JP2023062597A - Cardiotoxicity evaluation method - Google Patents

Abstract

To provide a cardiotoxicity evaluation method with high accuracy.SOLUTION: Provided is a cardiotoxicity evaluation method comprising: culturing cardiomyocytes in a medium in a culture vessel, in which at least the culture surface is composed of an alicyclic structure-containing polymer, until the beating of the cardiomyocytes is confirmed; subsequently adding an agent to be evaluated to the medium to contact the agent to the cultured cardiomyocytes; and subsequently monitoring by calcium imaging the beating of the cultured cardiomyocytes to evaluate the cardiotoxicity of the agent.SELECTED DRAWING: Figure 2

Description

TECHNICAL FIELD The present invention relates to a method for evaluating cardiotoxicity, and more particularly to a method for evaluating drug-induced cardiotoxicity of a new drug for prediction of side effects and determination of dosage to humans before clinical trials.

In recent years, studies have progressed on cardiotoxicity evaluation methods (MEA methods) using MEA (Micro Electrode Arrays). This evaluation method is based on the knowledge that the FPD (Field Potential Duration) appearing in the waveform of the extracellular action potential obtained from the myocardial cell sheet by MEA corresponds to the QT prolongation in the electrocardiogram.
However, the above evaluation method has the problem that the operation is complicated and it takes time to obtain the data.

In view of the above problems, the present inventors measured biomarkers of heart disease secreted from cardiomyocytes using a culture vessel made of an alicyclic structure-containing polymer to determine the cardiotoxicity of the drug to be evaluated. We propose a method of evaluation. (Patent Document 1, Patent Document 2). According to such a cardiotoxicity evaluation method, since it is not necessary to prepare a cardiomyocyte sheet, the operation is simplified and the time required to obtain data can be shortened.

By the way, many methods using cell image analysis have been developed as methods for observing cultured cells, and are also used for observing cultured cardiomyocytes (for example, Patent Document 3).

WO2020/066396 WO 2020/129774 Japanese Patent Application Laid-Open No. 2014-076000

However, as a result of further studies by the present inventors, it was found that data varies between cells provided by different providers (research institutions/manufacturers, etc.), which can affect the accuracy of evaluation. In addition, it was found that even with the same provider, variations in data similarly occur due to differences in production lots of cells.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described circumstances, and an object of the present invention is to reduce the influence of cells used for evaluation and to provide a highly accurate cardiotoxicity evaluation method.

The present invention has been made based on the above findings, and an object of the present invention is to advantageously solve the above problems. One aspect of the present invention is that at least the culture surface is made of an alicyclic structure-containing polymer. Cardiomyocytes are cultured in a culture medium in the constructed culture vessel until pulsation of the cardiomyocytes is confirmed, then a drug to be evaluated is added to the culture medium, and the drug is cultured in the cardiomyocytes. and then monitoring the heartbeat of the cultured cardiomyocytes by calcium imaging to evaluate the cardiotoxicity of the drug.
Such a method reduces the variation in data due to individual differences in cells (differences between cells/cell groups caused by differences in culture environments and cell sources), and enables highly reproducible measurements. Furthermore, in addition to being able to be easily evaluated in a short time, a large amount of data can be obtained by one measurement.

Moreover, in the above aspect, the cardiotoxicity is preferably evaluated by the QT prolongation rate.
Here, the QT prolongation rate refers to the rate at which the pulsatile waveform is elongated (or shortened) in the horizontal direction due to the toxicity of the drug to be evaluated. Greater variability (positive or negative) in the rate of QT prolongation.

Moreover, in the above aspect, it is preferable that the alicyclic structure-containing polymer is a hydrogenated norbornene-based ring-opening polymer. This is because, among the alicyclic structure-containing polymers, by forming the culture surface of the culture vessel with a hydrogenated norbornene-based ring-opening polymer, variations in measured values are further reduced.

