Method for producing myocardial cell layer, myocardial cell layer, and use thereof

Abstract

The present invention addresses the problem of providing: a method for producing a myocardial cell layer, whereby it becomes possible to mature myocardial cells without using a complicated control device and maintain the myocardial cells for a long period; a myocardial cell layer; a kit for evaluating a drug candidate substance: a transplantation material; and a method for evaluating a drug candidate substance. According to the present invention, a method for producing a myocardial cell layer is provided, the method comprising the following steps (1) to (3). (1) A step for seeding myocardial cells induced from a pluripotent stem cell in a single layer on a seeding surface having a shape shown in (1-1) to form a myocardial cell layer: (1-1) when the extension of the seeding surface in the horizontal direction is defined as a flat diagram, the shape is one formed by hollowing out at least a portion of a first flat diagram having a uniform extension and having an arbitrary shape in the shape of a second flat diagram having a uniform extension and having another arbitrary shape while avoiding the contact of the second flat diagram with the outer periphery of the first flat diagram having the arbitrary shape, in which the average value of the length of the outer periphery of the first flat diagram and the length of the outer periphery of the second flat diagram satisfies the following formula: x ≧ v / 10 wherein x represents an average value of the length of the outer periphery of the first flat diagram and the length of the outer periphery of the second flat diagram and is expressed in cm, and v represents a speed of the propagation of the myocardial cells derived from the pluripotent stem cell and is expressed in cm∙s-1; (2) a step for allowing an electrical signal or a calcium signal to progress in the myocardial cell layer while circulating the signal; and (3) a step for performing the culture for 8 days or longer while maintaining the state mentioned in (2) above.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a mature cardiomyocyte layer that can be maintained for a long period of time.The present invention further relates to a mature cardiomyocyte layer that can be maintained for a long period of time.The present invention further relates to a kit for evaluating a drug candidate substance, a transplant material, and a method for evaluating a drug candidate substance, using the above-mentioned cardiomyocyte layer. [Background technology]

[0002] In recent years, methods for inducing differentiation of human induced pluripotent stem cells (human iPS cells) into cardiomyocytes have been improved, and issues such as cardiomyocyte purity and cost are being resolved. However, it is known that the cardiomyocytes obtained are immature cardiomyocytes of the human fetal stage. Stimulation methods using devices and physical stimulation methods (e.g., electrical or mechanical stimulation) have been reported as methods for maturing immature cardiomyocytes (Non-Patent Documents 1 to 6). In these reports, after constructing a striped or ring-shaped myocardial tissue with a three-dimensional structure, external mechanical or electrical stimulation is applied to mature the myocardial tissue. However, maturation methods that apply external mechanical or electrical stimulation have problems such as causing cell damage, making it impossible to maintain myocardial tissue over the long term, and making the control device complex and difficult to operate.

[0003] A simpler method is known, which involves constructing a three-dimensional cell ring and promoting the maturation of cardiomyocytes (Patent Document 1). However, cardiomyocytes matured by this method have a very high metabolic rate, and if the three-dimensional structure is left as it is, the supply of nutrients to the cells in the center of the tissue becomes insufficient, making it difficult to maintain the cells for a long period of time (Non-Patent Document 7). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nature volume 556, pages239-243(2018) [Non-patent document 2] Nature Methods volume 10, pages 781-787(2013) [Non-patent document 3] Nature Communications volume 11, Article number:75(2020) [Non-patent document 4] Scientific Reports volume 10, Article number:6919(2020) [Non-patent document 5] Circulation. 2017 May 9;135(19):1832-1847 [Non-patent document 6] Scientific Reports volume 4,Article number:4781(2014) [Non-Patent Document 7] Communications Biology volume 3, Article number:122(2020) [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2018 / 124210 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for producing a cardiomyocyte layer, which allows cardiomyocytes to mature and be maintained for a long period of time without using a complex control device, and a cardiomyocyte layer.A further object of the present invention is to provide a kit for evaluating a drug candidate substance, a transplant material, and a method for evaluating a drug candidate substance, which use the above-mentioned cardiomyocyte layer. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that by culturing cells as a two-dimensional cell layer (monolayer) rather than a three-dimensional cell ring as described in Patent Document 1, the problem of nutrient supply as well as cardiomyocyte maturation can be solved. Furthermore, while it was known that cardiomyocytes must be cultured in a closed loop shape (e.g., a ring shape) to mature, the present inventors have found that long-term cell maintenance is possible when a specific relationship is satisfied between the perimeter of the closed loop shape and the conduction velocity of the cardiomyocytes (a parameter related to cardiac contraction). The present invention was completed based on the above findings.

[0008] That is, according to the present invention, the following inventions are provided. <1> A method for producing a cardiomyocyte layer, comprising the following steps (1) to (3): (1) Seeding cardiomyocytes induced from pluripotent stem cells in a single layer onto a seeding surface having the shape shown in (1-1) below to form a cardiomyocyte layer; (1-1) When the horizontal extent of the sowing surface is considered as a plane figure, the shape is a shape in which at least a part of a first plane figure of any shape having a uniform extent is hollowed out with a second plane figure of another arbitrary shape having a uniform extent without contacting the periphery of the first plane figure of any shape, and the average value of the periphery length of the first plane figure and the periphery length of the second plane figure satisfies the following formula: x ≥ v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is; (2) propagating at least one circulating electrical signal or calcium signal within the myocardial cell layer; (3) Maintain the conditions in (2) above and culture for 8 days or more. <2> The pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells; <1> The manufacturing method described in <3> The expression level of β-MHC in the myocardial cell layer is 10% or more of the expression level in normal adult myocardial cells. <1> or <2> The manufacturing method described in <4> The expression level of Connexin-43 in the myocardial cell layer is 1.5 times or more than that of control cells not treated with circulation waves. <1> from <3> The manufacturing method according to any one of the above. <5> Conduction velocity in the myocardial cell layer is 10 cm s -1 That's all. <1> from <4> The manufacturing method according to any one of the above. <6> The amount of VEGF secreted from the myocardial cell layer is more than twice the amount secreted from control cells not treated with the circulation wave. <1> from <5> 10. The manufacturing method according to claim 9 . <7> The hypoxia tolerance of the myocardial cell layer is improved compared to control cells not treated with circulatory waves. <1> from <6> The manufacturing method according to any one of the above. <8> The first plane figure is a circle, and the second plane figure is a circle having a diameter smaller than that of the circle of the first plane figure. <1> from <7> The manufacturing method according to any one of the above. <9> The first plane figure has a shape obtained by cutting out a substantially rectangular shape from a circle so as to be in contact with the circumference of the circle, and the second plane figure has a shape of a Landolt ring, At least a part of the first planar figure is hollowed out so that the rectangle is inserted between the gaps of the Landolt ring by the second planar figure. <1> from <7> The manufacturing method according to any one of the above. <10> The first plane figure is concave and the second plane figure is a smaller concave shape than the first plane figure. <1> from <7> The manufacturing method according to any one of the above. <11> The expression level of one or more myocardial maturation markers selected from TNNI3, ITGA7, IGF1, SCN5A, and KCNJ11 in the myocardial cell layer is 1.3 times or more the expression level in control cells not treated with circulation waves. <1> from <10> The manufacturing method according to any one of the above. <12> Further comprising a step of co-culturing with fibroblasts, <1> from <11> The manufacturing method according to any one of the above. <13> Forming a cardiomyocyte layer on a biodegradable or non-biodegradable scaffold; <1> from <12> The manufacturing method according to any one of the above. <14> The seeding density of cardiomyocytes in step (1) is 2×10 5 ~1×10 6 pieces / cm 2 That is, <1> from <13> 1. The method according to claim 1 , <15> The pluripotent stem cells are pluripotent stem cells having a gene mutation associated with heart disease; <1> from <14> The manufacturing method according to any one of the above. <16> <1> from <15> A myocardial cell layer obtained by the manufacturing method described in any one of the above. <17> A cardiomyocyte layer having the following characteristics (1) to (5): (1) Cardiomyocytes derived from pluripotent stem cells, (2) It is a single layer. (3) At least one of the following conditions (3-1) to (3-8) is satisfied: (3-1) The expression level of β-MHC in the myocardial cell layer is 10% or more of that of normal adult myocardial cells; (3-2) The amount of VEGF secreted from the myocardial cell layer was more than twice that of control cells not treated with the circulation wave; (3-3) The expression level of Connexin-43 in the myocardial cell layer was more than 1.5 times higher than that of control cells not treated with circulatory waves; (3-4) The expression level of TNNI3 in the myocardial cell layer was more than twice that of control cells not treated with circulatory waves; (3-5) The expression level of ITGA7 in the myocardial cell layer was more than 1.2 times higher than that of control cells not treated with circulatory waves; (3-6) The expression level of IGF1 in the myocardial cell layer was more than 1.5 times that of control cells not treated with circulatory waves; (3-7) The expression level of SCN5A in the myocardial cell layer was more than 1.2 times higher than that of control cells not treated with circulatory waves; (3-8) The expression level of KCNJ11 in the myocardial cell layer was more than 1.3 times higher than that of control cells not treated with circulatory waves; (4) When the horizontal spread of the sowing surface is considered as a plane figure, the shape is such that at least a part of a first plane figure of any shape having a uniform spread is hollowed out by a second plane figure of another arbitrary shape having a uniform spread, without touching the periphery of the first plane figure of any shape, (5) The average value of the perimeter length of the first plane figure and the perimeter length of the second plane figure satisfies the following formula. x ≥ v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is; <18> <16> or <17> A kit for evaluating a drug candidate substance, comprising the cardiomyocyte layer according to claim 1. <19> <16> or <17> A transplant material comprising the cardiomyocyte layer described in . <20> (1) <16> or <17> contacting the cardiomyocyte layer described in the above with a candidate substance; (2) detecting an electrical signal or a calcium signal in the myocardial cell layer contacted with the candidate substance; and (3) comparing the electrical signal or calcium signal detected in step (2) with a control electrical signal or calcium signal; A method for evaluating a drug candidate substance, comprising: [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a cardiomyocyte layer that can mature cardiomyocytes without using a complicated control device and maintain them for a long period of time. The cardiomyocyte layer of the present invention can be used as a transplant material or for evaluating candidate pharmaceutical substances. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 shows the geometry of the PDMS sheet. [Figure 2] FIG. 2 shows the shape of the culture vessel. [Figure 3] Figure 3 shows the relationship between the pulsation frequency and the stability of the circulation wave when cardiomyocytes B are seeded in a culture vessel. [Figure 4] Figure 4 shows the relationship between the beating frequency and the stability of the circulation wave when cardiomyocytes C of various purities were seeded in a culture vessel. [Figure 5] FIG. 5 shows the results of measuring the expression of β-MHC in cardiomyocytes A. [Figure 6] FIG. 6 shows the results of measuring the expression of Connexin-43 in cardiomyocytes A. [Figure 7] FIG. 7 shows the results of measuring the conduction velocity of cardiomyocyte A. [Figure 8] FIG. 8 shows the results of measuring the amount of VEGF secreted by cardiomyocytes A. [Figure 9] FIG. 9 shows the results of measuring the hypoxia tolerance of cardiomyocytes A. [Figure 10] FIG. 10 shows the results of confirming the circulation wave and pulsation frequency when cardiomyocytes C were cultured on a scaffold. [Figure 11] FIG. 11 shows the results of confirming the expression of myocardial maturation markers when cardiomyocytes C were cultured on a scaffold. [Figure 12] FIG. 12 shows the results of measuring the electrical signals when cardiomyocytes B were cultured on different scaffolds. [Figure 13] FIG. 13 shows the results of measuring the electrical signal, QT interval, and beating frequency when cardiomyocytes C were cultured on a scaffold. [Figure 14] FIG. 14 shows the results of evaluating the drug E-4031 using cardiomyocytes C. [Figure 15] FIG. 15 shows the results of evaluating the drug ranolazine using cardiomyocytes C. [Figure 16] FIG. 16 shows the results of evaluating the drug mexiletine using cardiomyocytes C. [Figure 17] FIG. 17 shows the results of evaluating the drug isoproterenol using cardiomyocytes C. [Figure 18] FIG. 18 shows the results of evaluating the drug aspirin using cardiomyocytes C. [Figure 19] FIG. 19 shows the results of evaluating the drug verapamil using cardiomyocytes C. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0012] <Method for producing myocardial cell layer> The method for producing a cardiomyocyte layer of the present invention comprises the following steps (1) to (3). (1) Seeding cardiomyocytes induced from pluripotent stem cells in a single layer onto a seeding surface having the shape shown in (1-1) below to form a cardiomyocyte layer; (1-1) When the horizontal extent of the sowing surface is considered as a plane figure, the shape is a shape in which at least a part of a first plane figure of any shape having a uniform extent is hollowed out with a second plane figure of another arbitrary shape having a uniform extent without contacting the periphery of the first plane figure of any shape, and the average value of the periphery length of the first plane figure and the periphery length of the second plane figure satisfies the following formula: x ≥ v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is; (2) propagating at least one circulating electrical signal or calcium signal within the myocardial cell layer; (3) Maintain the conditions in (2) above and culture for 8 days or more.

