a conceptual diagram for explaining CAD30 and CAD80. [Figure 27] 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. [Figure 28] 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. [Figure 29] 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. [Figure 30] 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. [Figure 31] Figure 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. [Figure 32] 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. [Figure 33]Figure 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. [Figure 34] 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. [Figure 35] 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. [Figure 36] 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. [Figure 37] 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. [Figure 38] 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. [Figure 39] 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. [Figure 40] 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. [Figure 41] 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. [Figure 42]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.

[0010] 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 represents intensity, the vertical axis on the right represents movement, and the horizontal axis represents time (frame), and the graphs show fluorescence. In Figures 7 to 9, the vertical axis represents intensity, the horizontal axis represents time (frame), and the graphs show fluorescence. In Figures 28, 30, 32, 34, 36, 38, 40, and 42, the vertical axis of A represents CAD30, and the vertical axis of B represents CAD80. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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 the 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 indicators 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. A 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).

[0012] [Method for evaluating the effect of drugs on cardiomyocytes] The drug in one embodiment 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 embodiment of the present invention allows for accurate evaluation of the effect on cardiomyocytes.

[0013] Isoproterenol is a nonselective beta-agonist with tachycardiac and inotropic effects. Pentamidine is an antibiotic with arrhythmia-inducing properties. Bepridil is a calcium channel blocker used to treat angina pectoris, but its side effects include potentially fatal ventricular arrhythmias known as torsade de pointes (TdP) and QT prolongation. Milrinone is a phosphodiesterase III inhibitor with tachycardiac and inotropic effects. Verapamil is an L-type calcium channel blocker. E-4031 is an hERG potassium channel blocker. Terfenadine is an antiallergic drug known to cause QT prolongation. Astemizole is an antiallergic 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 and has bradycardic effects.

[0014] 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, 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. In one embodiment of the present invention, 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 using 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 using 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 using conventional proarrhythmia models using cardiomyocytes differentiated from iPS cells are even more preferred, with bepridil, risperidone, terfenadine, and ranolazine being even more preferred.

[0015] 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.

[0016] The effects of a drug include the main effects and side effects of the drug. The main effect of a drug on cardiomyocytes is an effect that is originally expected from the pharmacological effects of the drug, such as bradycardic effect (negative chronotropic effect), tachycardic effect (positive chronotropic effect), cardiotonic effect (positive inotropic effect), and anaphylactic effect (negative inotropic effect). The main effect of a drug on cardiomyocytes is preferably a tachycardic effect or a bradycardic effect, since it can be detected by the method of the present invention even at low drug concentrations.

[0017] Side effects of a drug on cardiomyocytes refer to pharmacological effects of the drug that are unrelated to treatment or that 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 reentry arrhythmias. Myocardial injury may be irreversible or reversible myocardial injury.

[0018] 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 afterdepolarizations (EADs). 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.

[0019] A method according to one embodiment 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 less likely to be detected. Furthermore, substances that may be cardiotoxic in later in vivo tests 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 less likely to be detected. In particular, using a substance with reported or suspected in vivo cardiotoxicity as a drug allows for easier detection of cardiotoxicity than in vivo tests, and allows for the analysis of the mechanism of cardiotoxicity.

[0020] [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.

[0021] 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), trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), embryonic germ cells (EG cells), multipotent germline stem cells (mGS cells), induced pluripotent stem cells (iPS cells), and Muse cells (Multi-lineage differentiating Stress Enduring cells). Pluripotent stem cells are preferably ES cells or iPS cells.

[0022] 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 the protein-free cardiac differentiation (PFCD) method.

[0023] 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.

[0024] The origin of the cardiomyocytes is not particularly limited and may be mammalian, bird, 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.

[0025] 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.

[0026] Cardiomyocytes may be used in the form of single cells, cardiomyocyte clusters, or cardiomyocyte sheets.

[0027] Since oxygen can be efficiently supplied to the cardiomyocytes in the culture vessel (X), either adherent cardiomyocytes or floating cardiomyocytes may be used, but adherent cardiomyocytes are preferred.

[0028] [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.

[0029] 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.

[0030] 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.

[0031] 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 part or all of the bottom surface among the bottom surface, side surface, and top surface is formed from a base material containing a 4-methyl-1-pentene polymer. When at least a part or all of the bottom surface is formed from a base material containing a 4-methyl-1-pentene polymer, oxygen can be efficiently supplied to the culture medium via the 4-methyl-1-pentene polymer, facilitating efficient proliferation and differentiation of cells in the culture medium. Furthermore, it becomes easier to culture cells at high density while maintaining their functions.

[0032] 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, the entire bottom surface is preferably formed from a substrate containing a 4-methyl-1-pentene polymer.

[0033] 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 appropriately selected depending on the shape of the culture vessel, but by adjusting the thickness within the above range, it is easy to obtain an appropriate oxygen concentration in the culture medium necessary for cell proliferation and differentiation, and it is also easy to obtain sufficient strength as a culture vessel.

[0034] 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. Generally, culture vessels having a well-like depression on the bottom surface 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 surface is formed from a substrate containing a 4-methyl-1-pentene polymer, the shape is stable and sufficient oxygen is supplied to cells, even in plates having 1, 6, 12, 24, 48, 96, 384, 1536, or other wells.

[0035] 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), and a flat bottom with a curved edge. 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 by general injection molding or press molding, or it can be produced by first preparing 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 for two-dimensional cell culture, and a round bottom (U bottom) or conical bottom (V bottom) is usually desirable for three-dimensional cell culture.

[0036] 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.

[0037] 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 act 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.

[0038] The natural polymer material, synthetic polymer material, or inorganic material is not particularly limited, and 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.

[0039] The natural polymer material, synthetic polymer material, or inorganic material may be used after being processed by vitrification, etc. Examples of processing by vitrification, etc., 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.