Further, in the above aspect, the surface free energy of the culture surface is preferably 30 to 37 mN/m. By doing so, the cells can be allowed to adhere to the culture surface without applying unnecessary stress to the cells, thereby further suppressing variations in the measured values.
Here, the surface free energy is a criterion determined by measuring how much ink or the like adheres to the surface of plastic or metal. The unit is mN/m, and the higher the surface free energy, the higher the adhesiveness.

ADVANTAGE OF THE INVENTION According to this invention, the influence which the cell used for evaluation has can be reduced and a highly accurate cardiotoxicity evaluation method can be provided.

FIG. 1 is a diagram explaining a method of calculating the interbeat distance (D' _0.1 ), which is required for calculating the QT prolongation rate. FIG. 2 is a graph comparing drug (E-4031) concentrations at which QT prolongation (+) was observed. FIG. 3 is a graph comparing drug (Nifedipine) concentrations at which QT prolongation (-) was observed.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described in detail.
In the method of the present invention, cardiomyocytes are cultured in a medium in a culture vessel having at least a culture surface composed of an alicyclic structure-containing polymer until pulsation of the cardiomyocytes is confirmed. A drug to be evaluated is added to the drug, the drug is brought into contact with the cultured cardiomyocytes, and then the heartbeat of the cultured cardiomyocytes is monitored by calcium imaging to evaluate the cardiotoxicity of the drug. Let it be one.
By using such an evaluation method, variations in data due to differences in cells used for evaluation, manufacturers, and production lots can be reduced. Therefore, even in long-term tests using many drugs, highly accurate evaluation can be performed without being affected by differences in the cells used.

(monitoring)
The method of monitoring cardiomyocyte pulsation by calcium imaging is appropriately selected depending on the device to be employed. Cardiomyocyte beating can be confirmed by acquiring and continuously observing the Ca ²⁺ signal.

(cardiotoxicity evaluation)
Evaluation of cardiotoxicity is preferably based on the QT prolongation rate.
The QT prolongation rate is calculated from data obtained by calcium imaging (pulsation chart: vertical axis (Y) is signal intensity, horizontal axis (X) is time (seconds)) as shown below and in FIG. .
(1) Baseline correction: The minimum value (“5” in FIG. 1) within one beat of the beat chart is subtracted from the entire beat chart to set the baseline to zero (vertical axis: Y′).
(2) Normalization: Correction is made so that the maximum value of the vertical axis becomes 1 (vertical axis: Y'').
(3) Calculation of distance between beats (D′ _0.1 ): Calculate the distance between two points on the beat chart that cross Y″=0.1.
Specifically, when deriving quadratic functions of curves centered at two points of intersection between the pulse chart and Y''=0.1, and substituting 0.1 for Y in the two quadratic functions are set to X1 and X2 in descending order, and the difference between them (X2-X1) is calculated to calculate the distance between the two points (distance between beats).
(4) Calculate the value of the interbeat distance (D' _0.1 ) before and after the addition of the drug,
QT prolongation rate = (interbeat distance after drug addition/interbeat distance before drug addition) x 100
Calculate and evaluate as
For more precise evaluation, the value of the interbeat distance (D' _0.1 ) obtained under each condition (drug concentration) is calculated from the interbeat peak distance using the Fridericia correction formula described later. Calculate the "corrected QT interval" by correcting,
QT prolongation rate = (corrected QT interval after drug addition/corrected QT interval before drug addition) x 100 and evaluated.

(Cells and their culture method)
Cells used in the present invention are cardiomyocytes, and examples thereof include pluripotent stem cell-derived cardiomyocytes (particularly induced pluripotent stem cell-derived cardiomyocytes), embryonic stem cell-derived cardiomyocytes, and human-derived cardiomyocytes. be done.

In the present invention, "confirming that the cardiomyocytes are beating (culturing until)" means that almost all of the seeded cardiomyocytes in the culture container can be confirmed to be beating. cells need not be synchronized. From the viewpoint of the accuracy of evaluation results, it is more preferable that the pulsation of the cultured cells as a whole be synchronized.
From this, in the present invention, the timing of "adding the drug to be evaluated to the medium" is the time at which cardiomyocyte beating is observed, which is described in the attached culture protocol, in the case of commercially available cardiomyocytes. It is preferable that the cells are cultured as above.

