JP2021129518A - Cardio toxicity evaluation method - Google Patents

Abstract

To provide a cardio toxicity evaluation method capable of performing correct evaluation at a small cell amount.SOLUTION: A foothold for adhering droplets formed in a medium including cardiac muscle cell is formed to a culture surface, then, the droplets are discharged to the foothold for adhering the droplets to the culture surface for inoculating the cardiac muscle cell to a culture vessel with the medium, then a medicine is added to the culture vessel, then the medicine is brought into contact with the cardiac muscle cell, then, variation of a pulsation number of the cardiac muscle cell is measured for evaluating cardio toxicity of the medicine. In the cardio toxicity evaluation method, at least the culture surface of the culture vessel is formed of an alicyclic structure including polymer, and surface free energy of the culture surface is 30-37 mN/m.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for evaluating drug-induced cardiotoxicity for predicting side effects and determining the dose to humans before clinical trials with respect to a novel drug, in particular, the beating of myocardial cells caused by the administration of the drug. It relates to a method of assessing cardiotoxicity by measuring changes in numbers.

One of the methods for diagnosing heart disease is a method for measuring the number of heartbeats of cardiomyocytes. Patent Document 1 describes that in vitro differentiated cardiomyocytes are used for drug screening to evaluate their efficacy and toxicity. Further, Patent Document 2 describes that cells are accurately ejected to a ejection destination by a droplet ejection device using an inkjet technique developed as a printing apparatus.

Special Table No. 2007-537429 Japanese Unexamined Patent Publication No. 2015-158489

However, in general biomarker measurement for drug screening, a predetermined number or more of cells are required in order for the biomarker to be expressed to a measurable level by an analytical instrument. Therefore, analysis by a multi-well plate such as a 96-well plate requires a large number of cells such as the required number of cells × the number of wells.

Further, even when the droplet ejection device ejects the droplet containing the cell to the ejection destination such as each well of the multi-well plate for analysis, each droplet is placed at the ejected position. So far, it has not been considered to fix and analyze as it is.

The present invention has been made in view of the above-mentioned circumstances, and an object of the present invention is to provide a cardiotoxicity evaluation method capable of accurately evaluating with a small amount of cells.

The present inventor uses a culture vessel composed of (i) an alicyclic structure-containing polymer and having a surface free energy of the culture surface within a specific range, and (ii) with respect to the culture surface of the culture vessel. By forming a scaffold for adhesion of droplets formed in a medium containing myocardial cells, ejecting the droplets onto the scaffold, and fixing the droplets on the culture surface, a small amount of cells can be used. We have found that accurate cardiotoxicity evaluation is possible, and have completed the present invention.
Here, the surface free energy is a standard determined by measuring how much ink or the like adheres to the surface of plastic or metal. The unit is expressed in mN / m, and the higher the value of the surface free energy, the higher the adhesiveness.

The present invention has been made based on the above findings, and an object of the present invention is to solve the above problems advantageously. One aspect of the present invention is a droplet formed in a medium containing myocardial cells. A scaffold for adhering the cells is formed on the culture surface, and then the droplets are ejected onto the scaffold to attach the droplets to the culture surface, whereby myocardial cells are seeded in the culture vessel together with the medium. Then, a drug is added into the culture vessel, the drug is brought into contact with the myocardial cells, and then the change in the beat rate of the myocardial cells is measured to evaluate the cardiotoxicity of the drug. This is a cardiotoxicity evaluation method in which at least the culture surface of the culture container is composed of an alicyclic structure-containing polymer and the surface free energy of the culture surface is 30 to 37 mN / m.
By doing so, the droplets formed in the medium containing cardiomyocytes stably adhere to the scaffold, so that the droplets do not collapse. Therefore, tests and analyzes such as drug screening can be performed for each droplet in a state where a constant cell density is maintained.

Further, in the above aspect, it is preferable that the cardiomyocyte is a pluripotent stem cell-derived cardiomyocyte. In particular, artificial pluripotent stem cell-derived cardiomyocytes are preferable. This is because there are no ethical problems and a stable supply can be expected, so even if the measurement takes a long period of time, highly accurate measurement can be performed.