[0040] 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.

[0041] 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 viewpoint of adhesiveness of cardiomyocytes, the coating agent is preferably diluted with a solvent such as water, PBS, or culture medium before use, 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 a special basal culture medium described later in the Examples.

[0042] After being used for coating, the coating agent may be removed from the culture vessel (X) or may be left in the culture vessel (X), but from the viewpoint of adhesiveness of cardiomyocytes, it is preferable to leave it in the culture vessel (X).

[0043] At least the culture surface of the culture vessel (X) may be processed. Examples of surface processing include surface modification such as forming an uneven structure, hydrophilization, and hydrophobicization.

[0044] 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 of two or more. When performing surface modification treatments, it is preferable to perform them on at least the culture surface.

[0045] It is preferable to subject at least the culture surface of the culture vessel (X) to hydrophilic treatment, and more preferably to corona treatment or plasma treatment. By subjecting the surface of the culture vessel (X) to hydrophilic 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 hydrophilic treatment, it becomes easier to coat a coating agent onto the culture surface of the culture vessel (X). In particular, it becomes easier to uniformly load and adhere the coating agent onto the culture surface of the culture vessel (X). Furthermore, even after 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.

[0046] The culture vessel (X) may be disinfected or sterilized to prevent contamination. The disinfection or sterilization method is not particularly limited, and examples thereof include physical disinfection methods such as steam circulation, boiling, intermittent irradiation, and ultraviolet light; chemical disinfection methods 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.

[0047] The culture surface of the culture vessel (X) has a water contact angle of preferably 50° or more, more preferably 55° or more, and even more preferably 60° or more. The culture surface of the culture vessel (X) has a water contact angle of preferably 100° or less, more preferably 90° or less, and even more preferably 84° or less.

[0048] By adjusting the water contact angle of the culture surface of the culture vessel (X) to be equal to or less than the upper limit or equal to or more than the lower limit, for example, cardiomyocytes can be more easily attached to the culture surface and more easily proliferate 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 close contact. 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, and can be used for cell culture while maintaining a stable initial state.

[0049] 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. The water contact angle can be measured, for example, in accordance with Japanese Industrial Standard JIS-R3257 (test method for wettability of substrate glass surface), by dropping a water droplet of 4 μL or less, which can be considered as a sphere, under constant temperature and humidity conditions of 25±5° C. and 50±10% onto the surface of a measurement sample made of the same material as the culture vessel (X), and measuring the angle of the contact interface between the measurement sample and the water droplet within 1 minute immediately after the water droplet contacts the surface of the measurement sample by the sessile drop method.

[0050] The method for producing the culture vessel (X) is not particularly limited, and neither is the equipment used for production. 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 a 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 a 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 a 4-methyl-1-pentene polymer and the other substrate may be integrally formed, or may be bonded to each other via an adhesive or pressure-sensitive adhesive.

[0051] Specific examples of methods for forming the film or sheet include the usual inflation method and T-die extrusion method. The production is usually carried out under heating. When the T-die extrusion method is used, the extrusion temperature is preferably 100°C to 400°C, and more preferably 200°C to 300°C. The roll temperature is preferably 45°C to 75°C, and more preferably 55°C to 65°C.

[0052] Alternatively, the film or sheet may 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 limitations on the solvent used, and hydrocarbon solvents such as cyclohexane, hexane, decane, and toluene may be used. Two or more solvents may 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.

[0053] [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."

[0054] A copolymer of 4-methyl-1-pentene and another monomer, which is an example of a 4-methyl-1-pentene polymer, 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 and is resistant to tearing, cracking, and bending even when used as a substrate.

[0055] The 4-methyl-1-pentene polymer is preferably at least one polymer selected from a 4-methyl-1-pentene homopolymer and a copolymer of 4-methyl-1-pentene with at least one olefin selected from ethylene and an α-olefin 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 an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0056] 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 appropriately selected depending on the physical properties required for the substrate. For example, from the viewpoints 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.

[0057] The olefins can be used alone or in combination of two or more. When two or more different olefins are combined, from the viewpoint of material strength, the olefins preferably have two or more carbon atoms, more preferably ten or more carbon atoms. When two or more different α-olefins are combined, 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.

[0058] 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 uniform culture surface with excellent processability can be obtained, and the substrate has a good balance between toughness and strength, resulting in less deflection.

[0059] The 4-methyl-1-pentene polymer may contain structural units other than the structural units derived from 4-methyl-1-pentene and the structural units derived from ethylene and the α-olefin 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 the 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.

[0060] Examples of monomers from which other structural units are derived include cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins, and halogenated olefins. Examples of the cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins, and halogenated olefins include the compounds described in paragraphs

[0035] to

[0041] of JP 2013-169685 A.

[0061] The 4-methyl-1-pentene polymers may be used alone or in combination of two or more.

[0062] 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 satisfy the above requirements can be preferably used. These commercially available products can be used alone or in combination of two or more.

[0063] 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 undergo hydrolysis 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 (usually 90% or higher) 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 for maintaining cardiomyocytes and are less likely to change 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.

[0064] 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 in GPC is preferably orthodichlorobenzene. Measurement conditions include, but are not limited to, those shown in the Examples below.

[0065] By setting the weight-average molecular weight (Mw) to the above upper limit or less, in the molding method of the 4-methyl-1-pentene polymer described below, the occurrence of defects such as gelling in the film produced by melt molding is easily suppressed, and a film with a uniform surface can be easily produced. Furthermore, when produced by a solution casting method, the solubility in a solvent is improved, and defects such as gelling in the film are easily suppressed, and a film with a uniform surface can be easily produced.