The medium for culturing the above-mentioned cells is not particularly limited as long as the cardiomyocytes can be cultured and maintained, and a commercially available cardiomyocyte culture medium can be used.
Additives can also be added to the medium. Additives include minerals, metals, vitamin components, and the like.
These additives can be used singly or in combination of two or more.

There is no particular limitation on the method of seeding the cells in the culture vessel. For example, at least the culture surface is coated with an extracellular matrix or the like, if necessary. After that, the cells suspended in the medium are seeded in the culture vessel using a pipette or the like, and the vessel is shaken as necessary to evenly scatter the cells in the culture vessel, and then left standing in the incubator. Extracellular matrices include, for example, naturally-derived or synthetic materials such as gelatin, fibronectin, vitronectin, and laminin.

(Culture vessel)
As the culture vessel of the present invention, any shape can be used as long as the alicyclic structure-containing polymer can be used as the material for the culture vessel. Shapes of culture vessels include dishes, plates, microchannel chips, bags, tubes, scaffolds, cups, jars and fermenters.
At least the culture surface of the culture vessel should be composed of the alicyclic structure-containing polymer. For example, in the case of a 96-well plate, the inner bottom surface of each well may be formed of an alicyclic structure-containing polymer. In the case of a bag, for example, when it is composed of a laminate of films made of different polymer materials, the innermost layer (inner surface of the bag) may be formed of a film made of an alicyclic structure-containing polymer.
Alternatively, the entire culture vessel may be made of the alicyclic structure-containing polymer. For example, in the case of culture dishes, flasks, or plates having a plurality of wells, the entire container can be made of the alicyclic structure-containing polymer by molding the entire container with the alicyclic structure-containing polymer. .
The alicyclic structure-containing polymer is preferably hydrogenated norbornene ring-opening polymer.

The culture vessel used in the present invention is preferably sterilized.
There are no particular restrictions on the sterilization method, and heating methods such as high-pressure steam and dry heat methods; radiation methods such as gamma rays and electron beams, and irradiation methods that irradiate high frequencies; ethylene oxide gas (EOG). Filtration method using a sterilizing filter; and other methods generally employed in the medical field can be selected according to the shape of the molded body and the cells to be used. Among them, the gas method is preferable because the surface free energy of the culture surface can be easily maintained within a specific range.

(Surface free energy of culture surface)
The surface free energy of the culture surface of the culture vessel used in the present invention is preferably in the range of 30-37 mN/m. In order to control the surface free energy of the culture surface within the above range, it is important not to subject the culture surface to surface treatment such as plasma treatment, which is usually performed to improve cell adhesiveness. The surface free energy is preferably in the range of 32-36 mN/m.

The surface free energy is evaluated by measuring whether several test inks, each having a corresponding surface free energy value in a given range, can maintain adhesion to the culture surface for a given time. be done. More specifically, if the line of ink drawn on the culture surface does not change for 2 seconds without forming water droplets, the value of the surface free energy of the culture surface is the same as the value of the surface free energy imparted to the ink used. Means equal or greater than. The ink components are mixtures of organic solvents (2-ethoxyethanol, 2-propanol) and basic dyes (formamide), and these liquids are mixed in different ratios to produce inks with different surface free energy values. to make.
As a specific method for measuring the surface free energy, for example, first, using ink to which a surface free energy value of 38 mN/m is applied, a line is drawn on the measurement surface, and the line of ink does not become a water droplet. If it does not change for a second, the surface free energy is equal to or greater than 38 mN/m. In this case, next, similar measurements are performed using ink with a surface free energy value of 40 mN/m. If the line of ink does not turn into a drop and does not change for 2 seconds, then such measurements are made using higher valued inks until the line of ink turns into a drop within 2 seconds.
In the above measurement, if the ink is already transformed into water droplets at about 38 mN/m, the same measurement is performed using ink having a value lower than 38 mN/m. For example, a line is drawn on the measurement surface with ink having a surface free energy value of 35 mN/m. values (35-38 mN/m).