In the above aspect, it is preferable that a plurality of the scaffolds are formed in a dot shape with respect to one culture surface.
By doing so, for example, when the culture surface is one well of a multi-well plate (for example, a 96-well plate), a plurality of scaffolds are formed in one well so as not to contact each other, and each scaffold is formed. By arranging the droplets in, a plurality of tests can be performed for each droplet. This allows multiple tests to be performed in one well.

Further, in the above aspect, it is preferable that the scaffold is formed of a material having a peptide sequence having an affinity for the alicyclic structure-containing polymer.
By doing so, the scaffolding of the cells can be designed according to the type of cardiomyocytes, and the adhesion efficiency of the cardiomyocytes to the culture surface can be improved.
Since the alicyclic structure-containing polymer has a hydrophobic surface (has a predetermined surface free energy), it repels a material having a peptide sequence or an aqueous solution containing cardiomyocytes, so that droplets do not spread to other than the target location. .. Therefore, the adhesion of the droplets to the culture surface can be stabilized.

Further, in the above aspect, it is preferable that the alicyclic structure-containing polymer is a norbornene-based ring-opening polymer hydride. Among the alicyclic structure-containing polymers, by forming the culture surface of the culture vessel with the norbornene-based ring-opening polymer hydride, the efficiency of adhesion of cardiomyocytes to the culture surface can be further enhanced.

According to the present invention, it is possible to provide a cardiotoxicity evaluation method that can be accurately evaluated with a small amount of cells.

FIG. 1 is a graph showing the number of beats per minute of cardiomyocytes.

Hereinafter, embodiments of the present invention will be described in detail.
In the method of the present invention, in measuring the change in the number of beats of myocardial cells, a scaffold is formed on the culture surface, droplets of a medium containing myocardial cells are adhered onto the scaffold, and the surface of the inner surface of the culture vessel in which the cells are in contact ( Hereinafter, the surface free energy of the “culture surface”) is in a specific range, and the culture surface is characterized in that a culture container composed of an alicyclic structure-containing polymer is used.
By doing so, the test can be performed in units of droplets adhering to the culture surface via the scaffold, and thereby accurate cardiotoxicity evaluation can be performed even with a small number of cells. Further, even when the number of cells is sufficient, the number of tests (n number) can be increased many times with the same amount of cells.

In the present invention, the cells used are cardiomyocytes, for example, pluripotent stem cell-derived cardiomyocytes (in particular, artificial pluripotent stem cell-derived cardiomyocytes), embryonic stem cell-derived cardiomyocytes, human-derived cardiomyocytes, and the like. Can be mentioned.

In the present invention, the scaffold is for attaching droplets formed in a medium containing cardiomyocytes to a predetermined position on the culture surface of the culture vessel.
The scaffold is preferably formed of a material having a peptide sequence that has an affinity for an alicyclic structure-containing polymer (particularly, a norbornene-based ring-opening polymer hydride). Specifically, for example, it is preferable to use oligopeptides searched by the method described in WO 2018/117242. Examples of the oligopeptide used include, but are not limited to, oligopeptides containing the amino acid sequence of Thr-Val-Asp-Ser-Cys-Leu-Thr (SEQ ID NO: 1).
When used as a scaffold material, the concentration of the oligopeptide is preferably 1 to 500 μg / mL, more preferably 10 to 100 μg / mL because of good cell adhesion.

It is preferable that a plurality of scaffolds are formed in a dot shape with respect to one culture surface. The size of each dot forming the scaffold is preferably 100 to 1000 μm. When the size of the dots is smaller than 100 μm, when the droplets are ejected to the plurality of scaffolds formed on the plurality of culture surfaces (each well of the multi-well plate), the first droplet ejected is the amount of the liquid. This is because it dries while ejecting droplets to other scaffolds due to the small amount of water. Further, when the dot size exceeds 1000 μm, the scaffold becomes larger than necessary with respect to the size of the droplet. When the size of the droplet is adjusted to the scaffold, the number of cells per droplet increases, so that a large number of cells are required for the test. Therefore, the number of tests that can be performed per cell surface is reduced. More preferably, the size of each dot is 400-800 μm. The reason is that the size can maximize the number of tests performed at one time within the range where the droplets do not dry out. From the viewpoint of aligning the test conditions, it is preferable that the size of each dot is the same, but the size is not limited to this.