[0066] Furthermore, by setting the weight-average molecular weight (Mw) to the above-mentioned lower limit or more, 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 suppressed, and the toughness of the culture vessel tends to be sufficient, making it easier to suppress the occurrence of cracks during bending during molding and cutting.

[0067] 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 the 4-methyl-1-pentene polymer may be such that the respective Mw and Mw / Mn are within the above ranges.

[0068] 4-methyl-1-pentene polymer has an oxygen permeability coefficient of 100 to 2500 cm 3 ×mm / (m 2× 24h × atm), and 1000 to 2500 cm 3 ×mm / (m 2 × 24h × atm). When the oxygen permeability coefficient is within the above range, the cardiomyocytes maintain a good morphology, grow efficiently depending on the culture period, and detect the effects of drugs easily because of the excellent oxygen permeability.

[0069] Specifically, the oxygen permeability coefficient can be measured by the following method. A measurement sample is prepared from a substrate made of a 4-methyl-1-pentene polymer, and the oxygen permeability coefficient [cm ] at a temperature of 23°C and humidity of 0% is measured by a differential pressure gas permeability measurement method. 3 ×mm / (m 2 × 24h × atm)] is measured. The equipment used for the measurement is not particularly limited as long as it uses a differential pressure gas permeability measurement method, and an example is the differential pressure gas permeability measurement device MT-C3 manufactured by Toyo Seiki Seisakusho. The measurement sample is prepared by cutting a 90 × 90 mm test piece from a 50 μm thick substrate made of 4-methyl-1-pentene polymer, and the measurement section diameter is 70 mm (permeation area is 38.46 cm 2 Because the oxygen permeability is high, an aluminum mask is placed on the sample in advance to reduce the actual permeation area to 5.0 cm. 2 The measurement sample may or may not have undergone microfabrication or surface modification treatment, but it is preferable that no treatment has been performed. The oxygen permeability [cm 3 / (m 2 ×24h×atm)].

[0070] 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, and can be sterilized, making them extremely excellent culture vessels for culturing cardiomyocytes and evaluating the effects of drugs.

[0071] 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 present in the presence of the polymer to control the molecular weight or molecular weight distribution. The equipment used for the production is not limited. 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 viewpoint of polymerization efficiency, the polymerization 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.

[0072] When the substrate containing a 4-methyl-1-pentene polymer is a composition containing a 4-methyl-1-pentene polymer, the 4-methyl-1-pentene polymer is preferably contained in 100% by mass of the composition at 90% by mass or more but less than 100% by mass, more preferably at 95% by mass or more but less than 100% by mass, and particularly preferably at 99% by mass or more but less than 100% by mass. When 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.

[0073] [Process (A)] Step (A) is a step of seeding cardiomyocytes onto the culture surface of the culture vessel (X). The method for seeding 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), and then the culture vessel (X) is left to stand in an incubator.

[0074] The density at which cardiomyocytes are seeded is not particularly limited as long as the cardiomyocytes can be maintained or proliferated, but is preferably 0.1 × 10 5 cells / cm 2 ~10.0×10 5 cells / cm 2 and more preferably 0.3 × 10 5 cells / cm 2 ~5.0×10 5 cells / cm 2 and more preferably 0.5 × 10 5 cells / cm 2 ~3.0×10 5 cells / cm 2 is. 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.

[0075] The medium used in step (A) is not particularly limited as long as it allows cardiomyocytes to survive, and may be appropriately selected depending on the cell type used. Examples of media used for seeding include any basal cell culture medium, differentiation medium, and medium specifically designed for primary culture, such as serum-free medium (α), medium (β), Eagle's Miniature Essential Medium (EMMEM), 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 in the Examples), special basal medium, iCell Cardiomyocyte Maintenance Medium (FUJIFILM Cellular Dynamics), iCell Cardiomyocyte Thawing Medium (FUJIFILM Cellular Dynamics), and mixtures 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. Since cardiomyocytes tend to adhere to the culture vessel (X), the medium used in step (A) is preferably the iCell cardiomyocyte thawing medium, the serum-free medium (α) described later in the Examples, and more preferably the serum-free medium (α).

[0076] The amount of medium used in step (A) is not particularly limited and can be determined appropriately depending on the type of cell and medium. The culture conditions are not particularly limited and can be performed 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%.

[0077] [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, which is added as a nutrient necessary for cell survival during cell culture, and is also called Serum Protein Substitute (SPS), Serum Substitute Supplement (SSS), etc. The alternative serum is not particularly limited, and known alternative serums can be used. Commercially available alternative serums include Knockout Serum Replacement (ThermoFisher Scientific, No. 10828028), StemSure TM Examples include serum replacement (SSR, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), PL SOLUTION (manufactured by PL Bioscience), PL MATRIX (manufactured by PL Bioscience), and Artificial Serum (manufactured by 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).

[0078] The basal medium is not particularly limited as long as it is a medium for cell culture that does not contain animal serum. Examples of the basal medium include Grace's medium, IPL-41 medium, Schneider's medium, and Opti-PRO medium. TM SFM medium, Opti-MEM TM I medium, VP-SFM medium, CD293 medium, 293SFMII medium, CD-CHO medium, CHO-S-SFMII medium, FreeStyle TM 293 medium, CD-CHO, AGT TMExamples of suitable medium include 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.

[0079] The serum-free medium (α) is preferably a DMEM medium, an RPMI medium, or an MEM medium containing Knockout Serum Replacement, more preferably a DMEM medium containing Knockout Serum Replacement.

[0080] In addition to serum substitutes, the serum-free medium (α) may contain various growth factors, differentiation-inducing factors, antibiotics, hormones, amino acids, sugars, salts, minerals, metals, vitamins, and the like.

[0081] [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 carried out according to a known protocol.

[0082] Details of the medium used for the 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 is preferably the same as the medium used in step (A) because cardiomyocytes are more likely to proliferate therein.

