JP7176727B2 - Method for producing three-dimensional myocardial tissue - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law 1. Program abstract (web version/app version) of the 17th Annual Meeting of the Japanese Society for Regenerative Medicine; convention. co. jp/17jsrm/appli/ Publication date: February 23, 2018

Application of Article 30, Paragraph 2 of the Patent Law 2. The 17th Annual General Meeting of the Japanese Society for Regenerative Medicine Venue: Pacifico Yokohama (1-1-1 Minatomirai, Nishi-ku, Yokohama) Date: March 23, 2018

Application of Article 30, Paragraph 2 of the Patent Law 3. The 67th SPSJ Annual Meeting Abstracts page 17 of the paper version, page 1358 of the electronic version, and the program, The Society of Polymer Science, Japan Publication date: May 8, 2018

Application of Article 30, Paragraph 2 of the Patent Law 4. The 67th SPSJ Annual Meeting Venue: Nagoya International Conference Center (1-1 Atsuta Nishimachi, Atsuta-ku, Nagoya) Date: May 23, 2018

Application of Article 30, Paragraph 2 of the Patent Law 5. Proceedings of the 47th Symposium on Biomedical Polymers, pp. 27-28 and program, Society of Polymer Science, Biomedical Polymers Study Group Publication date: July 19, 2018

Application of Article 30, Paragraph 2 of the Patent Law 6. The 47th Symposium on Medical Polymers Venue: Conference room on the 11th floor of the Annex, Rinkai Fukutoshin Center, AIST (2-41-6 Aomi, Koto-ku, Tokyo) Date: July 19, 2018

Application of Article 30, Paragraph 2 of the Patent Law 7. 5th International Congress of Tissue Engineering and Regenerative Medicine 2018 Kyoto Abstract Poster Day 2 on page 535 and program Website address: http://www2. convention. co. jp/termis-wc2018/index. html publication date: August 9, 2018

TECHNICAL FIELD The present invention relates to an artificial biological tissue and a method for producing the same. In particular, the present invention relates to three-dimensional myocardial tissue and methods of making same.

In order to obtain a cardiomyocyte tissue having a three-dimensional structure, a culture method using a culture carrier or an aggregate containing cells is used. For example, it has been reported that a three-dimensional structure composed of cells and collagen gel can be obtained by mixing collagen gel and cardiomyocytes and pouring the mixture into a mold for molding (eg, Non-Patent Document 1). However, there is the problem that the cell density in the three-dimensional structure thus obtained is low.

A cell sheet technique has been reported to construct a three-dimensional cardiomyocyte tissue with a higher cell density (eg, Non-Patent Document 2). By stacking cell sheets, it is possible to construct a three-dimensional tissue with a high cell density. However, internal necrosis occurs due to the high cell density in the cell sheet, and the contractile force of the tissue itself is not sufficient.

In response to these problems, the cell layering method and cell accumulation method, which promote the adhesion between cells by coating the membrane surface of cells with proteins, which are cell adhesion factors, and build a three-dimensional structure with high cell density. have been reported (Patent Documents 1 and 2, Non-Patent Documents 3 and 4). Using these techniques, a three-dimensional myocardial tissue with a high cell density and a vascular network structure has been constructed (Non-Patent Document 5). Such myocardial tissue exhibits a response similar to that of a living body, and research toward drug discovery applications and evaluation of drug response are being promoted. In addition, since such myocardial tissue has a vascular network, engraftment and maturation have been confirmed when transplanted (Non-Patent Document 6). However, it cannot be said that the myocardial tissue obtained by the above technique can fully reproduce a living tissue from the viewpoint of contractile force.

Japanese Patent No. 4919464 Japanese Patent No. 5850419

Eschenhagen T et al. FASEB Journal. 11(8), 683-94, 1997. Shimizu T et al. Biomaterials. 24(13), 2309-16, 2003. Matsusaki M et al. Angewandte Chemie. 46(25), 1689-92, 2007. Nishiguchi A et al. Advanced materials.23(31), 3506-10, 2011. Amano Y et al. Acta Biomaterialia. 33, 110-21, 2016 Narita H et al. Scientific reports. 7(1), 13708, 2017.