The number of dot-shaped scaffolds formed per culture surface is, for example, when a 96-well plate is used as a culture vessel, the number of dot-shaped scaffolds formed on the bottom surface (culture surface) of the wells is When the distance between the centers of the dots is 800 μm, it can be a maximum of 19, preferably 15 to 16 dots, but is not limited to this. According to the number of tests, each dot may be formed at a distance between the centers where the formed dot-shaped scaffolds do not contact each other.

In the present invention, the medium for forming droplets containing cardiomyocytes is not particularly limited as long as the cardiomyocytes can be cultured and maintained, and a commercially available medium for cardiomyocyte culture can be used. can.
Additives can also be added to the medium. Examples of the additive include minerals, metals, vitamin components and the like.
These additives can be used alone or in combination of two or more.

The total number of cells contained in the droplets composed of the medium containing cardiomyocytes is preferably 100 to 5000 cells / dot, which is the total number of droplets attached to one scaffold (dot). This is because if the number of cells is less than 100 cells / dot, the cells are likely to be scattered. Further, when the number of cells exceeds 5000 cells / dot, the space in which the cells can beat decreases, so that the number of beats decreases, which affects the assay result. More preferably, it is 200 to 1000 cells / dot because the space in which the cells can beat becomes an appropriate size. The size and amount of the droplets may be adjusted to the size of the scaffold, and are not particularly limited. For example, if the diameter of the dot (scaffold) is 600 μm, using LaboJet®-500Bio (manufactured by Microjet Jet), if droplets containing cells are ejected 54 times (32 per time). .4 nL), the surface of dots of the above size can be filled with droplets.

In the present invention, droplets are ejected onto the scaffold formed on the culture surface. The scaffolding material and droplets can be discharged by, for example, a discharge device (inkprint device) described in Japanese Patent Application Laid-Open No. 2015-158489, but the present invention is not limited to this, and for example, LaboJet (registered trademark)-. A device capable of ejecting scaffolding material and droplets at a predetermined position and in a predetermined amount, such as 500Bio (manufactured by Microjet Jet Co., Ltd.), may be used.

In the present invention, the cardiotoxicity of a drug is evaluated by the change in the pulsatile number of cardiomyocytes before and after administration of the drug or with or without administration. As a result, a plurality of samples in one well can be accurately evaluated with a small number of cells. Specifically, the change in the pulsatile number of cardiomyocytes is performed by measuring the pulsatile number of the myocardium by imaging. For example, calcium imaging (for example, confocal quantitative image cytometer: CellVoyager (registered trademark) CQ1 (manufactured by Yokogawa Electric Co., Ltd.)), live cell imaging (for example, live cell imaging system SI-8000 series (manufactured by Sony Corporation)). ) Etc., but the number of beats of cardiomyocytes can be measured, but the present invention is not limited to these.

In the present invention, the culture vessel is preferably a 96-well plate in consideration of its use for drug screening, but the culture vessel is not limited to this, and an alicyclic structure-containing polymer can be used as a material for the culture vessel. If there is, any shape can be used. Examples of the shape of the culture vessel include dishes, plates, microchannel chips, bags, tubes, scaffolds, cups, jars and fermenters, and the like.
Of the culture vessels, at least the culture surface may be composed of an alicyclic structure-containing polymer. For example, in the case of a 96-well plate, the bottom surface inside each well may be formed of an alicyclic structure-containing polymer. In the case of a bag, for example, in the case of a bag 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 an alicyclic structure-containing polymer. For example, in the case of a culture dish, a flask, or a plate having a plurality of wells, the entire container can be composed of the alicyclic structure-containing polymer by molding the entire container with the alicyclic structure-containing polymer. ..