[0013] Pluripotent stem cells refer to cells that have the pluripotency to differentiate into all cells that constitute an adult body and the ability to self-renew, maintaining this pluripotency even after cell division. Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and embryonic germ cells (EG cells). Preferably, the pluripotent stem cells are induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells).

[0014] The species of pluripotent stem cells is not particularly limited, but is preferably mammalian, more preferably rodent or primate, and even more preferably primate.Monkey or human pluripotent stem cells, particularly monkey or human ES cells and iPS cells, can be preferably used.In one embodiment, the pluripotent stem cells are human iPS cells.

[0015] Methods for inducing cardiomyocytes from pluripotent stem cells are known, and examples thereof include the methods described in International Application Publication Nos. 2013 / 111875, 2015 / 182765, 2020 / 027278, and Minami I, et al., Cell Rep. 2012 Nov 29;2(5):1448-60. Cardiomyocytes induced from pluripotent stem cells can be commercially available as cardiomyocytes derived from ES cells or iPS cells (e.g., iCell® Cardiomyocytes (manufactured by FUJIFILM Cellular Dynamics, Inc.) and iCell® Cardiomyocytes (manufactured by FUJIFILM Cellular Dynamics, Inc.)). 2 (manufactured by FUJIFILM Cellular Dynamics, Inc.)

[0016] Furthermore, pluripotent stem cells having a genetic mutation associated with cardiac disease may be used. Cardiac disease models can be established by inducing cardiomyocytes using pluripotent stem cells having a mutation in a gene associated with cardiac disease (e.g., the MYBPC3 gene, the DSG2 gene, the TNNI3 gene, etc.). Such cardiac disease models can be used in drug discovery research.

[0017] As an example, cardiomyocytes induced from pluripotent stem cells by the method described in International Patent Publication No. 2015 / 182765 can be used. Briefly, cardiomyocytes can be induced to differentiate from pluripotent stem cells by a method comprising the steps of: (a) culturing pluripotent stem cells in a medium containing a WNT signaling activator and a PKC activator; and (b) culturing the cells obtained in step (a) in a medium containing a WNT signaling inhibitor, an Src inhibitor, and an EGF receptor inhibitor.

[0018] The WNT signal activator refers to a substance that activates the WNT signal pathway. Examples of the WNT signal activator include GSK3β inhibitors such as BIO, CHIR99021, and TWS119. The WNT signal inhibitor refers to a substance that inhibits the WNT signal pathway. Examples of the WNT signal inhibitor include compounds such as the compound of formula (I) or its salt described in International Publication No. 2012 / 026491, IWP2, IWP4, XAV939, and IWR1.

[0019] The PKC activator refers to a substance that activates protein kinase C (PKC) or its downstream signal transduction pathway. Examples of the PKC activator include phorbol 12-myristate 13-acetate (PMA), prostratin, bryostatin 1, bryostatin 2, FR236924, (-)-indolactam V, PEP005, and phorbol 12,13-dibutyrate. 12,13-dibutyrate, SC-9, SC-10, 1-oleoyl-2-acetyl-sn-glycerol, 1-O-hexadecyl-2-O-arachidonyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, PIP2, resiniferatoxin, phorbol 12,13-dihexanoate, mezerein, ingenol 3-angelate 3-Angelate), RHC-80267, DCP-LA, Lipoxin A4, etc.

[0020] The term "Src inhibitor" refers to a substance that inhibits the tyrosine kinase Src or its downstream signal transduction pathway. Examples of Src inhibitors include A419259, SU6656, PP1, 1-naphthyl PP1, PP2, indirubin-3'-(2,3-dihydroxypropyl)-oximether, TX-1123, Src Kinase Inhibitor I (CAS 179248-59-0), AZM475271, bosutinib, herbimycin A, KB SRC 4, MNS, PD166285, and TC-S7003.

[0021] An EGF receptor inhibitor (also referred to as an EGFR inhibitor) refers to a substance that inhibits signal transduction from the EGF receptor. EGF receptor inhibitors include AG1478, gefitinib, afatinib, ARRY334543, AST1306, AZD8931, BIBU1361, BIBX1382, BPDQ, BPIQ-I, BPIQ-II, canertinib, CL-387,785, CUDC101, dacomitinib, vandetanib, EGFR Inhibitor III (N-(4-((3,4-dichloro-6-fluorophenyl)amino)-quinazolin-6-yl)-2-chloroacetamide, CAS 733009-42-2), and EGFR / ErbB-2 Inhibitor (4-(4-benzyloxyanilino)-6,7-dimethoxyquinazoline, CAS 733009-42-2). 179248-61-4), Erlotinib, GW583340, GW2974, HDS029, Lapatinib, WHI-P154, OSI-420, PD153035, PD168393, PD174265, Pelitinib, Compound 56, XL657, PP3, AG-490, AG555, Tyrphostin B42, Tyrphostin B44, AG556, AG494, AG825, RG-13022, DAPH, EGFR Inhibitor (Cyclopropanecarboxylic acid-(3-(6-(3-trifluoromethyl-phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide, CAS 879127-07-8), Erbstatin Analog (Methyl 2,5-dihydroxycinnamate, CAS 63177-57-1), JNJ28871063, Tyrophostin 47, Lavendustin A, Lavendustin C, Lavendustin C methyl ester, LFM-A12, TAK165, TAK285, Tyrophostin 51, Tyrophostin AG183, Tyrophostin AG528, Tyrophostin AG99, Tyrophostin RG14620, WZ3146, WZ4002, WZ8040, butein, Tyrophostin AG112, and the like.In some embodiments, the EGF receptor inhibitor is an EGF receptor inhibitor having a quinazoline skeleton, such as AG1478, gefitinib, afatinib, ARRY334543, AST1306, AZD8931, BIBU1361, BIBX1382, BPDQ, BPIQ-I, BPIQ-II, canertinib, CL-387,785, CUDC101, dacomitinib, vandetanib, EGFR Inhibitor III (CAS 733009-42-2), EGFR / ErbB-2 Inhibitor (CAS 179248-61-4), erlotinib, GW583340, GW2974, HDS029, lapatinib, WHI-P154, OSI-420, PD153035, PD168393, PD174265, pelitinib, Compound 56, or XL657. EGF receptor inhibitors are available from Santa Cruz Biotech and other companies.

[0022] In one example, the medium in step (b) contains two or more WNT signal inhibitors, one of which is any of the compounds of formula (I) above or a salt thereof, and the other WNT signal inhibitor is one or more compounds selected from IWP2, XAV939, and IWR1, particularly XAV939.

[0023] In one example, the WNT signaling activator is BIO or CHIR99021, particularly CHIR99021. In one example, the PKC activator is PMA or prostratin, in particular PMA. In one example, the Src inhibitor is A419259 or SU6656, particularly A419259. In one example, the EGF receptor inhibitor is AG1478 or gefitinib, particularly AG1478.

