Method for evaluating beating of cardiomyocytes and adhesion molecule

Abstract

To provide a method that allows evaluation of beating of cardiomyocytes in a state of improved cardiomyocyte beatability.SOLUTION: The present invention relates to a method for evaluating beating of cardiomyocytes in a culture vessel, the method comprising: coating a culture surface of the culture vessel with an adhesion molecule; adhering the cardiomyocytes to the coated culture surface in the presence of a culture medium; and culturing the cardiomyocytes until beating of the cardiomyocytes is observed. The culture surface of the culture vessel is composed of an alicyclic structure-containing polymer, the culture surface has a surface free energy of 30 mN / m or more and 37 mN / m or less, and the adhesion molecule comprises a peptide sequence having affinity for the alicyclic structure-containing polymer and a laminin cell adhesion activity peptide sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the pulsation of cardiomyocytes and an adhesion molecule having affinity for an alicyclic structure-containing polymer. [Background technology]

[0002] One method for diagnosing heart disease is to measure the pulsatility of cardiomyocytes, and in recent years, efforts have been made to improve methods for assessing the pulsatility of cardiomyocytes.

[0003] For example, Patent Document 1 discloses a method in which droplets of a medium containing cardiomyocytes are ejected onto a scaffold formed on a culture surface to seed the cardiomyocytes together with the medium into a culture vessel, and then a drug is added to the culture vessel to measure changes in the heart rate of the cardiomyocytes. According to Patent Document 1, this method can accurately evaluate cardiotoxicity using a small amount of cells. Furthermore, Patent Document 2 discloses a method for evaluating the cardiotoxicity of a drug by seeding cardiomyocytes together with a medium in a culture vessel, adding a drug to the medium in the culture vessel to bring the drug into contact with the cardiomyocytes, and then measuring a biomarker for cardiac disease secreted from the cardiomyocytes. According to Patent Document 2, this method can measure the biomarker for cardiac disease with high accuracy. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-129518 [Patent Document 2] International Publication No. 2020 / 066396 Summary of the Invention [Problem to be solved by the invention]

[0005] Here, from the viewpoint of facilitating evaluation of the pulsation of cardiomyocytes, it is preferable to improve the pulsation of cardiomyocytes. However, the conventional methods described above have room for further improvement in terms of improving the pulsation of cardiomyocytes.

[0006] Therefore, an object of the present invention is to provide a method capable of evaluating the pulsation of cardiomyocytes in a state in which the pulsatility of the cardiomyocytes is improved. Another object of the present invention is to provide an adhesion molecule that has affinity for alicyclic structure-containing polymers and that can improve the pulsatility of cardiomyocytes. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above-mentioned problems, and have found that by using a culture vessel made of an alicyclic structure-containing polymer and having a culture surface with a surface free energy within a predetermined range, and by carrying out predetermined steps, it is possible to evaluate the pulsation of cardiomyocytes in a state where the pulsation of cardiomyocytes is improved, and have completed the present invention.

[0008] That is, the present invention aims to advantageously solve the above-mentioned problems, and provides the following methods for evaluating the pulsation of cardiomyocytes ([1] to [7]) and the following adhesion molecule ([8]) having affinity for an alicyclic structure-containing polymer.

[0009] [1] A method for evaluating the pulsation of cardiomyocytes in a culture vessel, comprising the steps of coating a culture surface of the culture vessel with an adhesion molecule, and attaching the cardiomyocytes to the coated culture surface in the presence of a medium. and culturing the cardiomyocytes until pulsation of the cardiomyocytes is observed, wherein the culture surface of the culture vessel is made of an alicyclic structure-containing polymer, the surface free energy of the culture surface is 30 mN / m or more and 37 mN / m or less, and the adhesion molecule has a peptide sequence having affinity for the alicyclic structure-containing polymer and a laminin cell adhesion activity peptide sequence. By using a culture vessel made of an alicyclic structure-containing polymer and having a culture surface with a surface free energy within the above-mentioned range, and by going through the above-mentioned steps, it is possible to evaluate the pulsation of cardiomyocytes in a state where the pulsation of the cardiomyocytes is improved. In the present invention, the term "culture surface" refers to the surface of a culture vessel to which cultured cardiomyocytes can adhere. For example, when the culture vessel is a 96-well plate, the "culture surface" refers to the inner bottom surface of the well. In the present invention, the "surface free energy" of the culture surface can be measured using the method described in the examples of this specification. In the present invention, "(culturing until) pulsation of cardiomyocytes is confirmed" means that it is sufficient to confirm that almost all of the cardiomyocytes are pulsating in the culture vessel, and the pulsating cells do not necessarily have to be synchronized. From the viewpoint of further improving the pulsation of cardiomyocytes and enhancing drug responsiveness of cardiomyocytes, it is preferable that the pulsation of all cultured cells is synchronized. Furthermore, in the present invention, "having affinity for the alicyclic structure-containing polymer" means being able to bind to a surface composed of the alicyclic structure-containing polymer.

[0010] [2] The method described in [1] above, wherein the cardiomyocytes are cardiomyocytes derived from induced pluripotent stem cells. If induced pluripotent stem cell (iPS cell)-derived cardiomyocytes are used as cardiomyocytes, the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced.

[0011] [3] The method according to [1] or [2] above, wherein the peptide sequence having affinity for the alicyclic structure-containing polymer is Thr-Val-Asp-Ser-Cys-Leu-Thr (sequence number 1). By using the above sequence as a peptide sequence having affinity for an alicyclic structure-containing polymer (hereinafter sometimes abbreviated as "affinity peptide sequence"), the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced.

[0012] [4] The method according to any one of [1] to [3] above, wherein the laminin cell adhesion active peptide sequence is Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 2). Use of the above sequence as a peptide sequence with laminin cell adhesion activity can further improve the pulsatility of cardiomyocytes and enhance the drug responsiveness of cardiomyocytes.