(Culture surface coating)
The method of coating the culture surface of the culture vessel with the extracellular matrix is the same as the method of coating the culture vessel with a general cell substrate. A method is adopted in which the culture surface is allowed to stand for usually 10 minutes to 5 hours, preferably 30 minutes to 2 hours, the coating agent is brought into contact with the culture surface, and then the coating agent is removed. If the contact time is too short, the coating will be insufficient. On the other hand, protein adsorption to the alicyclic structure-containing polymer that constitutes the culture surface is low, and unlike polystyrene, the adsorption amount does not increase even if the contact time is lengthened. Therefore, it is not necessary to increase the contact time beyond the above mentioned time. In addition, after removing the coating agent, it is desirable to quickly add the medium in order to prevent drying.

Since the culture surface composed of the alicyclic structure-containing polymer easily repels the aqueous solution, the amount of the coating agent added to the culture vessel is 1.5 to 1.5 times the amount of the coating agent added to the general polystyrene cell culture vessel. It is desirable to add about 3 times more, and specifically, it is preferable to add 0.15 to 0.30 ml to 1 cm ² of the culture surface.

(Alicyclic structure-containing polymer)
The alicyclic structure-containing polymer is a resin having an alicyclic structure in its main chain and/or side chains, and from the viewpoint of mechanical strength, heat resistance, etc., preferably contains an alicyclic structure in its main chain. From the viewpoint of differentiation induction efficiency, those having no polar group are more preferable. Here, the polar group refers to a polar atomic group. Polar groups include amino groups, carboxyl groups, hydroxyl groups, acid anhydride groups, and the like.

Examples of the alicyclic structure include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. structures are preferred, and those having a cycloalkane structure are most preferred.

Although the number of carbon atoms constituting the alicyclic structure is not particularly limited, it is usually 4-30, preferably 5-20, more preferably 5-15. When the number of carbon atoms constituting the alicyclic structure is within this range, the mechanical strength, heat resistance, and moldability are well balanced, which is preferable.

The proportion of repeating units having an alicyclic structure in the alicyclic structure-containing polymer may be appropriately selected depending on the purpose of use, but is usually 30% by weight or more, preferably 50% by weight or more, more preferably 70% by weight. % or more. If the proportion of repeating units having an alicyclic structure in the alicyclic structure-containing polymer is too low, the heat resistance is poor, which is undesirable. The remainder other than the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer is not particularly limited and is appropriately selected according to the purpose of use.

Specific examples of alicyclic structure-containing polymers include (1) norbornene-based polymers, (2) monocyclic cyclic olefin-based polymers, (3) cyclic conjugated diene-based polymers, and (4) vinyl alicyclic carbonization. Examples include hydrogen-based polymers and hydrides of (1) to (4). Among these, norbornene-based polymers and their hydrides are preferred from the viewpoint of heat resistance, mechanical strength, and the like.

(1) Norbornene-based polymer Norbornene-based polymer is obtained by polymerizing a norbornene-based monomer, which is a monomer having a norbornene skeleton, and is obtained by ring-opening polymerization and addition polymerization. It is roughly divided into things.

Products obtained by ring-opening polymerization include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers capable of ring-opening copolymerization thereof, and these and hydrides. Products obtained by addition polymerization include addition polymers of norbornene-based monomers and addition polymers of norbornene-based monomers with other monomers copolymerizable therewith. Among these, ring-opening polymer hydrides of norbornene-based monomers are preferable from the viewpoint of heat resistance, mechanical strength and the like.