[0083] 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 performed 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%.

[0084] 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.

[0085] The culture in step (B) may be either adherent culture or suspension culture, since oxygen can be efficiently supplied in the culture vessel (X), but adherent culture is preferred.

[0086] Steps (A) and (B) are preferably carried out in a serum-free medium (α). When steps (A) and (B) are carried out in a serum-free medium (α), cardiomyocytes tend to adhere to the culture vessel (X).

[0087] [Process (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 step (B). By including step (C), the effect of a drug can be easily evaluated with high accuracy. The method for culturing cardiomyocytes is not particularly limited, and may be carried out according to known protocols.

[0088] The medium used for culturing in step (C) is not particularly limited as long as it allows cardiomyocytes to survive, and may be appropriately selected depending on the cell type used. Examples of media used for culturing include any basal cell culture medium, differentiation medium, and medium specifically designed for primary culture, such as serum-free medium (α), medium (β) described below, Eagle's Miniature Essential Medium (EMMEM), 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 (Funakoshi), iCell Cardiomyocyte Maintenance Medium (FUJIFILM Cellular Dynamics), iCell Cardiomyocyte Thawing Medium (FUJIFILM Cellular Dynamics), and mixtures 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 the 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.

[0089] 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. Of 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).

[0090] The amount of medium used in step (C) is not particularly limited and can be determined appropriately depending on the type of cell and 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 performed 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%.

[0091] 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.

[0092] [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 it is a medium containing 1 to 100 μg / mL of free fatty acids.

[0093] 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.

[0094] 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.

[0095] The method for measuring the content of free fatty acids in the medium (β) is not particularly limited, and for example, the free fatty acid concentration can be measured by a method utilizing a coupled enzyme reaction.

[0096] The medium (β) preferably 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 content of lysophosphatidylcholine is more preferably 1 to 50 μg / mL, and even more preferably 1 to 20 μg / mL. The content of triacylglyceride is more preferably 1 to 50 μg / mL, and even more preferably 1 to 20 μg / mL. The content of phosphatidylcholine is more preferably 1 to 50 μg / mL, and even more preferably 1 to 25 μg / mL. The content of phosphatidic acid is more preferably 1 to 50 μg / mL, and even more preferably 1 to 10 μg / mL. The content of cholesterol is more preferably 0.1 to 8 μg / mL, and even more preferably 0.1 to 5 μg / mL. The content of sphingomyelin is more preferably 0.1 to 8 μg / mL, and even more preferably 0.1 to 5 μg / mL. When the medium (β) contains the lipids within the above ranges, the effects of drugs can be evaluated more accurately.

[0097] The method for measuring the content of the lipids in the medium (β) is not particularly limited, and the content can be measured by a known method.

[0098] [Process (D)] Step (D) is a step of exposing the cultured cardiomyocytes to the drug. The cultured cardiomyocytes are the cardiomyocytes cultured in step (B) if step (C) is not present, and are the cardiomyocytes cultured in step (C) if step (C) is present. 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.

[0099] 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 performed 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%.

[0100] Details of the medium used in step (D) are the same as the medium used in step (B) when step (C) is not present, and the same as the medium used in step (C) when 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 is preferably the same as the medium used in step (D) because this allows for accurate evaluation of the drug action.

[0101] The method according to one aspect of the present invention preferably includes step (C), and steps (C) and (D) are carried out in medium (β). When steps (C) and (D) are carried out in medium (β), the action of a drug, particularly cardiotoxicity, can be evaluated more accurately, and arrhythmogenicity, 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 accurately and arrhythmogenicity, particularly EAD-like waveforms, can be more easily detected.

[0102] [Process (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.

[0103] The method for analyzing indicators of cardiomyocyte cellular function is 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 clamping. 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 periphery using a motion analyzer. Sarcomere length can be analyzed, for example, by visualizing α-actinin present in sarcomeres with GFP labeling. The intracellular concentration of calcium ions or their 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, measurement of ATP levels, etc. Indicators of mitochondrial function can be analyzed, for example, by mitochondrial toxicity, for example, by a swelling assay as an indicator of mitochondrial permeability transition (MPT), or by measuring the activity of electron transport chain enzyme complexes. The concentrations or changes in the 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, and the like.

[0104] By analyzing calcium ion waveforms, it is possible to detect, for example, QT prolongation, bradycardia (negative chronotropic effect), tachycardia (positive chronotropic effect), inotropy (positive inotropy), weakness (negative inotropy), early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), torsades de pointes (TdP), triggered activity arrhythmias, or reentrant arrhythmias. In step (E), preferably, early after-depolarizations (EADs) are detected by analyzing calcium ion waveforms. Also, in step (E), preferably, tachycardia or bradycardia is detected by analyzing calcium ion waveforms.

[0105] [Other processes: Process (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 drug's effect. 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. [Example]

[0106] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0107] [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 (Tosoh Corporation) Data analysis software: Empower3 (Waters) Detector: Differential refractometer - Serially connected columns: TSKgelGMH6-HT (2 columns) and TSKgelGMH6-HTL (2 columns) Column temperature: 140℃ ·Flow rate: 1.0mL / min Sample concentration: 1.5mg / mL

[0108] [Production Example 1] Production of base material TPX (registered trademark), a 4-methyl-1-pentene polymer (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 α-olefins having 16 carbon atoms and α-olefins having 18 carbon atoms is 2.8 mol%) was used, and the base layer was extruded into a T-die extruder equipped with a full-flight screw, and the extrusion temperature was set to 270°C, the roll temperature to 60°C, and the roll rotation speed was varied to obtain a 50 μm thick film 1.