It is desired to construct a three-dimensional myocardial tissue that has a high cell density, a large contractile force, and can sufficiently reproduce a living tissue. It is desired to apply such a three-dimensional myocardial tissue to the fields of regenerative medicine and drug discovery.

As a result of extensive research, the present inventors have found that the above problems can be solved by constructing a three-dimensional myocardial tissue in a culture vessel having an extracellular matrix gel on the inner wall, and have completed the present invention. rice field.

That is, the present invention provides the following.
(1) A method for producing three-dimensional myocardial tissue, which comprises culturing cardiomyocytes or a cell mixture containing cardiomyocytes in a container having an extracellular matrix gel on the inner wall thereof.
(2) A group in which the extracellular matrix consists of collagen, atelocollagen, gelatin, fibronectin, laminin, cadherin, tenascin, entactin, elastin, fibrin, proteoglycan, hyaluronic acid, chitin, chitosan, polyethylene glycol, polyvinyl alcohol, and derivatives of the above substances. (1) The method according to (1), which is one or more selected from
(3) The method according to (1) or (2), wherein the cell mixture contains myocardial cells and myocardial fibroblasts.
(4) The method according to (3), wherein the cell mixture further contains vascular endothelial cells.
(5) The method according to any one of (1) to (4), wherein the cardiomyocytes are pretreated by alternately layering an extracellular matrix.
(6) The method according to any one of (1) to (5), wherein the three-dimensional myocardial tissue has an average maximum contraction velocity of 150 μm/s or more and an average maximum relaxation velocity of 90 μm/s or more.
(7) A cultured three-dimensional myocardial tissue comprising cardiomyocytes or a cell mixture containing cardiomyocytes in a container having an extracellular matrix gel on the inner wall.
(8) A group in which the extracellular matrix consists of collagen, atelocollagen, gelatin, fibronectin, laminin, cadherin, tenascin, entactin, elastin, fibrin, proteoglycan, hyaluronic acid, chitin, chitosan, polyethylene glycol, polyvinyl alcohol, and derivatives of the above substances. The tissue according to (7), which is one or more selected from
(9) The tissue according to (7) or (8), wherein the cell mixture contains myocardial cells and myocardial fibroblasts.
(10) The tissue according to (9), wherein the cell mixture further contains vascular endothelial cells.
(11) The tissue according to any one of (7) to (10), wherein the cardiomyocytes are pretreated by alternately layering an extracellular matrix.
(12) The tissue according to any one of (7) to (11), which has an average maximum contraction velocity of 150 μm/s or more and an average maximum relaxation velocity of 90 μm/s or more.

INDUSTRIAL APPLICABILITY According to the present invention, a three-dimensional myocardial tissue having a high cell density and a large contractile force close to that of a living body and a method for producing the same are provided. The large contractile force of the three-dimensional myocardial tissue of the present invention is considered to be due to the fact that the extracellular matrix gel on the inner wall of the container does not inhibit the pulsating function of the myocardial tissue and retains the myocardial tissue. Since the three-dimensional myocardial tissue of the present invention can reproduce in vitro the same reaction as in vivo myocardium, more advanced regenerative medicine, drug discovery research, and the like can be realized.

FIG. 1 is a diagram schematically showing a method for constructing a three-dimensional myocardial tissue using a collagen gel culture vessel. FIG. 2 is a photograph showing a three-dimensional myocardial tissue (inside the dashed line) constructed on a collagen gel culture vessel. FIG. 3 is a graph showing the contractile behavior of a three-dimensional myocardial tissue constructed using a collagen gel culture vessel and a three-dimensional novel tissue constructed using a culture insert. FIG. 4 is a graph showing the maximal contraction rate and maximal relaxation rate of a three-dimensional myocardial tissue constructed using a collagen gel culture vessel and a three-dimensional novel tissue constructed using a culture insert. FIG. 5 is a fluorescence-stained image (horizontal sectional view) of a three-dimensional myocardial tissue having a vascular network cultured on a collagen gel culture vessel. Anti-CD31 antibody staining is shown in red, anti-actin filament antibody staining in green, and DAPI staining in blue.