Norbornene-based monomers that can be used in the synthesis of norbornene-based polymers include bicyclo[2.2.1]hept-2-ene (common name norbornene), 5-methyl-bicyclo[2.2.1]hepta -2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, 5-ethylidene-bicyclo[2 .2.1]hept-2-ene, 5-vinyl-bicyclo[2.2.1]hept-2-ene, 5-propenylbicyclo[2.2.1]hept-2-ene, 5-methoxycarbonyl -bicyclo[2.2.1]hept-2-ene, 5-cyanobicyclo[2.2.1]hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo[2.2.1]hepta -bicyclic monomers such as 2-ene;
tricyclo[4.3.0 ^1,6 . 1 ^2,5 ]deca-3,7-diene (commonly known as dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, and other tricyclic monocyclic Ammer;
Tetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene (tetracyclododecene), tetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 8-methyltetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 8-ethyltetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 8-ethylidenetetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 8,9-dimethyltetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 8-ethyl-9-methyltetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 8-ethylidene-9-methyltetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 8-methyl-8-carboxymethyltetracyclo[4.4.0.1 ^2,5 . 1 ^7,10 ]-3-dodecene, 7,8-benzotricyclo[4.3.0.1 ^2,5 ]dec-3-ene (common name methanotetrahydrofluorene: 1,4-methano-1,4 , 4a,9a-tetrahydrofluorene), 1,4-methano-8-methyl-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a,9a- Tetrahydrofluorene, tetracyclic monomers such as 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene;

Other monomers capable of ring-opening copolymerization with norbornene monomers include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, Monocyclic cycloolefin monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatetraene can be mentioned.
These monomers may have one or more substituents. Examples of substituents include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, an alkylidene group, and the like.

Other monomers capable of addition copolymerization with norbornene monomers include α-olefin monomers having 2 to 20 carbon atoms such as ethylene, propylene, 1-butene, 1-pentene and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo[9.2.1.0 ^2,10 . cycloolefinic monomers such as 0 ^3,8 ]tetradeca-3,5,7,12-tetraene (also referred to as 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); non-conjugated diene monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, 1,7-octadiene;

Among these monomers, α-olefin monomers are preferable, and ethylene is more preferable as other monomers capable of being addition-copolymerized with norbornene monomers.
These monomers may have one or more substituents. Examples of substituents include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, an alkylidene group, and the like.

A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and other monomers capable of ring-opening copolymerization with the monomer component is prepared by a known ring-opening polymerization. It can be obtained by polymerization in the presence of a catalyst. The ring-opening polymerization catalyst includes, for example, a metal halide such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide such as titanium, zirconium, tungsten, molybdenum, or acetylacetone. A catalyst consisting of a compound and an organoaluminum compound can be used.
Ring-opening polymer hydrides of norbornene-based monomers are usually obtained by adding a known hydrogenation catalyst containing transition metals such as nickel and palladium to the polymerization solution of the ring-opening polymer to form a carbon-carbon unsaturated bond. can be obtained by hydrogenating

Addition polymers of norbornene-based monomers, or addition polymers of norbornene-based monomers and other monomers copolymerizable therewith are obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst. It can be obtained by polymerization. As the addition polymerization catalyst, for example, a catalyst comprising a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

(2) Monocyclic cyclic olefin polymer As the monocyclic cyclic olefin polymer, for example, addition polymers of monocyclic cyclic olefin monomers such as cyclohexene, cycloheptene and cyclooctene can be used. can.
(3) Cyclic conjugated diene-based polymer Examples of cyclic conjugated diene-based polymers include polymers obtained by 1,2- or 1,4-addition polymerization of cyclic conjugated diene-based monomers such as cyclopentadiene and cyclohexadiene; Its hydride and the like can be used.
(4) Vinyl Alicyclic Hydrocarbon Polymer Examples of the vinyl alicyclic hydrocarbon polymer include polymers of vinyl alicyclic hydrocarbon monomers such as vinylcyclohexene and vinylcyclohexane, and hydrogenated products thereof; styrene; , hydrides of aromatic ring portions of polymers of vinyl aromatic monomers such as α-methylstyrene; The vinyl alicyclic hydrocarbon polymer may be a copolymer of these monomers and other copolymerizable monomers.

There are no particular restrictions on the molecular weight of the alicyclic structure-containing polymer, but the weight-average molecular weight in terms of polyisoprene measured by gel permeation chromatography of a cyclohexane solution (or a toluene solution if the polymer does not dissolve) is usually 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, particularly preferably 10,000 to 100,000. When the weight-average molecular weight is within this range, mechanical strength and moldability are highly balanced, which is preferable.