[0109] The film 1 was used as a measurement sample and measured for oxygen permeability coefficient [cm] under an environment of temperature 23°C and humidity 0% using a differential pressure gas permeability measuring device MT-C3 manufactured by Toyo Seiki Seisakusho. 3 ×mm / (m 2 × 24h × atm)] was measured. The measurement area was 70 mm (the transmission area was 38.46 cm 2 Because the oxygen permeability coefficient was expected to be large, an aluminum mask was placed on the sample in advance to reduce the actual permeation area to 5.0 cm. 2 The oxygen permeability coefficient was 1912 cm 3 ×mm / (m 2 ×24h×atm).

[0110] The film 1 was subjected to plasma treatment 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).

[0111] The water contact angle was measured using the 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, was dropped onto the surface of the measurement sample under constant temperature and humidity conditions of 25±5°C and 50±10%. The angle of the contact interface between the measurement sample and the water droplet was measured using the sessile drop method within 1 minute after the water droplet made contact with the measurement sample surface. The water contact angle of the plasma-treated film 1 was 60.3°.

[0112] [Production 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 container frame made of polystyrene (also called PS) 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 of ​​one well was approximately 0.32 cm. 2 It was.

[0113] As controls for the T-plate, a 96-well plate made of TCPS (Tissue Culture Polystylene) (CellBIND, Corning, product number 3300, also called "C-plate") and a 96-well plate made of PDMS (Polydimethylsiloxane) (VECELL, product number V96WGPB, also called "V-plate") with an oxygen permeability coefficient of 19121 [cm 3 ×mm / (m 2 × 24h × atm)], bottom thickness 350 μm) TCPS 96-well plate (Greiner, product number 655090, also referred to as "G plate") was used.

[0114] [Preparation of medium and reagents] We used a human iPS cell-derived cardiomyocyte GCaMP transfectant (Myoridge, product number G-011106) that had been cryopreserved in liquid nitrogen. These cells were 37 days old after initiating differentiation of human iPS cells using the protein-free cardiac differentiation (PFCD) method, and were transfected with a gene to constitutively express GCaMP as a calcium sensor.

[0115] The reagents used were as follows. All were stored refrigerated. CDI maintenance medium (iCell cardiomyocyte maintenance medium, FUJIFILM Cellular Dynamics, 557-33591) CDI Thawing / Planting Medium (iCell Cardiomyocyte Thawing Medium, FUJIFILM Cellular Dynamics, 550-33581)

[0116] Special maintenance medium (CarmyA Maintenance Medium UG, manufactured by Myoridge, ME-01A00241, supplied as a liquid medium and supplements, which must be 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. CarmyA special seeding medium (CarmyA seeding medium kit UG, manufactured by Myoridge, ME-02A00211. Corresponds to serum-free medium (α)) Special basal medium (serum-free medium included in CarmyA Seeding Medium Kit UG, manufactured by Myoridge, ME-02A00211)

[0117] iMatrix-511silk (product number 387-10131, manufactured by Nippi) CultureSure TM Y-27632 (product number 034-24024, Fujifilm Wako Pure Chemical Industries, Ltd., lot number KCG7025)

[0118] Isoproterenol (Tokyo Chemical Industry Co., Ltd.) Bepridil (Kissei Pharmaceutical Co., Ltd.) Pentamidine (Kissei Pharmaceutical Co., Ltd.) E-4031 Kissei Pharmaceutical Co., Ltd. Verapamil (Kissei Pharmaceutical Co., Ltd.) Risperidone (Fujifilm Wako Pure Chemical Industries, Ltd.) Terfenadine (Sigma-Aldrich) Ranolazine (manufactured by Tokyo Chemical Industry Co., Ltd.) DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0119] [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).

[0120] [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: Cryopreservation of cardiomyocytes, pre-coating of plates, seeding onto 96-well plates for testing, and culturing Days 1-5: Culture medium was replaced with 150 μL of CDI maintenance medium every other day. Days 7-13: Culture medium was replaced with 200 μL of CDI maintenance medium every other day. Day14: Gene Expression Analysis

[0121] <iMatrix-511silk Coating Method for Test Plates> 70 μL of iMatrix-511silk was added to 9 mL of PBS(-) and diluted to make a coating solution. The coating solution was added to a 96-well test plate at 100 μL / well (use concentration 1.21 μg / cm 2 ) and left to stand in a 37°C incubator for 1 hour. Before seeding the cells, it was removed by aspiration.

[0122] <Revival 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. It was used after warming to 37°C at the time of use. Frozen cardiomyocytes were thawed in a 37°C water bath and suspended in the thawing medium. Centrifuged at 300×g for 5 minutes and the supernatant was removed. The cells were suspended in the thawing medium, adjusted to 2×10 6 ~5×10 6 cells / mL, the cell count was performed, and the cells were seeded at 8×10 4 cells / well in a 96-well test plate coated with iMatrix-??511silk and left to stand in a 37°C, 5% CO2 incubator. The experiment was performed in triplicate.

[0123] <RNA Extraction and RT-qPCR> cTnT, MYL2, Kir2.1, PGC1α (PPARGC1A) and GAPDH were used as analysis targets.

[0124] (1) Reagents and Instruments RNA extraction: miRNeasy Mini Kit (product number 217004, manufactured by Qiagen) cDNA synthesis: ReverTraAce(R) qPCR RT MasterMix with gDNA Remover (product number FSQ-301, manufactured by TOYOBO) qPCR reaction: PowerUp SYBRGreen MasterMix (product number A25776, ThermoFisher) QuantStudio6 Flex Real-time PCR system (ThermoFisher) Nanophotometer spectrophotometer C40 (manufactured by Wakembie Tech Co., Ltd.)

[0125] (2) RNA extraction After removing the culture supernatant from the 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.