In one aspect, the present invention provides a method for producing three-dimensional myocardial tissue, characterized by culturing cardiomyocytes or a cell mixture containing cardiomyocytes in a container having an extracellular matrix gel on the inner wall.

Extracellular matrices are known. Extracellular matrix is a substance that is secreted from living cells and accumulated outside the cells, and is involved in the regulation of cell adhesion, proliferation and differentiation. The extracellular matrix used for the container in the present invention may be one that exists in vivo, or one that does not exist in vivo but has the same functions and properties as those that exist in vivo. . In the present specification, these are collectively referred to as extracellular matrices. Preferred extracellular matrices for use in containers in the present invention are cell-adhesive and gellable. Moreover, those having biocompatibility are also preferable. Preferred examples of extracellular matrices used in the container of the present invention include collagen, atelocollagen, gelatin, fibronectin, laminin, cadherin, tenascin, entactin, elastin, fibrin, proteoglycan, hyaluronic acid, polysaccharides such as chitin and chitosan, or , polyethylene glycol, polyvinyl alcohol and other water-soluble synthetic polymers, and derivatives of the above substances, but are not limited to these. Collagen, gelatin, and fibrin are extremely excellent in both cell adhesiveness and gelling ability, and are also biocompatible, so they are particularly preferably used for the container of the present invention. Derivatives of the above substances include all derivatives as long as they do not impair cell adhesiveness, gelling ability and biocompatibility. Methods for making derivatives are known to those skilled in the art.

Extracellular matrix gel is a gel of extracellular matrix. The gel may consist of extracellular matrix components or may contain extracellular matrix components. Methods for producing extracellular matrix gels are known. For example, a type I collagen gel may be obtained by placing an aqueous type I collagen solution under physiological conditions. If the extracellular matrix to be used is difficult to gel or does not gel, it may be gelled using a known gelling agent such as agarose, carrageenan, sodium alginate and gelatin.

In the present invention, the extracellular matrix in the extracellular matrix gel may be of one type, or of two or more types.

The characteristics and physical properties of the extracellular matrix gel, such as gel strength and gel thickness, can be determined according to the shape of the container, the cells used, the desired contraction speed and size of the three-dimensional myocardial tissue, and the like. It is preferable to determine the characteristics and physical properties of the extracellular matrix gel so as to retain the myocardial tissue without inhibiting the beating function of the myocardial tissue. It is also preferable to determine the characteristics and physical properties of the extracellular matrix gel so that the three-dimensional myocardial tissue does not drop out of the gel. Such determinations are within the skill of those skilled in the art. For example, gel strength may be adjusted by adjusting the concentration of extracellular matrix components.

The container having the extracellular matrix gel on the inner wall may have the extracellular matrix gel on the entire inner wall, or may have the extracellular matrix gel on a part of the inner wall. The interior walls of the container include the bottom, sides, and, if present, the top of the interior of the container. For example, the entire bottom and all sides of the container may be covered with extracellular matrix gel or made of extracellular matrix. Also, for example, a portion of the bottom surface and a portion of the side surface of the container may be covered with extracellular matrix gel, or may be made of extracellular matrix. Also, for example, the entire bottom surface and part of the side surface of the container may be covered with extracellular matrix gel, or may be made of extracellular matrix. Also, for example, part of the bottom surface and all of the side surfaces of the container may be covered with extracellular matrix gel, or may be made of extracellular matrix. A container whose bottom surface is entirely covered with extracellular matrix gel or made of extracellular matrix is preferred. More preferably, the entire bottom surface and all of the side surfaces are covered with extracellular matrix gel or are made of extracellular matrix.

In order to produce the above container, an extracellular matrix gel may be applied to the inner wall of an existing culture container made of glass or plastic. Alternatively, as shown in the examples of the present application, a container may be produced by solidifying while pressing a mold against the extracellular matrix gel, and removing the mold after solidification. As for the container manufactured by forming the recess in the extracellular matrix gel, the recess is referred to as the inner wall of the container.