The alicyclic structure-containing polymer is a resin having an alicyclic structure in the main chain and / or side chain, and preferably contains an alicyclic structure in the main chain from the viewpoint of mechanical strength, heat resistance, and the like. 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. Examples of the polar group include an amino group, a carboxyl group, a hydroxyl group, an acid anhydride group and the like.

Examples of the alicyclic structure include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. From the viewpoint of mechanical strength and heat resistance, the cycloalkane structure and the cycloalkane structure can be mentioned. The structure is preferable, and the one having a cycloalkane structure is most preferable.

The number of carbon atoms constituting the alicyclic structure is not particularly limited, but is usually 4 to 30, preferably 5 to 20, and more preferably 5 to 15. When the number of carbon atoms constituting the alicyclic structure is within this range, the mechanical strength, heat resistance, and moldability characteristics are highly balanced and suitable.

The ratio of the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer may be appropriately selected according to 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 excessively small, the heat resistance is poor, which is not preferable. The rest of the polymer containing an alicyclic structure other than the repeating unit having an alicyclic structure is not particularly limited and is appropriately selected according to the purpose of use.

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

(1) Norbornene-based polymer The 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 or addition polymerization. It is roughly divided into things.

As a product obtained by ring-opening polymerization (COP: cycloolefin polymer), a ring-opening polymer of a norbornene-based monomer and a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization thereof are opened. Examples include ring polymers and hydrides thereof. Examples of the product obtained by addition polymerization (COC: cycloolefin copolymer) include an addition polymer of a norbornene-based monomer and an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith. Can be mentioned. Among these, a ring-opening polymer hydride of a norbornene-based monomer is preferable from the viewpoint of heat resistance, mechanical strength and the like.

Examples of norbornene-based monomers that can be used in the synthesis of norbornene-based polymers include bicyclo [2.2.1] hepta-2-ene (common name: norbornene) and 5-methyl-bicyclo [2.2.1] hepta. -2-ene, 5,5-dimethyl-bicyclo [2.2.1] hepta-2-ene, 5-ethyl-bicyclo [2.2.1] hepta-2-ene, 5-ethylidene-bicyclo [2] .2.1] Hept-2-ene, 5-vinyl-bicyclo [2.2.1] hepta-2-ene, 5-propenylbicyclo [2.2.1] hepta-2-ene, 5-methoxycarbonyl -Bicyclo [2.2.1] hepta-2-ene, 5-cyanobicyclo [2.2.1] hepta-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 (trivial name dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, etc. Quantum;
Tetracyclo [4.4.0.1 ^2,5 . ^{17 and 10} ] -3-dodecene (tetracyclododecene), tetracyclo [4.4.0.1 ^2,5 . ^{17 and 10} ] -3-dodecene, 8-methyltetracyclo [4.4.0.1 ^2,5 . ^{17 and 10} ] -3-dodecene, 8-ethyltetracyclo [4.4.0.1 ^2,5 . ^{17 and 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 . ^{17 and 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 ] deca-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; and the like.

Other monomers that can be ring-opened and copolymerizable with norbornene-based monomers include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, and 1,5-cyclodecadene. Examples thereof include monocyclic cycloolefin-based monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatelane.
These monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, an alkylidene group and the like.

Other monomers that can be additionally copolymerized with the norbornene-based monomers include α-olefin-based 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.} 0 ^3,8 ] Cycloolefin monomers such as tetradeca-3,5,7,12-tetraene (also called 3a, 5,6,7a-tetrahydro-4,7-methano-1H-indene); 1, Non-conjugated diene monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, 1,7-octadene; and the like can be mentioned.