The glass transition temperature of the alicyclic structure-containing polymer may be appropriately selected depending on the purpose of use, but is usually 50 to 300°C, preferably 100 to 280°C. When the glass transition temperature is within this range, heat resistance and moldability are highly balanced, which is preferable.
The glass transition temperature of the alicyclic structure-containing polymer in the invention is measured according to JIS K7121.

The alicyclic structure-containing polymer may be used alone or in combination of two or more.
In addition, the alicyclic structure-containing polymer may contain compounding agents commonly used in thermoplastic resin materials, such as soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, and release agents. , coloring agents such as dyes and pigments, plasticizers, antistatic agents, optical brighteners, and other compounding agents can be added in amounts normally employed.
Further, the alicyclic structure-containing polymer may be mixed with a polymer other than the soft polymer (hereinafter simply referred to as "other polymer"). The amount of the other polymer to be mixed with the alicyclic structure-containing polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, more preferably 100 parts by mass with respect to 100 parts by mass of the alicyclic structure-containing polymer. It is below.
If the ratio of various compounding agents and other polymers to be blended with the alicyclic structure-containing polymer is too high, the cells will be difficult to float. is preferred.
The method of mixing the alicyclic structure-containing polymer with the compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Moreover, there is no particular restriction on the order of blending. Examples of the compounding method include a method of kneading the resin in a molten state using a mixer, a single-screw kneader, a twin-screw kneader, a roll, a Brabender, an extruder, or the like; Examples include a method of removing the solvent by a coagulation method, a casting method, or a direct drying method.
When using a twin-screw kneader, after kneading, it is usually extruded in a molten state into a rod shape, cut into appropriate lengths with a strand cutter, and pelletized for use in many cases.

The method for forming the container composed of the alicyclic structure-containing polymer can be arbitrarily selected according to the desired shape of the culture container. Examples of molding methods include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendar molding, and roll molding. , cutting molding, spinning, etc., and these molding methods can be combined, and post-treatment such as stretching can be carried out as required after molding.

EXAMPLES The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples.

(Example I)
Culture vessel Zeonex (registered trademark) 790R (manufactured by Nippon Zeon Co., Ltd., norbornene-based ring-opening polymer hydride: hereinafter simply referred to as "790R") was used as the alicyclic structure-containing polymer, and injection molding was performed to obtain a A well plate containing 96 cylindrical wells with a bottom area of 32 cm ² was obtained as a culture vessel. Thereafter, the culture vessel was sterilized with ethylene oxide gas (hereinafter, this culture vessel is referred to as "790R 96-well plate"). The surface free energy of the well inner bottom surface of the 790R 96-well plate (the surface in contact with cells, that is, the culture surface) was 34 mN/m. Here, the surface free energy was measured using a surface energy value evaluation ink (manufactured by Arcotest).

Coating To each well of a 790R 96-well plate, 150 μL of a human fibronectin (manufactured by Corning, Model No. 356008) solution adjusted to 10 μg/mL using sterilized water was added and allowed to stand at 37° C. for 2 hours. The human fibronectin solution was removed from each well, and the inner bottom surface of the 790R 96-well plate was coated with human fibronectin.

Cell culture iCell (registered trademark) Cardiomyocytes ² (manufactured by FUJIFILM Cellular Dynamics, model number C1016) was suspended in the attached cardiomyocyte thawing medium (manufactured by FUJIFILM Cellular Dynamics, model number M1001) to obtain 7.5 × 10 ⁴ cells. /well, and cultured for 4 hours at 37°C in a 5% _CO2 atmosphere.
After that, the cardiomyocyte thawing medium was removed from each well of the 790R 96-well plate, and 150 μL of cardiomyocyte maintenance medium (manufactured by FUJIFILM Cellular Dynamics, model number M1003) was newly added and placed at 37° C. in a 5% CO ₂ atmosphere. was cultured in
Two days after seeding the cells, half of the cardiomyocyte maintenance medium in each well of the 790R 96-well plate was replaced.