[0024] In one example, cells are cultured in suspension in steps (a) and (b). In one example, step (a) is carried out for 1 to 3 days, and then step (b) is carried out immediately after the end of step (a) or 1 to 2 days after the end of step (a), for 2 to 13 days, preferably 3 to 10 days, more preferably 4 to 10 days, and even more preferably 4 to 8 days.

[0025] As another example of inducing cardiomyocytes from pluripotent stem cells, cardiomyocytes induced from pluripotent stem cells by the method described in International Patent Publication No. 2020 / 027278 can be used. Briefly, cardiomyocytes can be differentiated by a method including the steps of: (c) forming multiple embryoid bodies by spheroid culture of pluripotent stem cells; and (d) subjecting the multiple embryoid bodies obtained in step (c) to three-dimensional suspension culture to induce differentiation into cardiomyocytes.

[0026] The spheroid culture in step (c) can be carried out for typically 3 to 5 days, preferably 4 days, and the three-dimensional suspension culture in step (d) can be carried out for typically 9 to 15 days, preferably 10 to 14 days, more preferably 11 to 13 days, and even more preferably 12 days.

[0027] In step (c), a medium containing BMP-4, activin A, and bFGF can be used. More specifically, for example, the culture method can be performed under the following conditions. Day 0~:BMP-4 final concentration 2ng / ml Day 1~:Activin A final concentration 12ng / ml, BMP-4 final concentration 10ng / ml, bFGF final concentration 10ng / ml In step (d), a medium containing VEGF, IWP-3, SB431542, Dorsomorphin, and bFGF can be used. More specifically, for example, the culture method can be performed under the following conditions. Day 4~: VEGF final concentration 10ng / ml, IWP-3 final concentration 1μM, SB431542 final concentration 5.4μM, Dorsomorphin final concentration 3μM Day 8~: VEGF final concentration 10ng / ml, bFGF final concentration 5ng / ml

[0028] At least a portion of the cardiomyocytes induced from pluripotent stem cells may express a gene encoding a calcium sensor protein. The calcium sensor protein is not particularly limited as long as it emits a signal, such as luminescence, in response to changes in intracellular calcium concentration. Examples include luminescent proteins such as aequorin and aequorin-GFP fusion protein, and fluorescent proteins such as cameleon, FIP-CB SM, TN-L15, YC6.1, F2C, Camgaroo, G-CaMP, and Pericam. In one embodiment, the calcium sensor protein is G-CaMP, a calcium sensor protein genetically engineered to green fluorescent protein (EGFP), calmodulin (CaM), or myosin light chain fragment (M13), or a variant thereof, such as G-CaMP, GCaMP1.6, GCaMP2, GCaMP3, GCaMP6f, GCaMP6m, or GCaMP6s, particularly GCaMP3.

[0029] In the present invention, cardiomyocytes induced from pluripotent stem cells are seeded in a monolayer on a seeding surface having the shape shown in (1-1) below to form a cardiomyocyte layer. (1-1) When the horizontal extent of the sowing surface is considered as a plane figure, the shape is a shape in which at least a part of a first plane figure of any shape having a uniform extent is hollowed out with a second plane figure of another arbitrary shape having a uniform extent without contacting the periphery of the first plane figure of any shape, and the average value of the periphery length of the first plane figure and the periphery length of the second plane figure satisfies the following formula: x ≥ v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is.

[0030] In the present invention, the problem of nutrient supply can be solved by culturing cardiomyocytes induced from pluripotent stem cells in a monolayer (two-dimensional) culture, which has the advantages of being easy to collect and store, and easy to integrate into three-dimensional (3D) scaffold materials.

[0031] When cardiomyocytes induced from pluripotent stem cells are cultured in a monolayer (two-dimensional) culture, the cardiomyocytes may be co-cultured with cells other than the cardiomyocytes, such as fibroblasts. It is preferable to co-culture cardiomyocytes with cells other than cardiomyocytes, such as fibroblasts, rather than culturing them alone. When co-cultured, the ratio of cardiomyocytes to all cells (purity of cardiomyocytes) is preferably 50-90%, more preferably 60-90%, and even more preferably 70-90%.

[0032] To mature cardiomyocytes induced from pluripotent stem cells, the cardiomyocyte layer must be shaped like a closed loop (for example, but not limited to, a ring). Specifically, a closed loop shape refers to a shape in which, when the horizontal extent of the cardiomyocyte layer is considered as a planar figure, at least a portion of a first planar figure of any shape with uniform expansion is hollowed out by a second planar figure of another arbitrary shape with uniform expansion, without contacting the periphery of the first planar figure. By culturing cardiomyocytes induced from pluripotent stem cells in the closed loop shape described above, spontaneous fast propagation of action potentials is induced (i.e., spontaneous traveling waves (TW) are induced), and by continuing to culture in this state, the cardiomyocytes mature.

[0033] Cardiomyocytes induced from pluripotent stem cells may differ in characteristics and purity depending on the method of producing the pluripotent stem cells and the method of inducing differentiation used. One of the important characteristics regarding the formation and maturation of cardiomyocytes induced from pluripotent stem cells is the conduction velocity of the cardiomyocytes (a parameter related to the cardiac beat rate). In the present invention, it was found that cardiomyocytes with high conduction velocity require a large closed loop circumference.

[0034] More specifically, if the horizontal extent of the sowing surface is considered to be a plane figure, the shape is such that at least a portion of a first plane figure of any shape having a uniform extent is hollowed out with a second plane figure of another arbitrary shape having a uniform extent, without contacting the periphery of the first plane figure of any shape, and the average value of the periphery length of the first plane figure and the periphery length of the second plane figure must satisfy the following formula. x ≥ v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is. Preferably, the formula x≧v / 9, x≧v / 8, x≧v / 7, or x≧v / 6 is satisfied, and more preferably, the formula x≧v / 6 is satisfied.

[0035] As long as the average value of the perimeter length of the first plane figure and the perimeter length of the second plane figure satisfies the above formula, the shape of the closed loop is not particularly limited.

[0036] As described above, the shape of the closed loop of the myocardial cell layer is such that at least a portion of a first planar figure is hollowed out with a second planar figure of another arbitrary shape having uniform extent, without contacting the outer periphery of the first planar figure of the arbitrary shape.

[0037] A first example of a closed loop shape is when the first plane figure is a circle and the second plane figure is a circle having a smaller diameter than the circle of the first plane figure.

[0038] A second example of a closed loop shape is when the first planar figure is a shape obtained by cutting an approximately rectangular shape out of a circle so that it is tangent to the circumference of the circle, the second planar figure is a Landolt ring shape, and at least a portion of the first planar figure is hollowed out by the second planar figure so that the rectangle is inserted between the gaps in the Landolt ring.

[0039] A third example of a closed loop shape is a case where the first plane figure is a concave shape and the second plane figure is a concave shape that is smaller than the first plane figure.

[0040] Other examples of closed loop shapes include when the first plane figure is (1) a plane figure made up of curves (e.g., a circle, an ellipse), (2) a plane figure made up of straight lines (e.g., a triangle, a rectangle, a pentagon, a hexagon), (3) a plane figure made up of curves and straight lines (e.g., a semicircle, a Reuleaux polygon), or (4) a figure obtained by superimposing the above (1) to (3), and the second plane figure is any of the above (1) to (4) that is smaller than the first plane figure.

[0041] A vessel for culturing cardiomyocytes induced from pluripotent stem cells has at least one convex portion on the inner bottom. The inner bottom of the culture vessel refers to the part that forms the inner bottom surface of the culture vessel and forms the surface on the side that receives and cultures cardiomyocytes. The convex portion on the inner bottom of the culture vessel is formed so that the cardiomyocyte layer can take the closed loop shape described above.

[0042] The convex portion present on the inner bottom of the culture vessel may be integral with the main body portion or may be separate. If separate, it may be adhered to the main body portion or may be placed on the main body portion without being adhered to the main body portion. For adhesion, polymers exemplified as the material for the convex portion or commercially available biocompatible adhesives, such as silicone adhesives, can be used.

[0043] The main body of the culture vessel may be any vessel commonly used for cell culture, and is not particularly limited. The shape of the main body may be a Petri dish, plate, bottle, chamber, multiwell plate (e.g., 6-, 12-, 24-, 48-, 96-, or 384-well plate), etc. The material of the main body is not particularly limited. Specific examples include inorganic materials such as metal, glass, and silicone, and organic materials such as plastics (e.g., polystyrene resin, polyethylene resin, polypropylene resin, ABS resin (acrylonitrile-butadiene-styrene resin), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, polyvinyl chloride resin, and polytetrafluoroethylene tetrafluoroethylene resin). The main body may be a commonly available cell culture vessel, or may be manufactured by a method known to those skilled in the art. For example, a main body made of a plastic material can be manufactured by a conventional molding method, such as injection molding. In one embodiment, the main body is a plastic Petri dish or multiwell plate.

[0044] The material of the protrusions is not particularly limited as long as it does not adversely affect cell culture. For example, it may be selected from the same materials as those of the main body portion described above, and may be the same or different from the material of the main body portion. Furthermore, the material of the protrusions may be selected from the materials of the main body portion described above, as well as polymers such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyamide (PA), polymethyl glutarimide (PMGI), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene vinyl acetate (PEVA), and polyethylene oxide (PEO), but is not limited to these. For example, the material of the protrusions is PDMS.

[0045] The inner surface of the body portion may be cell-adhesive or non-cell-adhesive. At least the inner surface portion of the body portion may be made of a cell-adhesive or non-cell-adhesive material. The material of the body portion may be a cell-adhesive or non-cell-adhesive material, or at least the inner surface portion of the body portion may be coated with a cell-adhesive or non-cell-adhesive material. In one example, the inner surface of the body portion is non-cell-adhesive.

[0046] The surface of the convex portions may be cell-adhesive or non-cell-adhesive. At least the surface portion of the convex portions may be made of a cell-adhesive or non-cell-adhesive substance. The material of the convex portions may be a cell-adhesive or non-cell-adhesive substance, or at least the surface portion of the convex portions may be coated with a cell-adhesive or non-cell-adhesive substance. In one embodiment, the surface of the convex portions is non-cell-adhesive.