Among these, as the other monomer that can be additionally copolymerized with the norbornene-based monomer, an α-olefin-based monomer is preferable, and ethylene is more preferable.
These monomers may have one or more substituents. Examples of the substituent 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 another monomer capable of ring-opening copolymerization thereof, comprises a known ring-opening polymerization of a monomer component. It can be obtained by polymerization in the presence of a catalyst. Examples of the ring-opening polymerization catalyst include a catalyst composed of a metal halide such as ruthenium and osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide or acetylacetone such as titanium, zirconium, tungsten and molybdenum. A catalyst composed of a compound and an organoaluminum compound can be used.
A ring-opening polymer hydride of a norbornene-based monomer is usually obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to the polymerization solution of the ring-opening polymer to form a carbon-carbon unsaturated bond. Can be obtained by hydrogenating.

An addition polymer of a norbornene-based monomer or an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith contains a monomer component in the presence of a known addition polymerization catalyst. It can be obtained by polymerization. As the addition polymerization catalyst, for example, a catalyst composed of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

(2) Monocyclic Cyclic Olefin Polymer As the monocyclic cyclic olefin polymer, an addition polymer of a monocyclic cyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used. can.
(3) Cyclic-conjugated diene-based polymer Examples of the cyclic-conjugated diene-based polymer include polymers obtained by addition-polymerizing cyclic conjugated diene-based monomers such as cyclopentadiene and cyclohexadiene with 1,2- or 1,4-addition polymerization. The hydride or the like can be used.
(4) Vinyl alicyclic hydrocarbon polymer Examples of the vinyl alicyclic hydrocarbon polymer include a polymer of a vinyl alicyclic hydrocarbon-based monomer such as vinylcyclohexene and vinylcyclohexane and a hydride thereof; , Hydrocarbons of the aromatic ring portion of a polymer of a vinyl aromatic monomer such as α-methylstyrene; and the like. The vinyl alicyclic hydrocarbon polymer may be a copolymer of these monomers and other monomers copolymerizable.

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

The glass transition temperature of the alicyclic structure-containing polymer may be appropriately selected depending on the intended 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 molding processability are highly balanced and suitable.
The glass transition temperature of the alicyclic structure-containing polymer in the present invention was measured based on JIS K 7121.

The alicyclic structure-containing polymer can be used alone or in combination of two or more.
Further, the alicyclic structure-containing polymer includes a compounding agent usually used in a thermoplastic resin material, for example, a soft polymer, an antioxidant, an ultraviolet absorber, a light stabilizer, a near-infrared absorber, and a mold release agent. , Colorants such as dyes and pigments, and compounding agents such as plasticizers, antistatic agents, and fluorescent whitening agents can be added in an amount usually used.
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 other polymers mixed with the alicyclic structure-containing polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, and more preferably 100 parts by mass with respect to 100 parts by mass of the alicyclic structure-containing polymer. It is as follows.
If the ratio of various compounding agents and other polymers to be blended with the alicyclic structure-containing polymer is too large, it becomes difficult for cells to float. Therefore, all of them are blended within a range that does not impair the properties of the alicyclic structure-containing polymer. Is preferable.
The method for 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. In addition, there are no particular restrictions on the order of compounding. As a compounding method, for example, a method of kneading the resin in a molten state using a mixer, a uniaxial kneader, a biaxial kneader, a roll, a brabender, an extruder, etc. Examples thereof include a method of removing the solvent by a coagulation method, a casting method, or a direct drying method.
When a twin-screw kneader is used, after kneading, it is usually extruded into a rod shape in a molten state, cut into an appropriate length with a strand cutter, and pelletized.

The method for molding the container composed of the alicyclic structure-containing polymer can be arbitrarily selected according to the desired shape of the culture container. Examples of the molding method include an injection molding method, an extrusion molding method, a cast molding method, an inflation molding method, a blow molding method, a vacuum molding method, a press molding method, a compression molding method, a rotary molding method, a calendar molding method, and a rolling molding method. , Cutting molding method, spinning and the like, and these molding methods can be combined, and post-treatment such as stretching can be performed after molding as needed.