The size and shape of the container can be appropriately selected and changed according to the size and shape of the three-dimensional myocardial tissue, the use of the tissue, etc., and are not particularly limited. For example, the bottom surface of the container may be circular with a diameter of about 100 μm to several cm.

Cardiomyocytes can be any type of cardiomyocyte. Examples of animal species from which cardiomyocytes are derived include humans, monkeys, dogs, cats, cows, horses, pigs, sheep, and goats, but are not limited to these animal species. Commercially available cardiomyocytes may be used. Cardiomyocytes differentiated from iPS cells or ES cells may also be used. Alternatively, one obtained from the heart of a living organism and cultured may be used. Methods for differentiating iPS cells or ES cells into cardiomyocytes and methods for obtaining and culturing cardiomyocytes from cardiac tissue are known. For example, by using the three-dimensional myocardial tissue of the present invention produced using human iPS-derived myocardial cells, it is possible to accurately and early evaluate the cardiotoxicity and side effects of drugs on the human heart.

A cell mixture containing cardiomyocytes contains cells other than cardiomyocytes. Non-myocardial cells include, but are not limited to, fibroblasts, preferably myocardial-derived fibroblasts, vascular endothelial cells, and the like. Myocardial-derived fibroblasts and vascular endothelial cells are commercially available, and these commercially available products may be used. Fibroblasts may be obtained by harvesting and culturing from myocardium. Vascular endothelial cells may be obtained by obtaining them from blood vessels and culturing them. Methods for obtaining and culturing fibroblasts and vascular endothelial cells are known.

In the method for producing three-dimensional myocardial tissue of the present invention, three-dimensional myocardial tissue can be constructed using only cardiomyocytes. preferably. A three-dimensional myocardial tissue with a vascular network can be obtained by using a mixture of myocardial cells, myocardial-derived fibroblasts, and vascular endothelial cells.

Cultivation of cells in the method for producing three-dimensional myocardial tissue of the present invention includes seeding of cells in the container as a pre-step. Cell seeding can be performed by a known method. Usually the cell mixture is seeded as a cell suspension. For example, cells may be seeded by dropping the cells using a pipette. Alternatively, seeds may be seeded using various 3D printers. Printer methods include inkjet printers, micro-extrusion printers, and laser-assisted printers. Another method is seeding using a coating needle. A printer method and a method using a coating needle are suitable for producing a three-dimensional myocardial tissue microchip.

When using a cell mixture containing cardiomyocytes, the proportion of cardiomyocytes and cells other than cardiomyocytes can be appropriately determined. In the case of a mixture of cardiomyocytes and myocardial-derived fibroblasts, cardiomyocytes:fibroblasts=2 to 4:1, preferably 2.5 to 3.4:1, for example, 3:1. In the case of a mixture of cardiomyocytes, myocardial-derived fibroblasts and vascular endothelial cells, cardiomyocytes: fibroblasts: vascular endothelial cells = 2-4: 1: 0.2-0.6, preferably 2.5-3 .5:1:0.3 to 0.5, eg 3:1:0.4.

Any number of cells may be seeded as long as a three-dimensional myocardial tissue can be obtained. The number of cells can be determined according to the size, shape, application of the three-dimensional myocardial tissue, and the size, shape, etc. of the container. In general, the number may be such that several to several tens of layers of cells are accumulated on the bottom of the container, or the number is such that the thickness of the seeded cell layer is several tens of μm to several hundreds of μm.

In the method for producing a three-dimensional myocardial tissue of the present invention, it is preferable to subject cells constituting the tissue to a pretreatment of alternately layering an extracellular matrix. Such pretreated cells are called LbL cells. Such pretreatment induces adhesion between cells and allows the construction of a three-dimensional tissue in a short period of time. It is preferable to subject the cardiomyocytes to such a pretreatment. Cells other than myocardial cells may also be subjected to the above pretreatment. For example, when producing a three-dimensional myocardial tissue using cardiomyocytes, cardiac fibroblasts and vascular endothelial cells, the cardiomyocytes and cardiac fibroblasts may be pretreated as described above.