[0013] [5] The method according to any one of [1] to [4] above, wherein the peptide sequence having affinity for the alicyclic structure-containing polymer is Thr-Val-Asp-Ser-Cys-Leu-Thr (sequence number 1), and the laminin cell adhesion active peptide sequence is Tyr-Ile-Gly-Ser-Arg (sequence number 2). If the adhesion molecule has both the peptide sequence shown in SEQ ID NO: 1 and the peptide sequence shown in SEQ ID NO: 2, the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced.

[0014] [6] The method according to any one of [1] to [5] above, wherein the molecular weight of the adhesion molecule is 1,000 or more and 4,000 or less. If the molecular weight of the adhesion molecule is within the above range, the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced. In the present invention, the "molecular weight" of an adhesion molecule is measured using, for example, a mass spectrometer. It is possible.

[0015] [7] The method according to any one of the above [1] to [6], wherein the alicyclic structure-containing polymer is a hydrogenated norbornene ring-opening polymer. If a norbornene ring-opening polymer hydride is used as the alicyclic structure-containing polymer, the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced.

[0016] [8] An adhesion molecule having affinity for an alicyclic structure-containing polymer, comprising Thr-Val-Asp-Ser-Cys-Leu-Thr (sequence number 1) and Tyr-Ile-Gly-Ser-Arg (sequence number 2). By using an adhesion molecule having both SEQ ID NO: 1 and SEQ ID NO: 2, the pulsatility of cardiomyocytes can be improved. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide a method capable of evaluating the pulsation of cardiomyocytes in a state in which the pulsatility of the cardiomyocytes is improved. Furthermore, according to the present invention, it is possible to provide an adhesion molecule that has affinity for an alicyclic structure-containing polymer and that can improve the pulsatility of cardiomyocytes. DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, embodiments of the present invention will be described in detail. The method for evaluating the pulsation of cardiomyocytes of the present invention can be used to evaluate the pulsation of cultured cardiomyocytes in a culture vessel. The method of the present invention can also be used to evaluate changes in the pulsation of cardiomyocytes due to the addition of a drug (drug responsiveness of cardiomyocytes). The adhesion molecule of the present invention can be used when culturing cardiomyocytes in a culture vessel. More specifically, the adhesion molecule of the present invention can be used to coat the culture surface of a culture vessel used for culturing cardiomyocytes.

[0019] (Method for evaluating cardiac muscle cell pulsation) The method for evaluating the pulsation of cardiomyocytes of the present invention comprises the steps of coating the culture surface of a culture vessel with adhesion molecules having a predetermined sequence (step A), adhering cardiomyocytes to the culture surface coated with the adhesion molecules in step A in the presence of a medium (step B), and culturing the cardiomyocytes adhered in step B until pulsation is observed (step C). Note that the method of the present invention may further comprise steps (other steps) other than the above-mentioned steps A to C.

[0020] Since the method of the present invention includes the above-mentioned steps A to C, the method allows evaluation of the pulsation of cardiomyocytes in a state in which the pulsation of cardiomyocytes is improved. Although the reason why the above-mentioned effects are obtained by using the method of the present invention is not clear, it is presumed to be as follows.

[0021] The method of the present invention comprises step A of coating the culture surface of a culture vessel with adhesion molecules, and step B of adhering cardiomyocytes to the culture surface coated with the adhesion molecules. First, in step A, the adhesion molecules can adhere to the culture surface composed of an alicyclic structure-containing polymer via an affinity peptide sequence. Next, in step B, the adhesion molecules can adhere to receptors present on the surface of cardiomyocytes via a laminin cell adhesion activity peptide sequence. By going through the above two steps, cardiomyocytes can be adhered to the culture surface via adhesion molecules. In addition, the culture vessel used in the method of the present invention has a culture surface composed of an alicyclic structure-containing polymer, and the surface free energy of the culture surface is 30 mN / m or more and 37 mN / m or less. By having a culture surface composed of an alicyclic structure-containing polymer and having a surface free energy within the above range, the affinity of the adhesion molecules for the culture surface can be adjusted within an appropriate range, and as a result, the culture surface can be coated with adhesion molecules at an appropriate density. By adhering cardiomyocytes to a culture surface coated with the appropriate density in this manner, the cardiomyocytes can be favorably attached to the culture surface without excessive stress on the cardiomyocytes. Therefore, the cardiomyocytes can be cultured until pulsation is observed without reducing the pulsation of the cardiomyocytes (Step C). For the above reasons, it is believed that the method of the present invention makes it possible to evaluate the pulsation of cardiomyocytes in a state in which the pulsatility of the cardiomyocytes is improved.

[0022] <Process A> In step A, the culture surface of the culture vessel is coated with the adhesion molecules by attaching the adhesion molecules to the culture surface via an affinity peptide sequence.

[0023] <<Culture container>> Culture vessels of any shape can be used as long as they are made of alicyclic structure-containing polymers, including dishes, plates, microchannel chips, bags, tubes, scaffolds, cups, and jar fermenters. At least the culture surface of the culture vessel may be made 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 made of the alicyclic structure-containing polymer. In the case of a bag made of a laminate of films made of different polymer materials, the innermost layer (the inner surface of the bag) may be made of a film made of the alicyclic structure-containing polymer. Alternatively, the entire culture vessel may be made of the alicyclic structure-containing polymer. For example, in the case of a culture dish, a flask, or a plate having multiple wells, the entire vessel can be made of the alicyclic structure-containing polymer by molding the entire vessel from the alicyclic structure-containing polymer. The culture surface may have an uneven surface or may be smooth. According to the method of the present invention, even if the culture surface is smooth, the pulsation of cardiomyocytes can be evaluated while improving the pulsation of the cardiomyocytes. A part of the culture surface may be smooth, or the entire culture surface may be smooth.