[0126] (3) RT-qPCR Using 253 ng of the extracted RNA, cDNA was synthesized by reverse transcription according to the protocol provided with the PowerUpSYBRGreen MasterMix. Then, 6 ng of cDNA was used for qPCR using the standard method. Five standard curves were prepared by pooling 10 μL of each 10 ng / μL cDNA sample and diluting them 1 / 10.

[0127] <Evaluation of gene expression> Gene expression levels were analyzed using RNA extracted from the cells 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.

[0128] [Table 1]

[0129] [Table 2]

[0130] (result) The results of analyzing gene expression levels are shown in Figure 1. Gene expression levels are expressed as relative values, with the gene expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) set at 1, and are shown as ±SD (n = 3). When T plates were used (Reference Example 2), the expression levels of cTnT, MYL2, Kir2.1, and PGC1α (PPARGC1A) were higher than when C plates were used (Reference Example 1). These results demonstrate that culturing cardiomyocytes using T plates increases the expression of cardiomyocyte-specific genes and can improve cardiomyocyte function.

[0131] [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 them, 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: Cryopreservation of cardiomyocytes, pre-coating of plates, seeding onto 96-well plates for testing (step (A)), and culturing (step (B)) Day 1: Replace the medium with CDI maintenance medium and culture (Step (C)) Day 4-13: Culture by replacing the medium with a special maintenance medium every two days (Step (C)) Day 14: Using a special maintenance medium, drug addition and calcium transient analysis (step (D) and step (E))

[0132] <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 applied at 100 μL / well (working concentration 1 μg / cm). 2) into a 96-well test plate and allowed to stand in an incubator at 37°C for 1 hour. Without removing the coating solution, cardiomyocytes were seeded with the coating solution remaining.

[0133] <Awakening and seeding of frozen cardiomyocytes> Y-27632 was added to CarmyA 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 × g for 5 minutes, and the supernatant was removed. The cells were suspended in the special thawing and seeding medium and collected at 2 × 10 6 ~5×10 6 After adjusting the concentration to cells / mL, the number of cells was counted and 6 × 10 cells were placed in a 96-well test plate coated with iMatrix-511silk. 4 Cells were seeded at 200 μL / well and placed in a 37°C, 5% CO2 incubator. Experiments were performed in triplicate.

[0134] <Drug addition> 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. Examples 1 to 9 were prepared by adding bepridil to a T plate 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. Comparative Examples 1 to 9 were prepared by adding bepridil to C plate 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.

[0135] (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 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.

[0136] The addition of 0.1% DMSO slightly increased the fluorescence intensity in both the T-plate and C-plate, but did not affect the calcium ion waveform (Figure 2).

[0137] 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 clearly (Figure 5), and when the bepridil 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).

[0138] Bepridil is a calcium antagonist 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, EAD-like waveforms that trigger torsade depointes (TdP) were detected, demonstrating that the proarrhythmic effect of bepridil can be detected.

[0139] When 11.1 μM pentamidine was added, arrhythmia was detected 24 hours after addition on the T plate (Example 4) (FIG. 7A), but not on the C plate (Comparative Example 4) (FIG. 7B). When the pentamidine concentration was increased to 33.3 or 100 μM, arrhythmia was not detected even on the T plate (Examples 5 and 6, FIGS. 8A and 9A). When 100 μM pentamidine was added to the C plate (Comparative Example 6), acute arrhythmia was detected 10 minutes later (FIG. 9B).

[0140] Pentamidine is an antibacterial drug known to induce arrhythmias, and its mechanism of action is thought to be through the inhibition of membrane trafficking of the hERG (human ether-a-go-go related gene) protein. Therefore, prolonged exposure to pentamidine is required for its proarrhythmic effects to manifest. 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.

[0141] 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 also stably confirmed in the C plate (Comparative Examples 8 and 9) (Figures 11 and 12).

[0142] Isoproterenol is a β1, β2 receptor agonist and has the effect of increasing heart rate (tachycardiogenic effect). Compared to C-plates, T-plates were able to stably detect the effect of increasing heart rate (tachycardiogenic effect) even at low concentrations of isoproterenol. T-plates are thought to be able to evaluate the effects of drugs with higher sensitivity than C-plates.

[0143] [Reference Examples 3, 4, and 5] Study of test plates with high oxygen permeability (method) The influence on cardiomyocyte culture was examined using test plates made of PDMS, which have high oxygen permeability. The test plates used were G plates (made of TCPS, Reference Example 3), T plates (made of Reference Example 4), and V plates (made of PDMS, Reference Example 5).

[0144] <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 and seeding medium, frozen cardiomyocytes are thawed, pre-coated on plates, seeded onto 96-well plates for testing, and cultured. Day 1: Culture medium was replaced with special maintenance medium Day 3-13: Culture with special maintenance medium every two days. Day 6: Microscopic observation

[0145] Coating of the test plate with iMatrix-511silk, waking and seeding of 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 bright field microscope and photographed. They were also observed under a fluorescent microscope and video and still images were taken.

[0146] (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 synchronized pulsation-like waveforms. 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 no synchronized pulsation-like waveforms.

[0147] 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.

[0148] [Examples 10 to 17, Comparative Examples 10 to 17] Examination of culture media (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-coat the plate with a special basal medium, and then thaw and seed the frozen cardiomyocytes using a special thawing and seeding medium. Then seed them onto a 96-well plate for testing (step (A)), and culture them (step (B)). Day 1: Replace the medium with special maintenance medium or CDI maintenance medium and culture (Step (C)) Day 3 to 13: Culture by replacing the medium with special maintenance medium or CDI maintenance medium every two days (Step (C)) Day 14: Medium change, drug addition, calcium transient analysis (step (D), step (E))

[0149] <Culture medium> The test plates were coated with iMatrix-511silk in a special basal medium. The frozen cardiomyocytes were thawed and seeded in a special seeding medium. The medium used for medium replacement, drug addition, and calcium transient analysis after day 1 of seeding was a special maintenance medium or CDI maintenance medium.