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 the high-pressure steam method and dry heat method; radiation methods that irradiate radiation such as gamma rays and electron beams, and irradiation methods that irradiate high frequencies; ethylene oxide gas (EOG). It is possible to select from methods generally used in the medical field, such as a gas method in which gases such as those are brought into contact with each other; a filtration method using a sterilization filter; Of these, the gas method is preferable because it is easy to maintain the surface free energy of the culture surface within a specific range.

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

The surface free energy is evaluated by measuring how much the test ink to which a predetermined range is applied adheres to the culture surface. More specifically, if the line of ink drawn on the culture surface does not change for 2 seconds without becoming water droplets, it means that the value of surface free energy is equal to or higher than the range of ink used. do. The components of the ink are a mixture of an organic solvent (2-ethoxyethanol, 2-propanol) and a basic dye (formamide), and these liquids are mixed in different ratios to show various surface free energy values. To make.
As a specific method for measuring the surface free energy, for example, first use ink having a value of about 38 mN / m, draw a line on the measurement surface, and if the ink line does not become water droplets and does not change for 2 seconds, the surface free energy Is equal to or greater than 38 mN / m. In this case, next, the same measurement is performed using an ink of about 40 mN / m. If the ink line does not turn into water droplets and does not change for 2 seconds, such a measurement is performed using a higher value ink until the ink line turns into water drops within 2 seconds.
In the above measurement, if the ink of about 38 mN / m already turns into water droplets, the same measurement is performed using an ink having a value lower than 38 mN / m. For example, if a line is drawn on the measurement surface with ink having a value of about 35 mN / m and the ink line does not become water droplets within 2 seconds and does not change for 2 seconds, the surface free energy value is between two values (35 to 35). 38 mN / m).

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

(Example I)
As the alicyclic structure-containing polymer, Zeonoa (registered trademark) 1060R (manufactured by Zeon Corporation, norbornene-based ring-opening polymer hydride: hereinafter simply referred to as "1060R") was used by an injection molding method to obtain 0.32 cm ^2. A well plate containing 96 cylindrical wells having a bottom area of ZEON was obtained as a culture vessel. Then, the culture vessel was sterilized with ethylene oxide gas (hereinafter, this culture vessel is referred to as "1060R 96-well plate"). The surface free energy on the inner bottom surface of the well (the surface in contact with cells, that is, the culture surface) of the 1060R 96-well plate was 34 mN / m. Here, the surface free energy was measured using a surface energy value evaluation ink (manufactured by Arcotest).
For each well of the 1060R 96-well plate, use LaboJet®-500Bio (Microjet Jet) to prepare a norbornene-based ring-opened hydride prepared at 100 μg / mL with sterile water. On the other hand, an aqueous synthetic peptide solution having an affinity was applied in dots on the bottom surface of a plate made of 1060R with a diameter of 600 μm and a distance between center points of 800 μm to form cardiomyocyte scaffolds in dots. After drying the peptide solution, using the cardiomyocytes for thawing medium accessory, iCell (TM) Cardiomyocytes ² (Cellular Dynamics International, Inc., model number CMC-100-012-001) to 4.4 × 10 ⁵ Prepared in cells / mL, and using the above LaboJet®-500Bio, seeded 54 droplets of cell suspension per dot (scaffold) at an amount of 32.4 nL / droplet, and 5% CO. _The cells were cultured in 2 atmospheres at 37 ° C. for 4 hours.
Then, 100 μL of a new maintenance medium for cardiomyocytes was added to each well of a 96-well plate made of 1060R, and the cells _{were cultured under a 5% CO 2} atmosphere at 37 ° C.
Two days after seeding the cardiomyocytes, half the amount of the cardiomyocyte maintenance medium in each well of the 1060R 96-well plate was exchanged. Three days after seeding the cardiomyocytes, 10 μL of the culture supernatant was collected from each well of the 1060R well plate. Next, amiodarone Hydrochloride (Tokyo Chemical Industry Co., Ltd., model number A2530) and nifedipine (Nifedipine) were used in each well of the 1060R well plate using dimethylsporoxide (DMSO) (Nacalai Tesque, model number 13435-35). ) (Tokyo Chemical Industry Co., Ltd., model number N0528), Cyclosporin A (Tokyo Chemical Industry Co., Ltd., model number C2408) and doxorubicin (Toronto Research Chemical Co., Ltd., model number D558000). Was diluted to 1060R and added to a 96-well plate made of 1060R in an amount of 10 μL each. At this time, the drug concentration in each well was 10 μM.
Eighteen hours after the addition of the drug compound, cardiomyocytes in each well of the 1060R plate were calcium-stained with Cal-520AM (ATT Bioquest, model number 21130). Then, the calcium influx signal of the cardiomyocytes in each well was observed as the number of beats using a confocal quantitative image cytometer CellVoyager (registered trademark) CQ1 (manufactured by Yokogawa Electric Corporation). Moreover, as a control, a well to which 10 μL of DMSO was added was used.
The change in the number of beats of cardiomyocytes with and without the addition of each drug compound is shown in the graph of FIG. 1 (“COP” in FIG. 1). In the graph of FIG. 1, the number of beats per minute of the cardiomyocyte mass on each scaffold was plotted with an error bar representing the standard deviation from the average of the number of tests of n = 3 for each of the above drug compounds.