[0047] Examples of non-cell-adhesive substances for coating include celluloses such as MPC (2-methacryloyloxyethyl phosphorylcholine) polymer, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose; polyethylene oxide; carboxyvinyl polymer; polyvinylpyrrolidone; polyethylene glycol; polyamides such as polyacrylamide and poly-N-isopropylacrylamide; polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch, pectin, carrageenan, guar gum, gum arabic, and dextran; albumin and derivatives thereof.

[0048] Examples of substances having cell adhesive properties for coating include positively charged polymers such as poly-L-lysine and poly-L-ornithine, and extracellular matrices such as Matrigel (trademark), hyaluronic acid, laminin, fibronectin, vitronectin, and collagen.

[0049] An outer frame may be arranged around the convex portion. In this case, cardiomyocytes are seeded and cultured between the convex portion and the outer frame. The material of the outer frame can be selected according to the main body and convex portion of the culture vessel, and may be the same as or different from the main body or convex portion. The outer frame may be integral with the main body portion or may be separate. If separate, the outer frame may be adhered to the main body portion. For adhesion, the polymers exemplified as the material for the convex portion or commercially available biocompatible adhesives, such as silicone adhesives, can be used.

[0050] The inside of the outer frame may be any size so long as it can surround the convex portions. The inner dimensions of the outer frame can be selected so that there is a sufficient area between the convex portions and the outer frame for the formation of a cardiomyocyte layer. For example, the distance between the convex portions and the inner wall of the outer frame can be configured to be about 0.01 mm to about 10 mm, about 1 mm to about 9 mm, or about 4 mm to about 6 mm, e.g., about 2.5 mm. The shape and dimensions of the outside of the outer frame are not particularly limited as long as they fit within the main body of the culture vessel. Any number of outer frames may be present in one culture vessel. In one embodiment, one culture vessel has one convex portion and one outer frame. In one embodiment, one culture vessel has multiple convex portions, each of which is surrounded by one outer frame.

[0051] In some embodiments, the culture vessel does not include an outer frame. In such cases, the shape and dimensions of the sidewall of the culture vessel can be selected so that there is a sufficient area between the convex portion and the sidewall for the formation of a cardiomyocyte layer. For example, the distance between the convex portion and the sidewall can be configured to be about 0.01 mm to about 10 mm, about 1 mm to about 9 mm, or about 4 mm to about 6 mm, e.g., about 2.5 mm.

[0052] The height of the convex portion may be determined appropriately depending on the size of the main body portion, for example, about 0.1 mm to about 10 mm, 0.1 mm to about 5 mm, about 1 mm to about 5 mm, about 3 mm to about 5 mm, for example, about 2 to 4 mm. The height of the outer frame is not particularly limited, as long as it is sufficient to fill the interior of the outer frame with culture medium, and is determined appropriately depending on the size of the main body portion, for example. The height of the outer frame may be the same as or different from the height of the convex portion.

[0053] The culture medium, method, and conditions used for producing cardiomyocyte layers are not particularly limited as long as they are those generally used for culturing cardiomyocytes. For example, a medium containing 40% DMEM, 40% IMDM, 20% fetal bovine serum, 1% MEM non-essential amino acid solution, 0.1% penicillin-streptomycin, and 0.5% L-glutamine can be used.

[0054] The cardiomyocyte layer may be formed on a biodegradable or non-biodegradable scaffold. A cardiomyocyte layer can be produced by culturing cardiomyocytes using a culture vessel whose surface (culture surface) is coated with a biodegradable or non-biodegradable scaffold.

[0055] Biodegradable polymers can be used as biodegradable scaffolds. Examples of synthetic polymers include polyester polymers, polyamide polymers, and polycyanoacrylate polymers, with polylactic acid, polyglycolic acid, and glycolic acid-lactic acid copolymers being preferred. Biopolymers can also be used. Examples of biopolymers include polysaccharides, proteins, nucleic acids, and glycoproteins. Specifically, extracellular matrices such as collagen, laminin, fibronectin, elastin, tenascin, hyaluronic acid, fibrin, and proteoglycan can be used.

[0056] Non-biodegradable scaffolds can include polyethylene terephthalate, polypropylene, polystyrene, polycarbonate, nylon, Teflon, ABS, and the like. Examples of the shape of the biodegradable or biodegradable scaffold include films, meshes, woven fabrics, nonwoven fabrics, etc.

[0057] The seeding density of cardiomyocytes in step (1) is not particularly limited as long as it allows the formation of a cardiomyocyte layer, but is preferably 2×10 5 ~6×10 6 pieces / cm 2 and more preferably 4×10 5 ~1×10 6 pieces / cm 2 is.

[0058] The culture method includes adhesion culture. The medium may be changed, for example, once every 1 to 7 days, such as once every 4, 3, or 2 days. The culture period may be a period sufficient for the formation of a cardiomyocyte layer, and may be, for example, 0.5 days to 3 months, 1 day to 21 days, 2 days to 14 days, 5 days to 10 days, or 6 days to 8 days.

[0059] As described above, by culturing cardiomyocytes in a closed loop shape that satisfies the predetermined condition of x≧v / 10, spontaneous circulatory waves (TW) are induced within the myocardial cell layer, as described above. In other words, at least one electrical signal or calcium signal can be caused to circulate and progress (also referred to as a circulatory wave) within the myocardial cell layer. In the present invention, cardiomyocytes are cultured for 8 days or more (e.g., 8, 9, 10, 11, 12, 13, 14, or 14 days or more) while maintaining the state in which the electrical signal or calcium signal circulates and progresses, i.e., while maintaining the state in which the TW progresses.

[0060] The electrical signal refers to the change in membrane potential of the myocardial cells. The calcium signal refers to the Ca 2+ The term "circulation wave" refers to a wave of oscillation. Electrical and calcium signals circulate within the myocardial cell layer by traveling in a specific direction within the cell layer. In one embodiment, the electrical signals, calcium signals, and cardiac muscle pulsation exhibit a similar pattern. There may be one circulation wave (i.e., one electrical or calcium signal circulates within the myocardial cell layer), or there may be two, three, or more, and the number may fluctuate (e.g., decrease) during culture. Without being limited by theory, the generation of circulation waves tends to cause cardiomyocytes contained in the myocardial cell layer to adopt an oriented structure similar to that found in the living heart, and the maturity of the cardiomyocytes increases. Furthermore, the greater the number of circulation waves, the higher the orientation and maturity of the cardiomyocytes. Therefore, highly mature cardiomyocytes can be obtained with simple procedures. In the present invention, a myocardial cell layer in which at least one circulation wave is observed is selected.

[0061] Applying electrical or mechanical stimulation to enhance cardiomyocyte maturity is known in the art, and such steps may be performed in the above-described method, but are not required. In one embodiment, the cardiomyocyte layer is not subjected to electrical or mechanical stimulation at a frequency equivalent to the physiological heart rate. In one embodiment, the cardiomyocyte layer is not subjected to any electrical or mechanical stimulation.

[0062] In the present invention, electrical signals in the myocardial cell layer may be detected. By detecting the electrical signals, the function and maturity of cardiomyocytes in the myocardial cell layer can be evaluated.

[0063] Electrical signals can be detected, for example, by contacting the myocardial cell layer with a multi-electrode array. A multi-electrode array refers to an electrode in which a large number of microelectrodes, for example, 1 to 384, e.g., 64 or 96, are arranged on a substrate. Commercially available multi-electrode array (MEA) systems capable of measuring the extracellular potential of cultured cells include the multi-electrode array system from Multi-channel system MCS GmbH (Germany) and the MED system from AlphaMed Scientific. A multi-electrode array system uses a large number of microelectrodes arranged on a substrate to simultaneously observe electrical signals from multiple cells, or to electrically stimulate cells or tissues and observe their responses to the stimuli. Using a multi-electrode array system, cellular function can be evaluated without damaging the cells. Specifically, the multi-electrode array system measures action potential waveforms and can evaluate cardiomyocyte function based on the number of channels capable of detecting electrical signals, heart rate, potential amplitude, FPD, and the presence or absence of arrhythmia.

[0064] The myocardial cell layer can be easily removed from the culture vessel with tweezers or the like, and electrical signals can be measured by contacting the myocardial cell layer with the multi-electrode array. Alternatively, electrical signals from the myocardial cell layer can be measured by culturing the myocardial cells in a culture vessel in which a multi-electrode array and a convex portion are arranged so that at least a portion of the myocardial cell layer is formed on the multi-electrode array.

[0065] In the present invention, calcium signals in the myocardial cell layer may be detected. The calcium signals can be detected as changes in the intracellular calcium concentration of cardiomyocytes. By analyzing the calcium signals, the function and maturity of cardiomyocytes in the myocardial cell layer can be evaluated.

[0066] Ca 2+ Oscillations can be detected by standard methods, for example, using calcium detection reagents such as calcium-sensitive dyes. 2+ Cardiomyocytes are cultured in medium containing an ion-detecting dye, and changes in intracellular calcium levels are measured. 2+ Examples of ion-detecting dyes include fura-2, bis-fura2, indo-1, Quin-2, Quin-2AM, benzothiaza-1, benzothiaza-2, indo-5F, Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1, fluo-3, rhod-2, rhod-3, fura-4F, fura-5F, fura-6F, fluo-4, fluo-5F, fluo-5N, Oregon Green 488 BAPTA, calcium green, calcein, Fura-C18, calcium green-C18, calcium orange, calcium crimson, calcium green-5N, magnesium green, Oregon Green 488 BAPTA-1, Oregon Green 488 These dyes include, but are not limited to, BAPTA-2, X-rhod-1, Fura Red, Rhod-5F, Rhod-5N, X-Rhod-5N, Mag-Rhod-2, Mag-X-Rhod-1, Fluo-5N, Fluo-5F, Fluo-4FF, Mag-Fluo-4, aequorin, dextran conjugates, or derivatives of these dyes. Alternatively, if at least a portion of the myocardial cell layer expresses the gene for the calcium sensor protein listed above, intracellular Ca can be detected. 2+ Without the use of ion-detecting dyes, Ca 2+ Here, the myocardial cell layer is composed of myocardial cells that express calcium sensor protein genes, and these cells can detect calcium oscillations. 2+The vibrations may be detected in number or proportion.