[0024] [Alicyclic structure-containing polymer] The alicyclic structure-containing polymer is a resin having an alicyclic structure in the main chain and / or side chain. From the viewpoints of mechanical strength, heat resistance, etc., it is preferable that the main chain contains an alicyclic structure. From the viewpoint of differentiation induction efficiency, it is more preferable that the polymer does not have a polar group. Here, the polar group refers to a polar atomic group. Examples of polar groups include an amino group, a carboxyl group, a hydroxyl group, and an acid anhydride group.

[0025] Examples of the alicyclic structure include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. From the viewpoints of mechanical strength, heat resistance, and the like, a cycloalkane structure or a cycloalkene structure is preferred, and among these, those having a cycloalkane structure are most preferred.

[0026] 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 are well balanced, which is preferable.

[0027] The proportion of repeating units having an alicyclic structure in the alicyclic structure-containing polymer may be appropriately selected depending on the intended use, but is usually 30% by weight or more, preferably 50% by weight or more, and more preferably 70% by weight or more. If the amount is too small, the heat resistance will be poor, which is undesirable. The remainder other than the repeating units having an alicyclic structure in the alicyclic structure-containing polymer is not particularly limited and may be appropriately selected depending on the intended use.

[0028] Specific examples of the alicyclic structure-containing polymer include (1) norbornene polymers, (2) monocyclic olefin polymers, (3) cyclic conjugated diene polymers, (4) vinyl alicyclic hydrocarbon polymers, and hydrogenated products of (1) to (4). Among these, norbornene polymers and hydrogenated products thereof are preferred from the viewpoints of heat resistance, mechanical strength, etc.

[0029] -(1) Norbornene-based polymers- Norbornene polymers are obtained by polymerizing norbornene monomers, which are monomers having a norbornene skeleton, and are roughly classified into those obtained by ring-opening polymerization and those obtained by addition polymerization.

[0030] Examples of materials obtainable by ring-opening polymerization include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers copolymerizable therewith, and hydrogenated products of these. Examples of the polymers obtained by addition polymerization include addition polymers of norbornene-based monomers and addition polymers of norbornene-based monomers and other monomers copolymerizable therewith. Among these, from the viewpoints of further improving the pulsatility of cardiomyocytes and enhancing the drug responsiveness of cardiomyocytes, a norbornene-based monomer ring-opening polymer hydrogenation product (sometimes referred to as a "norbornene-based ring-opening polymer hydrogenation product") is preferred.

[0031] Norbornene monomers that can be used to synthesize norbornene polymers include bicyclo[2.2.1]hept-2-ene (commonly known as norbornene), 5-methyl-bicyclo[2.2.1]hept-2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, and 5-ethylidene-bicyclo[2.2.1]hept-2-ene. Bicyclic monomers such as 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]hept-2-ene; Tricyclo[4.3.0 1,6 .1 2,5 ] Tricyclic monomers such as deca-3,7-diene (commonly known as dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, and 2,3-dihydroxydicyclopentadiene; 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 ] tetracyclic monomers such as dec-3-ene (commonly known as methanotetrahydrofluorene: also known as 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, and 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene; and the like.

[0032] Other monomers that can be ring-opening copolymerized with norbornene-based monomers include monocyclic cycloolefin-based monomers such as cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, 1,5,9-cyclododecatriene, and 1,5,9,13-cyclohexadecatetraene. These monomers may have one or more substituents, such as alkyl groups, alkylene groups, aryl groups, silyl groups, alkoxycarbonyl groups, and alkylidene groups.

[0033] Other monomers that can be addition copolymerized 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, and tetracyclo[9.2.1.0]. 2,10 .0 3,8] Cycloolefin-based monomers such as tetradeca-3,5,7,12-tetraene (also known as 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); non-conjugated diene-based monomers such as 1,4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene; and the like.

[0034] Among these, as the other monomer capable of addition copolymerization with the norbornene-based monomer, an α-olefin-based monomer is preferred, and ethylene is more preferred. These monomers may have one or more substituents, such as alkyl groups, alkylene groups, aryl groups, silyl groups, alkoxycarbonyl groups, and alkylidene groups.

[0035] 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 therewith, can be obtained by polymerizing the monomer components in the presence of a known ring-opening polymerization catalyst. As the ring-opening polymerization catalyst, for example, a catalyst composed of a halide of a metal such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a catalyst composed of a halide or an acetylacetone compound of a metal such as titanium, zirconium, tungsten, or molybdenum, and an organoaluminum compound can be used. The hydrogenated ring-opening polymer of a norbornene-based monomer can usually be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to a polymerization solution of the ring-opening polymer and hydrogenating the carbon-carbon unsaturated bonds.

[0036] An addition polymer of a norbornene-based monomer or an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith can be obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst. As the addition polymerization catalyst, for example, a catalyst comprising a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

[0037] -(2) Monocyclic olefin polymers- As the monocyclic olefin polymer, for example, an addition polymer of a monocyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used.

[0038] -(3) Cyclic conjugated diene polymers- Examples of the cyclic conjugated diene polymer that can be used include polymers obtained by 1,2- or 1,4-addition polymerization of cyclic conjugated diene monomers such as cyclopentadiene and cyclohexadiene, and hydrogenated products thereof.

[0039] -(4) Vinyl alicyclic hydrocarbon polymer- Examples of vinyl alicyclic hydrocarbon polymers include polymers of vinyl alicyclic hydrocarbon monomers such as vinylcyclohexene and vinylcyclohexane, and hydrogenated products thereof; hydrogenated products of the aromatic ring moieties of polymers of vinyl aromatic monomers such as styrene and α-methylstyrene; etc. The vinyl alicyclic hydrocarbon polymer may be a copolymer of these monomers with other copolymerizable monomers.