Reagent adjustment Three days after seeding the cells, 10 μL of dimethyl sulfoxide (DMSO) (manufactured by Nacalai Tesque, model number 09659-14) was added to one commercially available reagent tube of Cal520 (manufactured by AAT Bioquest, model number 21130). 490 μL of sterilized water was added to prepare a Cal520 solution.
Pluronic F-127 (manufactured by BTI, model number 59000) was adjusted to 1 mg/mL with sterilized water to prepare a Pluronic F-127 solution.
A calcium staining solution was prepared using a cardiomyocyte maintenance medium (manufactured by FUJIFILM Cellular Dynamics, model number M1003) so that the Cal520 solution was 1% and the Pluronic-127 solution was 0.2%.

Data Acquisition Next, the culture supernatant was removed from each well of a 790R 96-well plate, and 150 μL of the above calcium staining solution was added to each well and allowed to stand at 37° C. for 1 hour to stain myocardial cells with calcium. After that, using a confocal quantitative image cytometer CQ1 (manufactured by Yokogawa Electric Corporation, hereinafter simply referred to as "CQ1"), the cardiomyocytes in each well were photographed at 20 fps (frames per second) for 30 seconds. , 600 image data were obtained, and the pulsation of cardiomyocytes was confirmed.
Next, E-4031 (manufactured by TOCRIS, model number 1808: HERG potassium channel blocker) solution was diluted with DMSO, 16.6 μL was added to predetermined wells of a 790R 96-well plate, and E-4031 in the wells. The final concentration was adjusted to 0.1 μM. Similarly, serial dilutions were performed to prepare final concentrations of E-4031 of 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM and 0.001 μM, and 16.6 μL was added to predetermined wells. rice field.
Samples of each final concentration were set to n=3, and the plate containing the samples was allowed to stand at 37° C. in a 5% CO ₂ atmosphere for 1 hour. After that, using CQ1, cardiomyocytes in each drug-added well were imaged at 20 fps for 30 seconds to obtain 600 image data.
600 images taken before and after adding the drug were combined to obtain moving image data.

Evaluation After that, using image analysis software ImageJ (manufactured by Wayne Rasband), the fluorescence brightness of the above video data was quantified, and a pulsation chart for each well (vertical axis: fluorescence brightness, horizontal axis: time (seconds)) was created. bottom.
Next, the minimum value within one beat of this pulsation chart was subtracted from the entire pulsation chart to correct the baseline to zero. Then, the distance between beats (D' _0.1 ) was calculated by normalizing the values on the vertical axis so that the maximum value was 1. Before and after the addition of E-4031, the QT prolongation rate (prolongation rate of interbeat distance) was determined at each concentration, and the standard deviation was calculated from the average of n=3 samples for each concentration. Table 1 shows the results.

(Example 2)
The above "culture vessel" to "evaluation" were performed in the same manner as in Example 1, except that Nifedipine (manufactured by Tokyo Chemical Industry Co., Ltd., model number N0528: calcium channel blocker) was used instead of E-4031. . Table 1 shows the results.
In addition, in E-4031 of Example 1, the QT prolongation rate is elongated in the positive direction with respect to the horizontal axis by its addition, whereas Nifedipine is exposed at a high concentration (for example, 0.010 μM or more ), the rate of QT prolongation is negatively prolonged (ie shortened).

(Example 3)
The above "culture vessel" to "evaluation" were performed in the same manner as in Example 1, except that the cell lot (manufacturing lot) of iCell (registered trademark) Cardiomyocytes ² was changed to CMC-105153. Table 1 shows the results.

(Example 4)
The above "culture vessel" to "evaluation" were performed in the same manner as in Example 1, except that the cell lot of iCell (registered trademark) Cardiomyocytes ² was changed to CMC-105256. Table 1 shows the results.

(Comparative example 1)
In the same manner as in Example 1, except that a polystyrene 96-well plate (Falcon (registered trademark), manufactured by Corning, model number 353916, surface free energy: 43 mN / m) was used instead of the 790R 96-well plate. "Coating" to "Evaluation" were performed. Table 1 shows the results.

(Comparative example 2)
The above "coating" to "evaluation" were performed in the same manner as in Example 2 (using Nifedipine) except that a polystyrene 96-well plate was used instead of the 790R 96-well plate. Table 1 shows the results.