[0150] <Drug addition> 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 T plates, special maintenance medium was used as the medium from the first day after seeding, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively. Examples 10 to 13 were used, and CDI maintenance medium was used as the medium from the first day after seeding, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively. Examples 14 to 17 were used. On the G plate, special maintenance medium was used as the medium from the first day of seeding 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 the first day of seeding 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 the first day of seeding onwards, and bepridil was added to final concentrations of 0.06 μM, 0.25 μM, 1 μM, and 4 μM, respectively.

[0151] (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 drug addition, and Post refers to after drug addition.

[0152] When 0.1% DMSO was added, the fluorescence intensity increased slightly in the T plate but did not affect the calcium ion waveform (Figure 14). However, in the G plate, a disturbance in the calcium ion waveform was observed (bottom of the special maintenance medium Post in Figure 15). The pulsatile 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 on the T plate from day 1 onwards, the pulsatile waveform was detected more stably and the signal was stronger than when the CDI maintenance medium was used.

[0153] No disturbance in the calcium ion waveform was detected when bepridil was added at 0.06 μM or 0.25 μM on the T plate (Examples 10, 11, 14, and 15) or when bepridil was added at 0.06 μM or 0.25 μM on the G plate (Comparative Examples 10, 11, 14, and 15) (Figures 16, 17, 18, and 19).

[0154] When special maintenance medium was used on T plates after day 1 of seeding, adding 1 μM bepridil (Example 12) resulted in a weak EAD (early after depolarization)-like waveform (Figure 20).When CDI maintenance medium was used on T plates after day 1 of seeding and adding 1 μM bepridil (Example 16), no EAD-like waveform was detected, but QT prolongation was detected (Figure 20). When special maintenance medium was used on G plates from day 1 onward, the addition of 1 μM bepridil (Comparative Example 12) resulted in a disruption of calcium ion waveforms, with no detectable pulsatile waveforms, and neither EAD-like waveforms nor QT prolongation was detected, although this varied depending on the well. When CDI maintenance medium was used on G plates from day 1 onward, 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 (Figure 21).

[0155] 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 (FIG. 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 (FIG. 23).

[0156] [Examples 18 to 21] Examination of medium replacement and verapamil immediately before drug addition Using T-plates as test plates, we investigated the effect of medium change immediately before drug addition. We also used T-plates as test plates to examine whether the effect of verapamil on cardiomyocytes could be evaluated. (method) <Exam Schedule> Day 1: Reagent preparation and setup Day 0: Pre-coat T-plates with a special basal medium, and then thaw and seed the frozen cardiomyocytes using a special thawing and seeding medium. Then seed them onto a 96-well plate for testing (step (A)), and culture them (step (B)). Day 1: Change the medium to the special maintenance medium and culture (Step (C)) Day 4 - 13: Change the medium to the special maintenance medium every two days and culture (Step (C)) Day 14: Change the medium, add the drug, and perform calcium transient analysis (Steps (D), (E))

[0157] <Details of Day 14> <Medium change> Medium change A: Remove the special maintenance medium in the plate and add a new special maintenance medium. Medium change B: Remove the special maintenance medium in the plate, add a new special maintenance medium, immediately remove it, and then add a new special maintenance medium again.

[0158] <Drug addition> To the plate subjected to medium change A or B, add the drug (verapamil) approximately 30 minutes after the medium change. The drug was dissolved in DMSO to prepare a 10 mM stock solution, and the 10 mM stock solution was diluted with the special maintenance medium to a concentration 10 times the target final concentration to obtain the drug solution for addition. The drug solution for addition was added to the measurement medium at a volume of one-tenth of the measured medium volume. For example, when adding verapamil at a final concentration of 10 nM, the 10 mM stock solution was diluted with the 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 the measurement medium. DMSO was added to the control group at a final concentration of 0.1%.

[0159] Those subjected to medium change A or B and added with verapamil at a final concentration of 10 nM were designated as Example 18 and Example 19, respectively. Those subjected to medium change A or B and added with verapamil at a final concentration of 100 nM were designated as Example 20 and Example 21, respectively.

[0160] <Calcium transient analysis> Perform calcium transient analysis 10 - 30 minutes after the drug addition.

[0161] (Results) The results of adding verapamil to a final concentration of 10 nM are shown in Figure 24, and the results of adding verapamil to 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 change A, Figure 24B shows the results before medium change B, Figure 24C shows the results after medium change A, and Figure 24D shows the results after medium change B. Figure 25A shows the results before medium change A, Figure 25B shows the results before medium change B, Figure 25C shows the results after medium change A, and Figure 25D shows the results after medium change B.

[0162] When 10 nM verapamil was added, no change in the waveform was observed in either medium exchange A or B (Figure 24). When 100 nM verapamil was added, in either medium exchange 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 inotropy) of verapamil was detected (Figure 25).

[0163] Verapamil is a calcium ion channel blocker. It is known to have a negative inotropic effect (which reduces the calcium ion concentration in cardiomyocytes and reduces the contractility of cardiomyocytes) and a QT shortening effect. Verapamil is a drug that has a low risk of clinical TdP, and is also predicted to have a low risk of TdP in conventional proarrhythmic models 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.

[0164] No difference was observed between medium changes A and B.

[0165] [Examples 22 to 53] Examination of other drugs Next, using T plates as test plates and a special maintenance medium as the medium from day 1 onwards, we investigated whether the effects of other drugs on cardiomyocytes could be evaluated. In Reference 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. However, there are exceptional drugs, namely, 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 embodiment 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. 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.