[0040] 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 250° C., and more preferably 100 to 200° C. When the glass transition temperature is within this range, heat resistance and moldability are well balanced, which is preferable. The glass transition temperature of the alicyclic structure-containing polymer in the present invention is measured in accordance with JIS K 7121.

[0041] The above alicyclic structure-containing polymers can be used either alone or in combination of two or more. Furthermore, to the alicyclic structure-containing polymer, compounding agents that are usually used in thermoplastic resin materials, such as soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, and fluorescent brighteners, can be added in usually employed amounts. The alicyclic structure-containing polymer may be mixed with other polymers (hereinafter simply referred to as "other polymers") other than the flexible polymer. The amount of the 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 or less, per 100 parts by mass of the alicyclic structure-containing polymer. If the ratio of various compounding agents or other polymers to the alicyclic structure-containing polymer is too high, cells will be less likely to float, so it is preferable to compound them in an amount that does not impair the properties of the alicyclic structure-containing polymer. The method of mixing the alicyclic structure-containing polymer with the compounding agent and other polymers is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Also, there is no particular limitation on the order of compounding. 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, etc., and a method of dissolving and dispersing the resin in a suitable solvent, and then removing the solvent by a coagulation method, a casting method, or a direct drying method. When a twin-screw kneader is used, after kneading, the material is usually extruded in a molten state into a rod shape, cut to an appropriate length with a strand cutter, and pelletized for use.

[0042] The method for molding a container made of an alicyclic structure-containing polymer can be selected arbitrarily depending on the desired shape of the culture vessel. 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, roll molding, cutting molding, spinning, etc. These molding methods can be combined, and post-processing such as stretching can be performed after molding as necessary.

[0043] The culture vessels used in the present invention are preferably sterilized. There are no particular limitations on the sterilization method, and it can be selected from methods commonly used in the medical field, such as heating methods such as high-pressure steam and dry heat; radiation methods using radiation such as gamma rays and electron beams, and irradiation methods using high-frequency waves; gas methods using gas such as ethylene oxide gas (EOG) in contact; and filtration methods using a sterilization filter, depending on the shape of the molded article and the cells used. Among these, sterilization methods are preferred because they make it easier to maintain the surface free energy of the culture surface within a predetermined range, which will be described later. The gas process is preferred.

[0044] The culture surface of the culture vessel may be subjected to a surface treatment such as plasma treatment. From the viewpoint of keeping the surface free energy of the culture surface within a predetermined range described below, it is preferable not to perform a surface treatment on the culture surface.

[0045] [Surface free energy] The surface free energy of the culture surface must be 30 mN / m or more and 37 mN / m or less, preferably 31 mN / m or more, more preferably 32 mN / m or more, even more preferably 33 mN / m or more, preferably 36 mN / m or less, and more preferably 35 mN / m or less. If the surface free energy of the culture surface is less than 30 mN / m, the culture surface will not be sufficiently coated with adhesion molecules, resulting in reduced pulsation and drug responsiveness of cardiomyocytes. On the other hand, if the surface free energy of the culture surface is more than 37 mN / m, the culture surface will be excessively coated with adhesion molecules, placing excessive stress on the cardiomyocytes and reducing the pulsation and drug responsiveness of the cardiomyocytes. The surface free energy of the culture surface can be adjusted by changing the type of alicyclic structure-containing polymer that constitutes the culture surface, whether or not the culture surface is subjected to a surface treatment, etc. Specifically, by not performing a surface treatment on the culture surface, it becomes easier to keep the surface free energy of the culture surface within the above-mentioned predetermined range.

[0046] <<Adhesion molecules>> The adhesion molecule has at least an affinity peptide sequence and a laminin cell adhesion activity peptide sequence, but may also have an amino acid sequence (other sequence) other than the affinity peptide sequence and the laminin cell adhesion activity peptide sequence.

[0047] [Affinity peptide sequence] The affinity peptide sequence is a peptide sequence that has affinity (adhesion) for the alicyclic structure-containing polymer. Examples of affinity peptide sequences include oligopeptide sequences discovered by the method described in WO 2018 / 117242. Among them, Thr-Val-Asp-Ser-Cys-Leu-Thr (SEQ ID NO: 1) is preferred from the viewpoint of further improving the pulsatility of cardiomyocytes and enhancing the drug responsiveness of cardiomyocytes. An adhesion molecule may have one type of affinity peptide sequence or two or more types of affinity peptide sequences, and may have only one affinity peptide sequence or two or more affinity peptide sequences.

[0048] [Laminin cell adhesion active peptide sequence] The laminin cell adhesion active peptide sequence is a cell adhesion active sequence derived from the cell adhesion molecule laminin. Examples of laminin cell adhesion active peptide sequences include Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 2), Pro-Asp-Ser-Gly-Arg (SEQ ID NO: 3), Arg-Tyr-Val-Val-Leu-Pro-Arg (SEQ ID NO: 4), Leu-Arg-Glu (SEQ ID NO: 5), Ile-Lys-Val-Ala-Val (SEQ ID NO: 6), Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (SEQ ID NO: 7), and Arg-Gly-Asp (SEQ ID NO: 8). Among these, SEQ ID NO: 2 is preferred from the viewpoints of further improving the pulsatility of cardiomyocytes and enhancing drug responsiveness of cardiomyocytes. The adhesion molecule may have one type of laminin cell adhesion active peptide sequence, or two or more types of laminin cell adhesion active peptide sequences, or may have only one laminin cell adhesion active peptide sequence, or two or more laminin cell adhesion active peptide sequences.

[0049] Furthermore, from the viewpoint of further improving the pulsatility of cardiomyocytes and enhancing the drug responsiveness of cardiomyocytes, it is preferable that the adhesion molecule has at least one of sequence numbers 1 and 2, and it is more preferable that it has both sequence numbers 1 and 2.