(Comparative Example 1)
Implementation except that a polystyrene 96-well plate (Falcon (registered trademark), Corning, model number 353916; surface free energy: 43 mN / m) was used instead of the 1060R 96-well plate used in Example 1. In the same manner as in Example 1, cell culture, amiodarone, nifedipine, cyclosporin A and doxorubicin were added, and the pulsatile number of myocardial cells 18 hours after the addition of the drug compound was measured and plotted in the graph in the same manner. (“Polystyrene” in FIG. 1).

From the results of FIG. 1, by using a well plate in which the value of the surface free energy of the culture surface is within a specific range and the culture surface is composed of an alicyclic structure-containing polymer, it is possible to use a well plate on each scaffold. It can be seen that the pulsatile number of the cardiomyocyte mass can be clearly measured. When the polystyrene 96-well plate was used, it is considered that the cardiomyocytes did not settle on the culture surface and the myocardial cell mass was not formed because the surface free energy of the culture surface was high. Therefore, it was not possible to clearly measure the number of beats by calcium imaging.

According to the cardiotoxicity evaluation method of the present invention, it is possible to measure the change in the number of heartbeats of cardiomyocytes caused by a drug indicating a heart disease with high accuracy, so that an accurate cardiotoxicity evaluation can be performed.
This makes it possible to accurately measure the toxicity of a compound that is already known to have cardiotoxicity. Therefore, by using the evaluation method of the present invention, for example, it is possible to evaluate the toxicity of another novel drug while using doxorubicin as a positive control, and by extension, to maximize the drug efficacy while reducing the toxicity. It is also possible to determine the dose of the drug.

Claims (5)

A scaffold for attaching droplets formed in a medium containing myocardial cells is formed on the culture surface, and then the droplets are ejected onto the scaffold to attach the droplets to the culture surface. By seeding the myocardial cells together with the medium in the culture vessel,
A drug is added into the culture vessel to bring the drug into contact with the cardiomyocytes, followed by
A cardiotoxicity evaluation method for evaluating the cardiotoxicity of the drug by measuring the change in the pulsatile number of the cardiomyocytes.
A cardiotoxicity evaluation method in which at least the culture surface of the culture vessel is composed of an alicyclic structure-containing polymer, and the surface free energy of the culture surface is 30 to 37 mN / m. The cardiotoxicity evaluation method according to claim 1, wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes. The cardiotoxicity evaluation method according to claim 1 or 2, wherein a plurality of the scaffolds are formed in dots on one culture surface. The cardiotoxicity evaluation method according to any one of claims 1 to 3, wherein the scaffold is formed of a material having a peptide sequence having an affinity for the alicyclic structure-containing polymer. The cardiotoxicity evaluation method according to any one of claims 1 to 4, wherein the alicyclic structure-containing polymer is a norbornene-based ring-opening polymer hydride.

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