[0067] Intracellular Ca 2+ The fluorescence intensity or luminescence of the ion-detecting dye or calcium sensor protein can be measured using, for example, a CCD camera, a luminometer, or any other suitable optical system. Commercially available optical systems include, for example, Qimaging (Exiblue). 2+ For vibration analysis, software such as ImageJ or Matlab may be used.

[0068] As an example, cardiac muscle cell beating, i.e., spontaneous contraction, may be detected using standard electrophysiological methods (e.g., patch clamping), elasticity detection devices (e.g., MicroTester (CellScale)), or a combination of these methods and devices.

[0069] In cardiomyocytes contained in the cardiomyocyte layer in which one circulation wave is observed, the expression level of β-MHC, a cardiomyocyte-specific marker indicating the maturity of cardiomyocytes, is preferably about 10% or more, about 15% or more, about 20% or more, about 25% or more, or about 30% or more of that of normal adult cardiomyocytes, and about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less of that of normal adult cardiomyocytes.

[0070] In the present invention, the expression of a myocardial maturation marker in cardiomyocytes may be detected. By analyzing the expression of the marker, the maturity of cardiomyocytes can be evaluated. In one embodiment, the above method further comprises the step of selecting a myocardial cell layer containing cardiomyocytes with high expression of the myocardial maturation marker.

[0071] Examples of myocardial maturation markers include TNNI3, integrin alpha 7 (ITGA7), IGF1, SCN5A, and KCNJ11. The expression of cardiomyocyte-specific markers can be detected by conventional biochemical or immunochemical methods (e.g., enzyme-linked immunosorbent assay, immunohistochemical assay, etc.). Alternatively, the expression of nucleic acids encoding cardiomyocyte-specific markers can be evaluated. The expression of nucleic acids encoding cardiomyocyte-specific markers can be confirmed by molecular biology methods commonly used in the past to amplify, detect, and analyze mRNA, such as reverse transcriptase polymerase chain reaction (RT-PCR) or hybridization analysis. Nucleic acid sequences encoding cardiomyocyte-specific markers are known and available through public databases such as GenBank, so marker-specific sequences to be used as primers or probes can be easily determined.

[0072] The expression level of myocardial maturation markers in the myocardial cell layer is preferably at least 1.3 times, more preferably at least 1.7 times, and even more preferably at least 2 times that of control cells not treated with perfusion waves. Alternatively, the expression levels of myocardial maturation markers in the myocardial cell layer are such that the FPKM (Fragments Per Kilobase of exon per Million mapped reads) values ​​for TNNI3 are at least 100, preferably at least 150, for ITGA7 are at least 35, preferably at least 40, for IGF1 are at least 0.2, preferably at least 0.4, for SCN5A are at least 18, preferably at least 20, and for KCNJ11 are at least 2.5, preferably at least 2.8.

[0073] In the present invention, the expression of Connexin-43 in cardiomyocytes may be detected. In one embodiment, the above method further comprises the step of selecting a layer of cardiomyocytes containing cardiomyocytes with high Connexin-43 expression. The expression of Connexin-43 can be analyzed in the same manner as the expression of cardiomyocyte-specific markers.

[0074] The expression level of Connexin-43 in the myocardial cell layer is preferably at least 1.5 times, more preferably at least 1.7 times, and even more preferably at least 2 times that of control cells not treated with perfusion waves. High expression levels of Connexin-43 are expected to improve excitation conduction velocity between myocardial cells and neighboring cells.

[0075] The conduction velocity in the myocardial cell layer (the conduction velocity of myocardial cells after treatment with spontaneous circulation waves) was 10 cm s -1 It is preferable that the value is equal to or greater than 12 cm s -1 More preferably, it is 14 cm s or more. -1 The above is even more preferable. A high conduction velocity is expected to result in high functionality as cardiomyocytes.

[0076] The amount of VEGF secreted from the myocardial cell layer is preferably at least twice, more preferably at least 2.5 times, and even more preferably at least 3 times the amount secreted from control cells not treated with circulatory waves. The amount of VEGF secreted from the myocardial cell layer can be measured by collecting the culture supernatant and using a standard method, such as enzyme-linked immunosorbent assay (ELISA). It is expected that increased VEGF secretion will promote angiogenesis and improve the survival rate of myocardial cells after transplantation.

[0077] The hypoxia tolerance of the myocardial cell layer is preferably improved compared to control cells not treated with circulatory waves. The hypoxia tolerance of the myocardial cell layer can be evaluated by measuring the cell viability after culturing the cardiomyocytes in a hypoxic environment. Improved hypoxia tolerance is expected to improve the engraftment rate of cardiomyocytes after transplantation.

[0078] <Cardiomyocyte layer> According to the present invention, there is provided a cardiomyocyte layer obtained by the above-described method for producing a cardiomyocyte layer according to the present invention.

[0079] The present invention further provides a cardiomyocyte layer having the following characteristics (1) to (5): (1) Cardiomyocytes derived from pluripotent stem cells, (2) It is a single layer. (3) At least one of the following conditions (3-1) to (3-8) is satisfied: (3-1) The expression level of β-MHC in the myocardial cell layer is 10% or more of that of normal adult myocardial cells; (3-2) The amount of VEGF secreted from the myocardial cell layer was more than twice that of control cells not treated with the circulation wave; (3-3) The expression level of Connexin-43 in the myocardial cell layer was more than 1.5 times higher than that of control cells not treated with circulatory waves; (3-4) The expression level of TNNI3 in the myocardial cell layer was more than twice that of control cells not treated with circulatory waves; (3-5) The expression level of ITGA7 in the myocardial cell layer was more than 1.2 times higher than that of control cells not treated with circulatory waves; (3-6) The expression level of IGF1 in the myocardial cell layer was more than 1.5 times that of control cells not treated with circulatory waves; (3-7) The expression level of SCN5A in the myocardial cell layer was more than 1.2 times higher than that of control cells not treated with circulatory waves; (3-8) The expression level of KCNJ11 in the myocardial cell layer was more than 1.3 times higher than that of control cells not treated with circulatory waves; (4) When the horizontal spread of the sowing surface is considered as a plane figure, the shape is such that at least a part of a first plane figure of any shape having a uniform spread is hollowed out by a second plane figure of another arbitrary shape having a uniform spread, without touching the periphery of the first plane figure of any shape, (5) The average value of the perimeter length of the first plane figure and the perimeter length of the second plane figure satisfies the following formula. x ≥ v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is.

[0080] The above (1), (2), (4) and (5) are as described in the description of the method for producing a cardiomyocyte layer.

[0081] The expression level of β-MHC in the myocardial cell layer is 10% or more, preferably 20%, and more preferably 30% of that in normal adult cardiomyocytes. The amount of VEGF secreted from the myocardial cell layer is at least two times, preferably three times, more preferably four times, that of control cells not treated with the circulation wave. The expression level of Connexin-43 in the myocardial cell layer is at least 1.5 times, preferably 2 times, and more preferably 2.5 times, that of control cells not treated with perfusion waves. The expression level of TNNI3 in the myocardial cell layer is at least 2 times, preferably 5 times, more preferably 10 times, that of control cells not treated with circulation waves. The expression level of ITGA7 in the myocardial cell layer is at least 1.2 times, preferably 1.3 times, and more preferably 1.5 times, that of control cells not treated with circulatory waves. The expression level of IGF1 in the myocardial cell layer is at least 1.5 times, preferably 2 times, more preferably 3 times, that of control cells not treated with circulation waves. The expression level of SCN5A in the myocardial cell layer is at least 1.2 times, preferably 1.3 times, and more preferably 1.5 times, that of control cells not treated with circulation waves. The expression level of KCNJ11 in the myocardial cell layer is at least 1.3 times, preferably 1.4 times, more preferably 1.5 times, that of control cells not treated with perfusion waves.

[0082] The above-described cardiomyocyte layer of the present invention can be used in the form of a cell sheet or the like as a transplant material for the heart.

[0083] <Method and kit for evaluating drug candidate substances> The present invention provides (1) contacting the cardiomyocyte layer of the present invention with a candidate substance; (2) detecting an electrical signal or a calcium signal in the myocardial cell layer contacted with the candidate substance; and (3) comparing the electrical signal or calcium signal detected in step (2) with a control electrical signal or calcium signal; The present invention provides a method for evaluating a drug candidate, comprising:

[0084] In step (1), the cardiomyocyte layer is contacted with a candidate substance, for example, by adding the candidate substance to the culture medium for the cardiomyocyte layer. Candidate substances include candidate substances for active pharmaceutical ingredients across all fields. In step (2), the electrical or calcium signal of the cardiomyocyte layer contacted with the candidate substance is detected using the above-described method for detecting an electrical or calcium signal. The control in step (3) may be the cardiomyocyte layer before contact with the candidate substance, or it may be a cardiomyocyte layer not contacted with the candidate substance. Alternatively, the control may be a cardiomyocyte layer contacted with a substance known to be non-cardiotoxic or with acceptable cardiotoxicity. If the result of step (3) shows that the electrical or calcium signal of the cardiomyocyte layer contacted with the candidate substance is significantly different from the electrical or calcium signal of the control, the drug candidate substance can be determined to be cardiotoxic. Furthermore, to confirm the validity of the assay system, a cardiomyocyte layer contacted with a substance that decreases or increases myocardial beating frequency or prolongs or shortens the QT interval can be used as a positive control.