[0050] The positional relationship between the affinity peptide sequence and the laminin cell adhesion activity peptide sequence in the adhesion molecule is not particularly limited; the affinity peptide sequence may be located on the N-terminus and the laminin cell adhesion activity peptide sequence on the C-terminus, or the laminin cell adhesion activity peptide sequence may be located on the N-terminus and the affinity peptide sequence on the C-terminus.

[0051] Other sequences that adhesion molecules may contain are not particularly limited, as long as they do not significantly affect the adhesiveness of the adhesion molecules to the culture surface and cardiomyocytes. For example, adhesion molecules may contain a linker sequence between the affinity peptide sequence and the laminin cell adhesive activity peptide sequence. Examples of linker sequences include a sequence of consecutive glycine residues (-Gly-Gly-) and a sequence of alternating glycine and serine residues (-Gly-Ser-Gly-Ser-). Alternatively, adhesion molecules may contain an affinity tag sequence at the N-terminus and / or C-terminus to facilitate purification or a sequence to improve solubility. The adhesion molecule may have only one type of other sequence, or may have two or more types of other sequences.

[0052] Here, the molecular weight of the adhesion molecule is preferably 1000 or more, more preferably 1500 or more, even more preferably 2000 or more, particularly preferably 2500 or more, and preferably 4000 or less, more preferably 3500 or less. When the molecular weight of the adhesion molecule is within the above range, the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced.

[0053] Furthermore, the number of amino acid residues in the adhesion molecule is preferably 10 or more, more preferably 15 or more, even more preferably 20 or more, particularly preferably 25 or more, and preferably 40 or less, more preferably 35 or less. When the number of amino acid residues in the adhesion molecule is within the above range, the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced.

[0054] Adhesion molecules can be synthesized by known methods or prepared as recombinant proteins, which can be obtained by overexpressing the desired adhesion molecule in a known host such as Escherichia coli and then purifying the resulting protein using known column chromatography or the like.

[0055] The method for coating the culture surface of a culture vessel with adhesion molecules is similar to the method for coating a general cell substrate on a culture vessel. Specifically, the adhesion molecules, dissolved in an appropriate buffer solution, are placed in the culture vessel and allowed to stand at a temperature close to the culture temperature, typically for 10 minutes to 5 hours, preferably 30 minutes to 2 hours, to allow the adhesion molecules to contact the culture surface. After this, any adhesion molecules that do not adhere to the culture surface are removed. If the contact time is too short, the coating will be insufficient. On the other hand, the alicyclic structure-containing polymer that constitutes the culture surface has low protein adsorption and does not form multilayer adsorption like polystyrene, so even if the contact time is extended, the amount of adsorption will not increase. Therefore, it is not necessary to extend the contact time beyond the time mentioned above.

[0056] Because the culture surface made of an alicyclic structure-containing polymer is prone to repelling aqueous solutions, it is desirable to add 1.5 to 3 times more adhesion molecules to the culture vessel than to a general polystyrene cell culture vessel. Specifically, the amount of adhesion molecules added to the culture vessel should be 1.5 to 3 times more than that added to a 1 cm of the culture surface. 2 It is preferable to add 0.3 to 0.7 ml to the solution.

[0057] <Process B> In step B, cardiomyocytes are allowed to adhere in the presence of a medium to the culture surface coated with the adhesion molecules in step A. In step A, the adhesion molecules adhere to the culture surface via the affinity peptide sequence, and in step B, the adhesion molecules bind to receptors on the surface of cardiomyocytes via the laminin cell adhesion activity peptide sequence, allowing cardiomyocytes to adhere to the culture surface via the adhesion molecules.

[0058] <<Cardiomyocytes>> Examples of cardiomyocytes include pluripotent stem cell-derived cardiomyocytes (especially induced pluripotent stem cell (iPS)-derived cardiomyocytes), embryonic stem cell-derived cardiomyocytes, human-derived cardiomyocytes, and primary cultured cardiomyocytes derived from the heart of an organism. Among these, induced pluripotent stem cell-derived cardiomyocytes are preferred from the viewpoints of further improving the pulsatility of cardiomyocytes and enhancing the drug responsiveness of cardiomyocytes. The cardiomyocytes described above may be used singly or in combination of two or more types. From the viewpoint of further improving the pulsatility of cardiomyocytes and enhancing the drug responsiveness of cardiomyocytes, it is preferable to use one type alone.

[0059] <<Culture Medium>> The medium is not particularly limited, and commercially available medium for culturing cardiomyocytes can be used. The medium may also contain additives, such as minerals, metals, and vitamins. These additives may be used singly or in combination of two or more.

[0060] There are no particular limitations on the method for adhering cardiomyocytes to a culture surface coated with an adhesion molecule. For example, cardiomyocytes suspended in the above-mentioned medium are seeded into a culture vessel using a pipette or the like, and the vessel is shaken as necessary to distribute the cardiomyocytes evenly throughout the culture vessel, and then the vessel is allowed to stand in an incubator. After standing in the incubator, the medium is removed and fresh medium is added to remove non-adhered cardiomyocytes.

[0061] The seeding density of cardiomyocytes may be adjusted appropriately depending on the type of cardiomyocytes, etc. The seeding density is usually 1.0 × 10 4 cells / well ~1.0 × 10 6 cells / well, preferably 1.0 × 10 4 cells / well ~1.0 × 10 5 cells / well.

[0062] <Process C> In step C, the cardiomyocytes adhered in step B are cultured until pulsation of the cardiomyocytes is observed.

[0063] The culture is usually carried out in the presence of a medium. For example, the medium described above in the section "Step B" can be used as the medium. The medium used in Step C may be the same as or different from the medium used in Step B.