[0166] (method) <Exam Schedule> Day 1: Reagent preparation and setup Day 0: Pre-coat T-plates with a special basal medium, and then thaw and seed the frozen cardiomyocytes using a special thawing and seeding medium. Then seed them onto a 96-well plate for testing (step (A)), and culture them (step (B)). Day 1: Replace the medium with a special maintenance medium and culture (Step (C)) Day 4-13: Culture by replacing the medium with a special maintenance medium every two days (Step (C)) Day 14: Medium change, drug addition, calcium transient analysis (step (D), step (E))

[0167] <Drug addition> The drug was added in the same manner as in Examples 18 to 21. Those to which verapamil was added to a final concentration of 60 nM, 250 nM, 1000 nM, and 4000 nM were designated as Example 22, Example 23, Example 24, and Example 25, respectively. Those to which E-4031 was added to a final concentration of 1 nM, 10 nM, 100 nM, and 1000 nM were designated as Example 26, Example 27, Example 28, and Example 29, respectively. Those to which bepridil was added to a final concentration of 60 nM, 250 nM, 1 μM, and 40 μM were designated as Example 30, Example 31, Example 32, and Example 33, respectively. Those to which bepridil was added to a final concentration of 312 nM, 1.25 μM, 5 μM, and 20 μM were designated as Example 34, Example 35, Example 36, and Example 37, respectively. Those to which risperidone was added to a final concentration of 1 nM, 10 nM, 0.1 μM, and 1 μM were designated as Example 38, Example 39, Example 40, and Example 41, respectively. Those to which risperidone was added to a final concentration of 60 nM, 250 nM, 1 μM, and 4 μM were designated as Example 42, Example 43, Example 44, and Example 45, respectively. Those to which trifluoperazine was added to a final concentration of 0.25 nM, 1 nM, 4 nM, and 16 nM were designated as Example 46, Example 47, Example 48, and Example 49, respectively. Those to which ranolazine was added to a final concentration of 10 nM, 100 nM, 1000 nM, and 10000 nM were designated as Example 50, Example 51, Example 52, and Example 53, respectively.

[0168] <Calcium transient analysis> Calcium transient analysis was performed in the same manner as in Examples 18 to 21.

[0169] <CAD30 and CAD80> CAD30 (CaD30, calcium transient duration at 30%) and CAD80 (CaD80, calcium transient duration at 80%) were calculated from the waveforms obtained by calcium transient analysis. The time when the minimum peak intensity value 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 when 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 when a 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.

[0170] (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 60 nM and 250 nM verapamil inhibited the increase in intensity. 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-attenuating effect (negative inotropic effect) and QT shortening effect of verapamil.

[0171] (About 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 clinical risk of TdP and is also predicted to have a high risk of TdP in conventional proarrhythmic models using cardiomyocytes differentiated from iPS cells. 29 and 30, QT prolongation was observed from 10 nM, and was more pronounced at 100 nM, 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.

[0172] (About Bepridil) Bepridil is a calcium channel blocker originally approved as an antianginal drug, but is also classified as a Class 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 by adding 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 bepridil concentration range. As shown in Figures 33 and 34, QT prolongation was detected by adding 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.

[0173] (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.

[0174] 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. At 250 nM in particular, EAD-like waveforms were also detected (arrow in Figure 37). The method according to one embodiment of the present invention was able to detect the proarrhythmic effect of risperidone.

[0175] (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.

[0176] 39 and 40, no concentration-dependent QT prolongation was detected with the addition of 0.25 nM to 16 nM terfenadine. However, at 4 nM, an abnormal waveform was detected (arrow in FIG. 39). The method according to one embodiment of the present invention was able to detect the proarrhythmic effect of terfenadine.

[0177] (About ranolazine) Ranolazine is a sodium ion channel blocker. Although ranolazine has a low risk of causing arrhythmia in vivo, it is known to cause QT prolongation in cardiomyocytes differentiated from iPS cells, leading to its being judged as having proarrhythmic effects. In other words, ranolazine is known to be judged as a false positive in conventional proarrhythmic models using cardiomyocytes differentiated from iPS cells.

[0178] 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 embodiment of the present invention, ranolazine was not evaluated as having a proarrhythmic effect. That is, according to the method of one embodiment 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.

[0179] Examples 30 to 53 demonstrate that one aspect of the present invention makes it possible to detect the effects of drugs that were previously difficult to detect using conventional in vitro test systems using cardiomyocytes differentiated from iPS cells.

[0180] <Incorporated 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 entireties.

Claims

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 (C) of culturing the cardiomyocytes obtained in the step (B) for an additional 3 to 30 days; (D) exposing the cultured cardiomyocytes to the drug; and 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. 2. The method of claim 1, wherein steps (A) and (B) are carried out in a serum-free medium (α) containing a serum replacement.

3. 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).

4. The method according to claim 1 or 2, wherein the entire culture surface of the culture vessel is formed from a base material containing a 4-methyl-1-pentene polymer.

5. The method according to claim 1 or 2, wherein the culture surface is coated with a coating agent containing laminin.

6. The method according to claim 1 or 2, wherein the cardiomyocytes are cardiomyocytes differentiated from induced pluripotent stem cells.

7. 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.

8. 3. The method of claim 1 or 2, wherein the effect is cardiotoxicity.

9. 9. The method of claim 8, wherein the cardiotoxicity is proarrhythmic.

10. The method of claim 8, wherein the cardiotoxicity is myocardial damage.

11. The method of claim 10 , wherein the indicator of cellular function is an indicator of mitochondrial function.

12. The method according to claim 1 or 2, wherein the indicator of cell function is a calcium ion waveform.

13. The method of claim 1 or 2, wherein in step (E), early after-depolarizations (EADs) are detected.

14. 3. The method of claim 1 or 2, wherein the effect is a tachycardic or bradycardic effect.

15. 15. The method of claim 14, wherein the indicator of cellular function is a calcium ion waveform.

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