[0085] As an example, drug candidates can be evaluated for the treatment of heart disease. Cardiac disease refers to arrhythmia or a circulatory system disease accompanied by arrhythmia. Arrhythmia includes tachyarrhythmia and bradyarrhythmia. In step (1), the cardiomyocyte layer is contacted with a candidate substance, for example, by adding the candidate substance to the culture medium of the cardiomyocyte layer. Candidate substances include substances expected to have a therapeutic effect on cardiac disease, such as substances expected to control myocardial beating frequency or QT interval. Prior to step (1), the cardiomyocyte layer may be contacted with a substance that decreases or increases myocardial beating frequency or prolongs or shortens the QT interval, thereby inducing arrhythmia. In step (2), the electrical or calcium signal of the cardiomyocyte layer contacted with the candidate substance is detected using the above-described method for detecting electrical or calcium signals. The control in step (3) may be the cardiomyocyte layer before contact with the candidate substance, or may be the cardiomyocyte layer not contacted with the candidate substance (negative control). Alternatively, the control may be the cardiomyocyte layer contacted with a substance that decreases or increases myocardial beating frequency or a substance that prolongs or shortens the QT interval (positive control). If desired, a control myocardial cell layer may be subjected to a treatment that induces arrhythmias similar to those of the test myocardial cell layer.

[0086] If the result of step (3) is that the electrical signal or calcium signal of the myocardial cell layer contacted with the candidate substance is significantly different from the electrical signal or calcium signal of the negative control, or shows a similar trend to the electrical signal or calcium signal of the positive control, it is determined that the candidate substance can be used to treat cardiac disease.

[0087] Substances that decrease myocardial beating rate and are used in the method for evaluating drug candidate substances include, for example, adrenergic agonists such as isoproterenol. Substances that increase myocardial beating rate include, for example, β-blockers such as propranolol. Substances that prolong the QT interval include HERG channel inhibitors such as E-4031. Drug candidate substances that can be used include calcium channel blockers such as ranolazine, sodium channel blockers such as mexiletine, and verapamil.

[0088] In the method for evaluating a drug candidate substance, calcium signals may be detected using a cardiomyocyte layer expressing a calcium sensor protein. In this case, the above method can be performed without using special detection reagents or electrodes. In another embodiment, electrical signals from the cardiomyocyte layer may be detected using a multi-electrode array.

[0089] The present invention provides a kit for carrying out the method for evaluating a drug candidate substance, which comprises the cardiomyocyte layer, and may further include a culture medium, a control substance, a calcium detection reagent, a multi-electrode array, instructions for use of the kit, etc.

[0090] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0091] <Cells used> Cardiomyocyte A: Cardiomyocytes (beating cardiac colonies) induced from pluripotent stem cells were used according to the method described in the Examples of International Publication No. 2015 / 182765 (particularly paragraphs 0064, 0065, and Table 2). Human iPS cells (cell line name: 253G1) were used as pluripotent stem cells.

[0092] Cardiomyocyte B: Cardiomyocytes were induced from pluripotent stem cells according to the method described in International Publication No. 2020 / 027278 (particularly Example 3). Human iPS cells (cell line name: 253G1) were used as pluripotent stem cells.

[0093] Cardiomyocyte C: Commercially available iCell® Cardiomyocytes 2 (FUJIFILM Cellular Dynamics, Inc.) was used. <Characteristics of the cells used>

[0094] Cardiomyocytes A, B, and C differ in the characteristics of the resulting myocardium, particularly the conduction velocity, due to the differences in the iPS cells or the methods of cardiac differentiation used to establish them. Specifically, the conduction velocity of each cardiomyocyte measured using a multi-electrode system (USB-ME64-System, Multi Channel Systems, Germany) is as follows: Conduction velocity of cardiomyocyte A: 8 cm / s; Conduction velocity of cardiac myocyte B: 30 cm / s; Conduction velocity of cardiac muscle cell C: 31 cm / s

[0095] Furthermore, the purity of each cardiomyocyte varies. The purity of each cardiomyocyte is as follows. The purity of cardiomyocytes was determined by measuring the percentage of cells expressing TnT2, a cardiomyocyte-specific marker, using a flow cytometer. For example, a cardiomyocyte purity of 90% means that 90% of the measured cells express TnT2. Purity of cardiomyocytes A: 90% Purity of cardiomyocytes in cardiomyocyte B: 50-90% Cardiomyocyte purity of Cardiomyocyte C: 100%

[0096] Example 1: Preparation of culture vessel A 4 mm thick PDMS (SYLGARD® 184, Dow Corning) sheet was cut out into the shapes shown in Figure 1 using a tissue puncher (Fisher Scientific) and a cutter. Specifically, for PDMS sheet 1, a 10 mm square PDMS sheet with an 8 mm diameter circular hole and a 3 mm diameter circular PDMS sheet were prepared as shown in Figure 1. PDMS sheets 2 to 4 were also prepared in the shapes shown in Figure 1. Silicone adhesive (one-component condensation-type RVT rubber (transparent) (Shin-Etsu Silicones)) was applied to one side of the cut PDMS sheets, and PDMS sheets 1, 3, and 4 were attached to an adhesive culture dish (product name), and PDMS sheet 2 was attached to the wells of a 24-well plate (product name) at the positions shown in Figure 2. After drying overnight, the sheets were sterilized by UV irradiation for 30 minutes. As a result of the above, culture vessels 1 to 4 shown in Figure 2 were prepared. In FIG. 2, the shaded area indicates the cell seeding surface, and the dotted line indicates the inner frame of the well of the adherent culture dish or 24-well plate.

[0097] If the horizontal extent of the seeding surface of culture vessel 1 is considered as a planar figure, its shape is obtained by cutting out a 3 mm diameter circle (second planar figure) from an 8 mm diameter circle (first planar figure) without touching the periphery of the first planar figure. The periphery of the first planar figure is 25.12 mm, and the periphery of the second planar figure is 9.42 mm, with the average of both being 17.27 mm. For culture devices 1 to 4, the periphery of the first planar figure, the periphery of the second planar figure, and the average of both are shown in Table 1.

[0098] [Table 1]

[0099] Example 2: Preparation of cardiomyocyte layer (using cardiomyocyte B) Cardiomyocyte B induced from pluripotent stem cells was isolated from 3-4 week old cardiomyocyte spheres using a dissociation solution (0.05% Trypsin / EDTA). The isolated cardiomyocyte B was cultured at 1 × 10 6 pieces / cm2 The cells were dispersed in 200 μl of serum-containing myocardial culture medium (medium containing IMDM (Sigma-Aldrich, I3390), 1% MEM non-essential amino acid solution (Sigma-Aldrich, M7145), 1% penicillin-streptomycin (GIBCO, 15140), 2 mmol / L L-glutamine (Sigma-Aldrich, G7513), 0.001% 2-mercaptoethanol (GIBCO), and 0.005 mol / L NaOH), and the suspension was spread on the cell seeding surface of culture vessels 1 to 4 prepared in Example 1 to form a myocardial cell layer.

[0100] Five hours after seeding cardiomyocytes B onto the seeding surface, 1 mL of myocardial culture medium was added. Two days later, the medium was replaced with serum-free myocardial culture medium, and thereafter, the medium was replaced once every four days.

[0101] The day cardiomyocytes B were seeded into culture vessels 1 to 4 prepared in Example 1 was designated Day 0 (Day 0), and the occurrence of TW within the cardiomyocyte layer was confirmed under a microscope on Day 2 (Day 2). Beating frequency was measured on Day 2 (Day 2), Day 6 (Day 6), Day 10 (Day 10), and Day 14 (Day 14). Beating frequency was measured by recording the beating of cardiomyocytes with a CCD camera (DP74; Olympus), and the beating frequency was analyzed using the Z-axis profile in ImageJ to obtain data. The measurement results are shown in Figure 3. In Culture Vessel 1, the TW of cardiomyocytes B remained stable until Day 6 (beating frequency was not measured after Day 10). In Culture Vessel 2, the TW remained stable until Day 10 (beating frequency on Day 14 was not measured). In Culture Vessels 3 and 4, the TW remained stable until Day 14.

[0102] Example 3: Preparation of cardiomyocyte layer (using cardiomyocyte A) Cardiomyocyte differentiation induction of cardiomyocyte A induced from pluripotent stem cells. Cardiac colonies 3-4 weeks after the start of culture were dissociated using a dissociation solution (0.1% collagenase type I, 0.25% trypsin, 1 U / ml DNase I, 116 mmol / L NaCl, 20 mmol / L HEPES, 12.5 mmol / L NaH2PO4, 5.6 mmol / L glucose, 5.4 mmol / L KCl, and 0.8 mmol / L MgSO4, pH 7.35). The resulting cardiomyocyte A cells were 1 x 10 6 pieces / cm 2 The mixture was dispersed in 200 μl of serum-containing myocardial culture medium (medium containing IMDM (Sigma-Aldrich, I3390), 1% MEM non-essential amino acid solution (Sigma-Aldrich, M7145), 1% penicillin-streptomycin (GIBCO, 15140), 2 mmol / L L-glutamine (Sigma-Aldrich, G7513), 0.001% 2-mercaptoethanol (GIBCO), and 0.005 mol / L NaOH), and seeded on the cell seeding surface of culture vessel 1 prepared in Example 1 to form a myocardial cell layer. The myocardial cell layer was cultured in the same manner as in Example 2. It was confirmed that the TW remained stable in culture vessel 1 until Day 14.

[0103] The period during which TW stability was maintained varied depending on the type of cardiomyocytes and the combination of culture vessels used. The results of Examples 2 and 3, along with the conduction velocity of cardiomyocytes (circled number 1) and the average perimeter of the seeding surface (circled number 2), are summarized in Table 2.

[0104] [Table 2]

[0105] From Table 2 (especially the results of No. 1 and No. 3), it can be inferred that for TW to be stable up to Day 10 or later, the value obtained by dividing circled number 1 by circled number 2 (circled number 1 / circled number 2) must be less than approximately 10. Furthermore, if the above relationship that circled number 1 / circled number 2 is less than approximately 10 is satisfied, it can be inferred that there are no restrictions on the shape of the seeding surface other than that it be a closed loop shape.