[0064] The culture conditions are not particularly limited and can be determined appropriately depending on the type of cardiomyocytes, etc. For example, cardiomyocytes can be cultured using a humidified incubator with a carbon dioxide concentration of about 5% and a temperature maintained constant within the range of 20°C to 37°C.

[0065] The culture period until pulsation of cardiomyocytes can be observed varies depending on the type of cardiomyocytes and culture conditions, but is usually 2 days to 1 week. From the viewpoint of suppressing a decrease in pulsatility due to long-term culture, the culture period is preferably within 5 days, more preferably within 4 days, and most preferably within 3 days. It is more preferable to keep it within 0.5 mm.

[0066] Here, it is preferable that step C further includes a step of adding a drug that improves the pulsatility of cardiomyocytes to the culture medium in the culture vessel. By including the above step in step C, the pulsatility of cardiomyocytes can be further improved and the drug responsiveness of cardiomyocytes can be enhanced.

[0067] Drugs that improve the pulsatility of cardiomyocytes include positive inotropic drugs such as isoproterenol, dobutamine, and milrinone. Among these, isoproterenol, a β-receptor agonist, is preferred. Isoproterenol is known to increase contractile force, shorten contraction time, increase contraction and relaxation rates, and increase beat rate in mature cardiomyocytes (Takeda et al., Tissue Eng Part C Methods. 2018, 24(1):56-67; International Publication No. 2021 / 100718). The above-mentioned drugs may be used singly or in combination of two or more.

[0068] <Other processes> Other steps include a step of sterilizing the culture vessel prior to the above-mentioned step A; and a step (step D) of measuring the pulsatility of the cardiomyocytes cultured until pulsation is observed in the above-mentioned step C by phase contrast image analysis.

[0069] <<Process D>> In particular, the method of the present invention preferably includes, as another step, a step (step D) of measuring the pulsation of cardiomyocytes cultured until pulsation is observed in the above-mentioned step C by phase-contrast image analysis. By further including step D, it is possible to observe live cardiomyocytes without staining that may be toxic to living cells. Therefore, it is possible to further improve the pulsation of cardiomyocytes and enhance the drug responsiveness of cardiomyocytes.

[0070] Phase-contrast images can be obtained by, for example, photographing cardiomyocytes contained in a culture vessel using a phase-contrast observation method with an inverted microscope and outputting the photographed phase-contrast images of cardiomyocytes. The obtained phase-contrast images can be analyzed using software commonly used in the field of cell biology, such as ImageJ (manufactured by Wayne Rasband).

[0071] By the image analysis described above, the contractile force, contraction time, contraction velocity, relaxation velocity, etc. of the cardiomyocytes are evaluated as the pulsatility of the cardiomyocytes.

[0072] (adhesion molecule) The adhesion molecule of the present invention has both the peptide sequence Thr-Val-Asp-Ser-Cys-Leu-Thr (SEQ ID NO: 1) and the peptide sequence Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 2), where SEQ ID NO: 1 is a peptide sequence (affinity peptide sequence) having affinity for the above-mentioned alicyclic structure-containing polymer, and SEQ ID NO: 2 is the above-mentioned peptide sequence exhibiting laminin cell adhesion activity.

[0073] The adhesion molecule of the present invention has both the peptide sequence shown in SEQ ID NO: 1 and the peptide sequence shown in SEQ ID NO: 2, and therefore, the use of the adhesion molecule of the present invention can improve the pulsatility of cardiomyocytes. Although the reason why the above-mentioned effects are obtained by using the adhesion molecule of the present invention is unclear, it is presumed to be as follows.

[0074] SEQ ID NO: 1 included in the adhesion molecule of the present invention has affinity for the alicyclic structure-containing polymer. The adhesion molecule of the present invention has a peptide sequence with laminin cell adhesion activity, which can bind to the culture surface of a culture vessel. On the other hand, SEQ ID NO: 2 contained in the adhesion molecule of the present invention is a peptide sequence with laminin cell adhesion activity, which can bind to a receptor present on the surface of cardiomyocytes. Therefore, if the culture surface of a culture vessel is coated with the adhesion molecule of the present invention and then cardiomyocytes are seeded, cardiomyocytes can be favorably adhered to the culture surface without excessive stress on the cardiomyocytes, and as a result, the pulsatility of the cardiomyocytes can be improved.

[0075] The positional relationship between sequence number 1 and sequence number 2 in the adhesion molecule is not particularly limited, and sequence number 1 may be located on the N-terminal side and sequence number 2 on the C-terminal side, or sequence number 2 may be located on the N-terminal side and sequence number 1 on the C-terminal side.

[0076] The molecular weight and number of amino acid residues of the adhesion molecule are not particularly limited as long as the desired effect is obtained. The preferred ranges of the molecular weight and number of amino acid residues of the adhesion molecule are the same as those described above in the section "Method for evaluating cardiac muscle cell pulsation."

[0077] The adhesion molecule may have an amino acid sequence other than SEQ ID NO: 1 and SEQ ID NO: 2. Examples of amino acid sequences other than SEQ ID NO: 1 and SEQ ID NO: 2 that the adhesion molecule may have include affinity peptide sequences other than SEQ ID NO: 1, laminin cell adhesive activity peptide sequences other than SEQ ID NO: 2, and other sequences, as described above in the section "Method for Evaluating Cardiomyocyte Pulsation." For example, the adhesion molecule may have a linker sequence between SEQ ID NO: 1 and SEQ ID NO: 2. Examples of linker sequences include a sequence of consecutive glycine residues (-Gly-Gly-) and a sequence of alternating glycine and serine residues (-Gly-Ser-Gly-Ser-). Alternatively, the adhesion molecule may have an affinity tag sequence at the N-terminus and / or C-terminus to facilitate purification or a sequence to improve solubility. The adhesion molecule may have only one amino acid sequence other than SEQ ID NO:1 and SEQ ID NO:2, or may have two or more amino acid sequences other than SEQ ID NO:1 and SEQ ID NO:2. [Example]

[0078] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. In the examples and comparative examples, the surface free energy, pulsation, and drug responsiveness of the culture surface were measured and evaluated by the following methods.