From the results in Table 1, in the example using the 790R 96-well plate made of the alicyclic structure-containing polymer, the variation in the QT prolongation rate before and after the addition of the drug was higher than in the comparative example using the polystyrene 96-well plate. is extremely small.

By the way, Ando H et al., "A new paradigm for drug-induced torsadogenic risk assessment using human iPS cell-derived cardiomyocytes." Journal of Pharmacological and Toxicological Methods, Mar-Apr 2017;84:111-127. Table 3-1 summarizes the +10% QT prolongation, +30% QT prolongation, and -10% QT prolongation concentrations for each drug.

Therefore, from the interbeat distance (D' _0.1 ) obtained in Examples 1, 3 and 4 using E-4031 and Comparative Example 1,
Fridericia's correction formula:
Corrected QT interval = interbeat distance (D' _0.1 )/beat peak to top distance ^1/3
was used to calculate the corrected QT interval, and the concentration of E-4031 at which +10% QT prolongation was observed and the concentration (μM) of E-4031 at which +30% QT prolongation was observed were calculated, respectively. The results are shown in FIG.
The “beat peak-to-top distance” refers to the distance between the peaks of each beat in a pulsation chart in which multiple beats are recorded. Calculated by described methods and known methods.
In this example, with respect to the above-mentioned "corrected QT interval", the concentration of the drug at which QT prolongation is observed by 10% compared to before addition of the drug is represented as +10% QT prolongation concentration, and 30% compared to before addition of the drug. The concentration of the drug that causes QT prolongation by 30% is designated as the +30% QT prolongation concentration, and the concentration of the drug that produces -10% QT prolongation is designated as the -10% QT prolongation concentration.

Similarly, in Example 2 and Comparative Example 2 using Nifedipine, the corrected QT interval was similarly calculated from the obtained interbeat distance (D' _0.1 ), and -10% QT prolongation was observed. Nifedipine concentration was calculated. The results are shown in FIG.

For comparison, the +10% QT prolongation concentration data (Free conc. FPDcF10) of E-4031 (siteD) and the +30% QT prolongation concentration data (Free conc. FPDcF30) was extracted and shown in FIG. 2 as a reference example (comparison standard data).
Similarly, data of −10% QT prolongation concentration of Nifedipine (Free conc. FPDc-10) was extracted and shown in FIG. 3 as a reference example (comparison standard data).

2 and 3, in the example using the 790 96-well plate, the drug concentrations at which QT prolongation rates of +10%, +30%, and -10% were observed were significantly higher than the drug concentrations derived by the MEA method. is found to be correlated with On the other hand, in a comparative example using a well plate having a culture surface made of polystyrene, no such correlation was observed.
From the above, when monitoring pulsations by calcium imaging, it is possible to determine with high accuracy the drug concentration at which a predetermined QT prolongation is observed by using a plate whose culture surface is an alicyclic structure-containing polymer. It could be confirmed. In addition, it was confirmed that by using the QT prolongation rate as an evaluation index, the effects caused by individual differences in cells can be minimized and accurate cardiotoxicity evaluation can be performed.

Claims (4)

Cardiomyocytes are cultured in a medium in a culture vessel in which at least the culture surface is composed of an alicyclic structure-containing polymer until pulsation of the cardiomyocytes is confirmed, and then
A drug to be evaluated is added to the medium, the drug is brought into contact with the cultured cardiomyocytes, and then
A cardiotoxicity evaluation method, comprising monitoring the heartbeat of the cultured cardiomyocytes by calcium imaging to evaluate the cardiotoxicity of the drug. The cardiotoxicity evaluation method according to claim 1, wherein the cardiotoxicity is evaluated by QT prolongation rate. 3. The cardiotoxicity evaluation method according to claim 1, wherein the alicyclic structure-containing polymer is a hydrogenated norbornene ring-opening polymer. The cardiotoxicity evaluation method according to any one of claims 1 to 3, wherein the culture surface has a surface free energy of 30 to 37 mN/m.

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