[0106] Example 4: Preparation of cardiomyocyte layer (using cardiomyocyte C) iCell® Cardiomyocytes 2 "iCell Cardiomyocytes 2 Cardiomyocytes were thawed according to the "User's Guide" to prepare cardiomyocyte C (100% cardiomyocyte purity). Furthermore, myocardial-derived fibroblasts (NHCF-V human cardiac fibroblasts, Lonza, CC-2904) were added to the cardiomyocyte C to prepare cardiomyocytes with purities of 25%, 50%, and 75%, respectively. Each of the prepared cardiomyocytes was then cultured at 1 × 10 6 pieces / cm 2 The cells were dispersed in a mixture of 100% and seeded on the cell seeding surface of the culture vessel 3 prepared in Example 1 to form a cardiomyocyte layer. Culture was performed in the same manner as in Example 2, and the beating frequency was measured to evaluate the stability of TW. The measurement results are shown in Figure 4. The beating frequency on Day 6 was 0.7 Hz for a cardiomyocyte purity of 25%, 1.7 Hz for a cardiomyocyte purity of 50%, 4 Hz for a cardiomyocyte purity of 75%, and 4.8 Hz for a cardiomyocyte purity of 100%, indicating a tendency for the beating frequency to increase with increasing myocardial purity. Furthermore, in the case of cardiomyocytes C with a purity of 100%, the TW was stable only up to Day 6 (the beating frequency was not measured after Day 9). Considering that the purity of cardiomyocytes B used in Example 2 was 50-90% and the purity of cardiomyocytes C used in Example 3 was 90%, it can be assumed that the appropriate purity of cardiomyocytes is 50-90%.

[0107] Example 5: Increased expression of myocardial-related genes by TW As in Example 2, a myocardial cell layer was formed using cardiomyocytes B and culture vessel 3. Two cases (TW-1, TW-2) in which the resulting TW was stably maintained until Day 14, and two cases (Control-1, Control-2) in which TW was stopped by changing the medium to 4°C on Day 2, yielding a total of four cases. The expression of each gene on Day 14 was examined. Gene expression was measured as follows.

[0108] Total RNA was purified using Trizol reagent (Life Technologies). RNA concentration was measured using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using SuperScript 3 First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen), and cDNA sequencing was measured using the Illumina Hiseq 4000 platform (Illumina). The results are shown in Table 3. In Table 3, "Adult" refers to normal adult cardiomyocytes (BioChain: P1244122, USA). Maintaining TW for a certain period of time revealed that myocardial-related genes in the cells constituting the myocardial cell layer increased, reaching values ​​closer to those of adults.

[0109] [Table 3]

[0110] Example 6: Increased expression of myocardial maturation β-MHC markers by TW As in Example 3, a cardiomyocyte layer was formed using cardiomyocytes A and culture vessel 1. Two types of TW were produced: one in which the resulting TW was stably maintained until Day 14 (TW), and one in which TW was stopped by changing the medium to 4°C on Day 2 (Control). The protein and gene expression levels of β-MHC, a specific marker associated with cardiomyocyte maturation, on Day 14 were examined using the following method. The results are shown in Figure 6. β-MHC expression was clearly elevated in TW compared to Control.

[0111] Immunostaining: Cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.5% v / v Triton X-100 for 1 hour, and then incubated in blocking solution for 16 hours. Subsequently, cells were incubated with a primary antibody (β-MHC (Santa Cruz Biotechnology: SC-53089) for 16 hours at 4°C. Then, cells were incubated with a secondary antibody (same as the primary antibody) for 1 hour at room temperature. Cell nuclei were stained with 300 nM DAPI (Wako) for 30 minutes at room temperature. After each step, cells were washed with PBS. Images of the stained cells were taken using a confocal microscope.

[0112] Western Blot: Proteins were recovered using SB10 lysis buffer (10% SDS, 5 mM EDTA, 50 mM Tris-HCl pH 7.4) (Sigma). Protein quantification was performed using a BCA protein assay kit (Thermo). β-MHC expression levels were compared by Western blotting. GAPDH was used as a positive control.

[0113] RT-PCR: Total RNA was purified using Trizol reagent (Life Technologies). RNA concentration was measured using a Nanodrop1000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using SuperScript 3 First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). PCR was performed using a StepOnePlus Real-Time PCR system (Life Technologies). Gene expression in normal adult cardiomyocytes (BioChain: P1244122, USA) was also measured.

[0114] Example 7: Increased expression of Connexin-43 by TW As in Example 3, cardiomyocyte layers were formed using cardiomyocytes A and culture vessel 1. Two types of cardiomyocytes were produced: one in which the resulting TW was stably maintained until Day 14 (TW), and one in which TW was stopped by changing the medium to 4°C on Day 2 (Control). The expression levels of Connexin-43 protein and gene on Day 14 were examined using the following method. The results are shown in Figure 6. Connexin-43 expression was clearly elevated in TW compared to Control.

[0115] Immunostaining: The same procedures as in Example 6 were carried out except that Connexin 43 (Santa Cruz: sc-9059) was used as the primary antibody and secondary antibody.

[0116] Western Blot: The same procedure as in Example 6 was carried out.

[0117] RT-PCR: The same procedures as in Example 6 were carried out except that gene expression in normal adult cardiomyocytes (BioChain: P1244122, USA) was not measured.

[0118] In addition, the conduction velocity of the control and TW was measured on Day 14. For TW, the medium was changed to 4°C on Day 14 to stop TW, and then the conduction velocity was measured. The results are shown in Figure 7. It was confirmed that increased expression of Connexin-43, a gap junction marker, also increased the conduction velocity between cells and neighboring cells.

[0119] Example 8: TW increases the secretion of angiogenic factors (VEGF) and improves hypoxia tolerance As in Example 3, a myocardial cell layer was formed using cardiomyocytes A and culture vessel 1. Two types of myocardial cells were produced: one in which the resulting TW was stably maintained until Day 14 (TW), and one in which TW was stopped by changing the medium to 4°C on Day 2 (Control). The culture supernatant was collected on Day 14, and the amount of angiogenic secretory factor (VEGF) was measured by ELISA (ELISA Kit, R&D Systems). The measurement results are shown in Figure 8. The amount of VEGF was clearly increased in TW compared to Control.

[0120] Similarly, two types of cardiomyocyte layers, TW and control, were prepared and cultured for an additional five days in a 5% hypoxic environment after Day 14, and cell viability was measured using a Live / Dead kit (ThermoFisher Scientific). The normal oxygen environment for cell culture is 30%. The measurement results are shown in Figure 9. After culturing in a hypoxic environment, the TW cells had a higher viability than the control cells. These data suggest that if cardiomyocytes maintained in TW for a certain period of time are used to treat heart failure, it is expected that angiogenesis will be promoted and the engraftment rate of cardiomyocytes after transplantation will be improved.

[0121] Example 9: Preparation of cardiomyocyte layer using scaffold material (using cardiomyocyte B) Upcell (registered trademark) (CellSeed) and an oriented biodegradable fiber material (polylactic acid-co-glycolic acid (Sigma-Aldrich: PLGA, P1941)) were prepared as scaffold materials. These were cut out to the same shape and size as the bottom of the wells of the 24-well plate used in Example 1 and attached to the wells of the 24-well plate using a silicone adhesive (one-component condensation type RVT rubber (transparent) (Shin-Etsu Silicones)). PDMS sheet 2 was prepared in the same manner as in Example 1 and attached to the scaffold material. After drying overnight, the scaffold was sterilized by UV irradiation for 30 minutes to prepare culture vessels with scaffold material (culture vessel 2 (Upcell), culture vessel 2 (biodegradable fiber material)).

[0122] A myocardial cell layer was formed in the same manner as in Example 2 using cardiomyocytes B, the above-mentioned culture vessel 2 (Upcell), and culture vessel 2 (biodegradable fiber material). The occurrence of TW within the myocardial cell layer was confirmed under a microscope on Day 2. It was also confirmed that the TW remained stable until Day 14. It was confirmed that the myocardial cell layer of the present invention can be applied to transplantation therapy for heart failure after cardiomyocytes are matured on a scaffold material.

[0123] Example 10: Preparation of cardiomyocyte layer using scaffold material (using cardiomyocyte C) An oriented biodegradable fiber material (polylactic acid-co-glycolic acid (PLGA)) was prepared as a scaffold material, cut out to the same shape and size as the bottom of the adhesive culture dish used in Example 1, and attached to the adhesive culture dish with a silicone adhesive (one-component condensation type RVT rubber (transparent) (Shin-Etsu Silicones)). PDMS sheet 3 was prepared in the same manner as in Example 1 and attached to the scaffold material. After drying overnight, it was sterilized by UV irradiation for 30 minutes to prepare a culture vessel with a scaffold material (culture vessel 3 (biodegradable fiber material)). A myocardial cell layer was formed in the same manner as in Example 4 using cardiomyocytes C (80% pure cardiomyocytes obtained in the same manner as in Example 4) and the above-mentioned culture vessel 3 (biodegradable fiber material). The occurrence of TW within the myocardial cell layer was confirmed under a microscope on Day 2. Beating frequency was also measured up to Day 14 (TW). As a control, TW was stopped by changing the medium to 4°C on Day 2 (Control), and the beating frequency was also measured in the same manner. The measurement results are shown in Figure 10. When culture vessel 3 (biodegradable fiber material) was used, the beating frequency remained stable at around 3-4 Hz up to Day 14.

[0124] Example 11: Preparation of cardiomyocyte layer using scaffold material (using cardiomyocyte B) Culture vessel 3 (biodegradable fiber material) was prepared in the same manner as in Example 10. A myocardial cell layer was formed in the same manner as in Example 2 using cardiomyocytes B and culture vessel 3 (biodegradable fiber material). Two types were produced: one in which the resulting TW was stably maintained until Day 14 (TW), and one in which TW was stopped by changing the medium to 4°C on Day 2 (Control). At the end of Day 14, immunostaining was performed using the method described below. The results are shown in Figure 11. In TW, the protein expression of markers associated with cardiomyocyte-specific maturation (TnnT2, MYL2) was elevated compared to controls, and TW samples exhibited mature myocardial structures with an oriented fiber structure. Immunostaining: The same procedures as in Example 6 were carried out, except that cTnT (Santa Cruz Biotechnology: SC-20025) and Connexin 43 (Santa Cruz: sc-9059), or MYL2 (Proteintech: 10906-1-AP) were used as the primary and secondary antibodies.