[0079] <Surface free energy> The surface free energy of the culture surface of the culture vessels prepared in the examples and comparative examples was measured. Specifically, the measurement was carried out using a surface energy value evaluation ink (manufactured by Arcotest) according to the following procedure. First, a line is drawn on the culture surface using ink with a surface free energy of 38 mN / m. If the ink line does not turn into a droplet and remains unchanged for two seconds, the surface free energy is 38 mN / m or higher. In this case, a similar measurement is then performed using ink with a surface free energy of 40 mN / m. If the ink line does not turn into a droplet and remains unchanged for two seconds, similar measurements are performed using inks with higher surface free energy until the ink line turns into a droplet within two seconds. In the above measurement, if the ink with 38 mN / m already turns into droplets, perform the same measurement using ink with a lower value than 38 mN / m. For example, if a line is drawn on the measurement surface with ink with a value of 35 mN / m and the ink line does not turn into droplets within 2 seconds and remains unchanged for 2 seconds, the surface free energy value is between these two values (35 mN / m or more but less than 38 mN / m). <Pulsatility> The cardiomyocytes after step C in each example and comparative example were evaluated. Using a microscope (Leica Microsystems, product name "DMi8"), phase-contrast images of cardiomyocytes in each well were captured at 20 fps for 60 seconds, obtaining video data of cardiomyocytes in each well before and after the addition of isoproterenol. The obtained video data was then analyzed using the MUSCLEMOTION plug-in tool (Sala et al., Circu. Res., 2018, vol. 122, pp. e5-e16) of the image analysis software ImageJ (Wayne Rasband), and the contractile force, contraction time, contraction velocity, and relaxation velocity of the cardiomyocytes in each well were quantified. The average values of the quantified contractile force, contraction velocity, and relaxation velocity for five wells were then calculated. Higher contractile force, contraction velocity, and relaxation velocity indicate better pulsatility. <Drug responsiveness> The cardiomyocytes after step C in each example and comparative example were evaluated. The average values of the contraction time, contraction velocity, and relaxation velocity for each isoproterenol concentration (0 nM, 10 nM, 100 nM, and 1000 nM, each in 3 wells) were calculated, and a significance test was performed against 0 nM isoproterenol (non-added group). Student's t-test was used to test for significance. The contraction time, contraction velocity, and relaxation velocity were each evaluated according to the following criteria. The lower the lowest isoproterenol concentration at which a significant difference was observed, the better the drug responsiveness. A: The lowest concentration of isoproterenol at which a significant difference was observed was 10 nM. B: The lowest concentration of isoproterenol at which a significant difference was observed was 100 nM. C: The lowest concentration of isoproterenol at which a significant difference was observed was 1000 nM. D: No significant difference was observed at any isoproterenol concentration.

[0080] Example 1 <Preparation of culture vessel> A hydrogenated ring-opened polymer of a norbornene-based monomer (manufactured by Zeon Corporation, product name "ZEONEX (registered trademark) 790R"; glass transition temperature (Tg): 163°C; hereinafter referred to as "790R") was used as the alicyclic structure-containing polymer, and a 0.32 cm2 polymer having a base area was injection molded. 2 A well plate having 96 cylindrical wells was obtained as a culture vessel. The culture vessel was then sterilized with ethylene oxide gas (hereinafter, this culture vessel will be referred to as the "790R 96-well plate"). The surface free energy of the bottom surface (culture surface) of the wells of this 790R 96-well plate was measured. The results are shown in Table 1. <Process A> Next, an adhesion molecule (molecular weight 3020; number of amino acid residues 29; hereinafter referred to as "adhesion molecule A") having an affinity peptide sequence of Thr-Val-Asp-Ser-Cys-Leu-Thr (SEQ ID NO: 1) and a laminin cell adhesion activity peptide sequence of Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 2) was dissolved in Dulbecco's phosphate-buffered saline to prepare an adhesion molecule A solution (10 μg / mL). This adhesion molecule A solution was applied to each well of a 96-well plate (manufactured by 790R) at 0.5 ml / cm. 2 After adding each solution individually, the mixture was incubated at 37°C for 45 minutes, and the culture surface of the 96-well plate (manufactured by 1060R) was coated with adhesion molecule A. <Process B> Next, iCell® Cardiomyocytes, which are induced pluripotent stem cell-derived cardiomyocytes, were placed in each well of a coated 790R 96-well plate. 2 (Cellular Dynamics International, model number C1016) were suspended in the provided thawing medium for cardiomyocytes and thawed at a concentration of 7.5 × 10 4 The cells were seeded at a seeding density of 100 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, and 150 μL of cardiomyocyte maintenance medium was added. The cells were then cultured at 37°C in a 5% CO2 atmosphere. <Process C> Two days after seeding the cardiomyocytes, half of the cardiomyocyte maintenance medium in each well of the 790R 96-well plate was replaced. Furthermore, three days after seeding the cardiomyocytes, isoprotein Isoproterenol hydrochloride (TOCRIS, model number 1747) was dissolved in purified water and diluted with medium. 16.6 μL of this solution was added to three wells of a 790R 96-well plate to adjust the final isoproterenol concentration in each well to 1000 nM. Further serial dilutions were performed to prepare triplicate wells containing 100 nM and 10 nM isoproterenol. Furthermore, 16.6 μL of fresh medium without isoproterenol was added to each of the three wells. This resulted in triplicate wells containing 1000 nM, 100 nM, 10 nM, and 0 nM isoproterenol (no isoproterenol). After the addition of isoproterenol, the cells were incubated for an additional 1 hour at 37°C in a 5% CO2 atmosphere. Pulsatility and drug responsiveness were then evaluated. The results are shown in Table 1.