[0125] Example 12: Relationship between the production of myocardial cell layer using scaffold material (using myocardial cell B) and electrical signal A culture vessel 3 (biodegradable fiber material) was prepared in the same manner as in Example 10. A polycarbonate (PC) porous membrane (Thermo Fisher, Nunc) was used as a scaffold material. TMCulture vessel 3 (PC) was prepared using Polycarbonate Cell Culture Inserts in Multi-Well Plates (140644) in the same manner as Culture vessel 3 (biodegradable fiber material). A myocardial cell layer was formed in the same manner as in Example 2 using cardiomyocytes B, Culture vessel 3 (biodegradable fiber material), and Culture vessel 3 (PC). The resulting TW was stably maintained until Day 14. After Day 14, the TW was stopped by replacing the medium with 4°C, and the electrical signals of spontaneous myocardial beating were measured using an MEA. Specifically, data on extracellular field potentials (FPs) were recorded using a multielectrode array (MEA) system (USB-ME64-System, Multi Channel Systems, Germany).

[0126] The measurement results are shown in Figure 12. Electrical signals could be measured despite the differences in scaffold materials. The biodegradable fiber material provided better contact with the MEA electrode tip, and the T wave amplitude of the obtained electrical signals was larger and easier to detect. This feature is advantageous for measuring drug-induced QT prolongation when applied to drug evaluation.

[0127] Example 13: Preparation of cardiomyocyte layer using scaffold material (using cardiomyocyte C) and changes in QT interval and beating frequency by TW Culture vessel 3 (biodegradable fiber material) was prepared in the same manner as in Example 10. Cardiomyocytes C (cardiomyocardial cells with 80% purity obtained in the same manner as in Example 4) and the above culture vessel 3 (biodegradable fiber material) were used to form a myocardial cell layer in the same manner as in Example 4. Two types were produced: one in which the resulting TW was stably maintained until Day 14 (TW), and one in which TW was stopped by changing the medium to 4°C on Day 2 (Control). After Day 14, TW was stopped by changing the medium to 4°C, and the electrical signal of spontaneous myocardial beating was measured using an MEA in the same manner as in Example 12.

[0128] The measurement results are shown in Figure 13. In the graph, "normal adult level" refers to normal adult cardiomyocytes (BioChain: P1244122, USA). In samples in which TW was maintained stably up to Day 14, the QT interval approached that of a normal adult. In addition, the pulsation frequency improved. The pulsation frequency was measured in the same manner as in Example 2.

[0129] Example 14: Drug Screening As in Example 13, cardiomyocyte layers were formed using cardiomyocytes C (80% pure cardiomyocytes obtained as in Example 4) and culture vessel 3 (biodegradable fiber material). Two types of cardiomyocytes were produced: one in which the resulting TW was stably maintained until Day 14 (TW), and one in which TW was stopped by changing the medium to 4°C on Day 2 (Control). For TW, TW was stopped by changing the medium to 4°C after Day 14. On Day 17, 1 ml of medium containing various concentrations of various drugs (E-4031, ranolazine, mexiletine, isoproterenol, aspirin (negative control)) was added to the cardiomyocytes. Five minutes later, electrical signals of spontaneous myocardial beating were measured using an MEA as in Example 12. This data was analyzed using an MC Rack (MultiChannel Systems) to measure the QT interval or pulsation rate and evaluate cardiotoxicity.

[0130] The results of evaluating E-4031 (a potassium channel blocker) as a drug are shown in Figure 14. The addition of E-4031 prolonged the QT interval in both TW and the control. The QT interval prolongation value in TW was smaller than that in the control.

[0131] The results of evaluating ranolazine (a calcium channel blocker) as a drug are shown in Figure 15. The control values ​​varied widely, while the TW values ​​varied little, demonstrating stability and reproducibility between samples.

[0132] The results of evaluating mexiletine (a sodium channel blocker) as a drug are shown in Figure 16. The control showed a large variation and poor reproducibility. The QT interval prolongation value for TW was smaller than that for the control.

[0133] The results of evaluating isoproterenol (a β-adrenergic receptor agonist) as a drug are shown in Figure 17. Addition of isoproterenol increased the myocardial pulsation rate. TW was more sensitive to isoproterenol than the control.

[0134] The results of evaluating aspirin (negative control) as a drug are shown in Figure 18. The QT interval prolongation and myocardial pulsation rate were almost the same in the control and TW. The QT interval in the control decreased with increasing concentration.

[0135] Example 15: Drug Screening A drug (verapamil) was evaluated using the same procedure as in Example 14. The results are shown in Figure 19. Addition of verapamil increased the myocardial pulsation rate. TW was more sensitive to verapamil than the control.

Claims

1. A method for producing a cardiomyocyte layer, comprising the following steps (1) to (3): (1) Seeding cardiomyocytes induced from pluripotent stem cells in a monolayer onto a seeding surface having the shape shown in (1-1) below to form a cardiomyocyte layer; (1-1) When the horizontal extent of the sowing surface is considered as a plane figure, the shape is a shape in which at least a part of a first plane figure of any shape having a uniform extent is hollowed out with a second plane figure of another arbitrary shape having a uniform extent without contacting the periphery of the first plane figure of any shape, and the average value of the periphery length of the first plane figure and the periphery length of the second plane figure satisfies the following formula: x≧v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is; (2) propagating at least one electrical signal or calcium signal in the myocardial cell layer; (3) The cells are cultured for 8 days or more while maintaining the conditions of (2).

2. The method of claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic pluripotent stem cells.

3. The method according to claim 1, wherein the expression level of β-MHC in the myocardial cell layer is 10% or more of the expression level in normal adult cardiomyocytes.

4. The method according to claim 1, wherein the expression level of Connexin-43 in the myocardial cell layer is 1.5 times or more that of control cells not treated with perfusion waves.

5. The conduction velocity in the myocardial cell layer is 10 cm s -1 The manufacturing method according to claim 1 .

6. The method of claim 1, wherein the amount of VEGF secreted from the myocardial cell layer is at least twice as much as the amount of VEGF secreted from control cells not treated with circulatory waves.

7. The method of claim 1, wherein the hypoxia resistance of the myocardial cell layer is improved compared to control cells not treated with circulatory waves.

8. 8. The manufacturing method according to claim 1, wherein the first planar figure is a circle, and the second planar figure is a circle having a smaller diameter than the circle of the first planar figure.

9. The first plane figure has a shape obtained by cutting out a substantially rectangular shape from a circle so as to be in contact with the circumference of the circle, and the second plane figure has a shape of a Landolt ring, 8. The manufacturing method according to claim 1, wherein at least a portion of the first planar figure is hollowed out so that the rectangle is inserted between the gaps of the Landolt ring by the second planar figure.

10. The manufacturing method according to claim 1 , wherein the first planar figure is a concave shape, and the second planar figure is a concave shape that is smaller than the first planar figure.

11. The manufacturing method described in any one of claims 1 to 7, wherein the expression level of one or more myocardial maturation markers selected from TNNI3, ITGA7, IGF1, SCN5A and KCNJ11 in the myocardial cell layer is 1.3 times or more the expression level in control cells not treated with circulatory waves.

12. The method of claim 1 , further comprising a step of co-culturing with fibroblasts.

13. The method according to claim 1 , wherein a layer of cardiomyocytes is formed on a biodegradable or non-biodegradable scaffold.

14. The seeding density of cardiomyocytes in step (1) is 2 × 10 5 ~1 x 10 6 pieces / cm 2 The method according to any one of claims 1 to 7, wherein

15. The production method according to any one of claims 1 to 7, wherein the pluripotent stem cells are pluripotent stem cells having a gene mutation associated with heart disease.

16. A myocardial cell layer obtained by the method of claim 1.

17. A cardiomyocyte layer having the following characteristics (1) to (5): (1) A method for producing a cardiac muscle cell comprising: (2) It is a single layer, (3) At least one of the following (3-1) to (3-8) is satisfied: (3-1) the expression level of β-MHC in the myocardial cell layer is 10% or more of that in normal adult cardiomyocytes; (3-2) The amount of VEGF secreted from the myocardial cell layer was more than twice that of control cells not treated with circulatory waves; (3-3) the expression level of Connexin-43 in the myocardial cell layer is 1.5 times or more than that of control cells not treated with circulation waves; (3-4) the expression level of TNNI3 in the myocardial cell layer is more than twice that of control cells not treated with circulation waves; (3-5) The expression level of ITGA7 in the myocardial cell layer is 1.2 times or more than that of control cells not treated with circulation waves; (3-6) The expression level of IGF1 in the myocardial cell layer is 1.5 times or more than that of control cells not treated with circulation waves; (3-7) The expression level of SCN5A in the myocardial cell layer is 1.2 times or more than that of control cells not treated with circulation waves; (3-8) The expression level of KCNJ11 in the myocardial cell layer is 1.3 times or more than that of control cells not treated with circulation waves; (4) When the horizontal spread of the sowing surface is considered as a plane figure, the shape is such that at least a part of a first plane figure of any shape having a uniform spread is hollowed out with a second plane figure of another arbitrary shape having a uniform spread without touching the periphery of the first plane figure of any shape, (5) The average value of the perimeter length of the first plane figure and the perimeter length of the second plane figure satisfies the following formula. x≧v / 10 In the formula, x represents the average value of the perimeter of the first planar figure and the perimeter of the second planar figure, and its unit is cm; v represents the conduction velocity of the cardiomyocytes induced from the pluripotent stem cells, and its unit is cm s -1 is;

18. A kit for evaluating a drug candidate substance, comprising the cardiomyocyte layer according to claim 16 or 17.

19. A transplant material comprising the cardiomyocyte layer according to claim 16 or 17.

20. (1) contacting the cardiomyocyte layer according to claim 16 or 17 with a candidate substance; (2) detecting an electrical signal or a calcium signal in the myocardial cell layer contacted with the candidate substance; and (3) comparing the electrical signal or calcium signal detected in step (2) with a control electrical signal or calcium signal; A method for evaluating a drug candidate substance, comprising:

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