[0081] Example 2 Except for using the culture vessel prepared as follows, various operations and evaluations were carried out in the same manner as in Example 1. The results are shown in Table 1. <Preparation of culture vessel> A hydrogenated ring-opening polymer of a norbornene-based monomer (manufactured by Zeon Corporation, product name "ZeonorFilm (registered trademark) ZF16-188"; Tg: 161°C; hereinafter referred to as "ZF16") and a hydrogenated ring-opening polymer of a norbornene-based monomer (manufactured by Zeon Corporation, product name "ZEONOR (registered trademark) 1060R"; Tg: 100°C; hereinafter referred to as "1060R") were used as the alicyclic structure-containing polymer to form a culture medium with a base area of 0.32 cm2, with the culture surface made of ZF16, by insert molding. 2 A well plate having 96 cylindrical wells was obtained as a culture vessel. The culture vessel was then sterilized with ethylene oxide gas.

[0082] Example 3 Various operations and evaluations were carried out in the same manner as in Example 1, except that an addition copolymer of norbornene and ethylene (manufactured by Polyplastics Co., Ltd., product name "TOPAS (registered trademark) 6013M-07"; Tg: 142°C; hereinafter referred to as "COC") was used instead of 790R as the alicyclic structure-containing polymer. The results are shown in Table 1.

[0083] (Comparative Example 1) Various procedures and evaluations were performed in the same manner as in Example 1, except that vitronectin (ThermoFisher Scientific, model number "A14700"), which has a cell adhesion activity peptide sequence (RGD sequence) but no affinity peptide sequence, was used instead of adhesion molecule A. The results are shown in Table 1.

[0084] (Comparative Example 2) Except for using COC instead of 790R as the alicyclic structure-containing polymer and using vitronectin instead of adhesion molecule A as the adhesion molecule, various procedures and evaluations were carried out in the same manner as in Example 1. The results are shown in Table 1.

[0085] (Comparative Example 3) Various operations and evaluations were performed in the same manner as in Comparative Example 1, except that a polystyrene 96-well plate (manufactured by Corning Incorporated, Falcon (registered trademark), model number "353916"; Tg: 100°C; sterilized, vacuum gas plasma surface treated) was used instead of the 790R 96-well plate as the culture vessel. The results are shown in Table 1.

[0086] In addition, in Table 1 shown below, "iPS" stands for induced pluripotent stem cell-derived cardiomyocytes, "PS" indicates polystyrene; "Tg" indicates the glass transition temperature, The term "affinity peptide sequence" refers to a peptide sequence having affinity for an alicyclic structure-containing polymer.

[0087] [Table 1]

[0088] From Table 1, it can be seen that in Examples 1 to 3, which used a culture vessel made of an alicyclic structure-containing polymer and having a culture surface with a surface free energy within a predetermined range and underwent predetermined processes, the pulsation of cardiomyocytes could be evaluated in a state where the pulsation of the cardiomyocytes was improved. On the other hand, in Comparative Examples 1 and 2, in which the adhesion molecules do not have affinity peptide sequences and laminin cell adhesion activity peptide sequences, and in Comparative Example 3, in which the adhesion molecules do not have affinity peptide sequences and laminin cell adhesion activity peptide sequences and the surface free energy of the culture surface is outside the specified range, it can be seen that the pulsatility of cardiomyocytes is reduced compared to Examples 1 to 3. [Industrial Applicability]

[0089] According to the present invention, it is possible to provide a method capable of evaluating the pulsation of cardiomyocytes in a state in which the pulsatility of the cardiomyocytes is improved. Furthermore, according to the present invention, it is possible to provide an adhesion molecule that has affinity for an alicyclic structure-containing polymer and that can improve the pulsatility of cardiomyocytes.

Claims

1. A method for evaluating the beating of cardiomyocytes in a culture vessel, comprising: Coating the culture surface of the culture vessel with adhesion molecules; Adhering the cardiomyocytes to the coated culture surface in the presence of a medium; Culturing the cardiomyocytes until pulsation of the cardiomyocytes is observed. Including, the culture surface of the culture vessel is made of an alicyclic structure-containing polymer, The surface free energy of the culture surface is 30 mN / m or more and 37 mN / m or less, The method, wherein the adhesion molecule has a peptide sequence having affinity for the alicyclic structure-containing polymer and a peptide sequence exhibiting laminin cell adhesion activity.

2. The method of claim 1, wherein the cardiomyocytes are induced pluripotent stem cell-derived cardiomyocytes.

3. 2. The method according to claim 1, wherein the peptide sequence having affinity for the alicyclic structure-containing polymer is Thr-Val-Asp-Ser-Cys-Leu-Thr (SEQ ID NO: 1).

4. The method of claim 1, wherein the laminin cell adhesion active peptide sequence is Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 2).

5. the peptide sequence having affinity for the alicyclic structure-containing polymer is Thr-Val-Asp-Ser-Cys-Leu-Thr (SEQ ID NO: 1); The method of claim 1, wherein the laminin cell adhesion active peptide sequence is Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 2).

6. The method of claim 1, wherein the molecular weight of the adhesion molecule is 1,000 or more and 4,000 or less.

7. The method according to any one of claims 1 to 6, wherein the alicyclic structure-containing polymer is a hydrogenated norbornene ring-opening polymer.

8. An adhesion molecule having affinity for an alicyclic structure-containing polymer, which has the structures Thr-Val-Asp-Ser-Cys-Leu-Thr (SEQ ID NO: 1) and Tyr-Ile-Gly-Ser-Arg (SEQ ID NO: 2).

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