In the pretreatment, cells are alternately layered with first and second extracellular matrices. Extracellular matrices and their application to cells are described in US Pat. No. 5,850,419, the contents of which are incorporated herein by reference. A typical example of the combination of the first and second extracellular matrices is that the first extracellular matrix is a protein containing an Arg-Gly-Asp sequence (RGD sequence) and the second extracellular matrix is the first cell A protein that interacts with the outer matrix. Here, the interaction means, for example, an electrostatic interaction, a hydrophobic interaction, a hydrogen bond, a charge transfer interaction, a covalent bond formation, a specific interaction between proteins, a , means that it is physically close to the polymer containing the RGD sequence to the extent that it can bind, adhere, adsorb, or transfer electrons. The first and second extracellular matrices are preferably biodegradable.

Specific examples of combinations of the first and second extracellular matrices include fibronectin and gelatin, fibronectin and ε-polylysine, fibronectin and hyaluronic acid, laminin and gelatin, vitronectin and gelatin, fibronectin and dextran sulfate, fibronectin and heparin, and elastin. and polylysine, fibronectin and collagen, laminin and collagen, vitronectin and collagen, RGD-bound collagen, or RGD-bound gelatin and collagen or gelatin. Combinations of fibronectin and gelatin, fibronectin and heparin, fibronectin and ε-polylysine, fibronectin and hyaluronic acid, fibronectin and dextran sulfate are preferred, and fibronectin and gelatin are more preferred. The first extracellular matrix and the second extracellular matrix may be of one type each, or two or more types may be used in combination within the range of interaction.

The method for alternately layering extracellular matrices on cells is not particularly limited. The cells may be immersed in Alternatively, for example, the aqueous solution containing the first extracellular matrix and the aqueous solution containing the second extracellular matrix may be alternately brought into contact with the cell surface of the monolayer. For these methods, see, for example, Akagi et al., Surface Vol. 51, No. 4, p. 1-11 (2013) and Matsuzaki et al., Pharmaceutical Sciences, Vol. 76, No. 5, p. 294-300 (2016).

The three-dimensional myocardial tissue of the present invention can be constructed by culturing cardiomyocytes or a cell mixture containing cardiomyocytes in the vessel. Cardiomyocytes or cell mixtures containing cardiomyocytes can be cultured using conventional animal cell culture methods. For example, it may be cultured at 20 to 40° C., preferably 35 to 39° C., more preferably 37° C. for 1 to 14 days, preferably several to 10 days. A known medium can also be used. Examples of media include, but are not limited to, Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RDMI, GlutaMax medium, and the like. The culture conditions and medium are not particularly limited and can be determined depending on the type of cells, the number of cells, the size and shape of the three-dimensional myocardial tissue, the size and shape of the container, and the like.

The thickness of the three-dimensional myocardial tissue of the present invention obtained by the above production method can be several tens of μm to several hundred μm.

The contractile force of the three-dimensional myocardial tissue of the present invention obtained by the above manufacturing method is extremely large, and can be about ten times or more than that of myocardial tissue obtained by conventional methods. The three-dimensional myocardial tissue of the present invention may have an average maximum contraction velocity of 50 μm/s or more, preferably 100 μm/s or more, more preferably 150 μm/s or more, and an average maximum relaxation velocity of 30 μm/s or more, preferably 60 μm/s. s or more, more preferably 90 μm/s or more. Since the three-dimensional myocardial tissue of the present invention has a high rate of contraction and relaxation, a response can be observed even when a small amount of test drug is given, for example, during drug screening. Furthermore, when a three-dimensional myocardial tissue is constructed using vascular endothelial cells in a cell mixture containing myocardial cells, a vascular network is formed in the three-dimensional myocardial tissue. Moreover, the cell density in the three-dimensional myocardial tissue of the present invention is high. Therefore, by using the three-dimensional myocardial tissue of the present invention, it becomes possible to reproduce in vitro the same reaction as in vivo.

The three-dimensional myocardial tissue obtained by the production method of the present invention may be used after being separated from the extracellular matrix gel, or may be used together with the extracellular matrix gel. The three-dimensional myocardial tissue of the present invention may be used as a heart-applied graft. The three-dimensional myocardial tissue of the present invention may also be used for tests such as drug screening and cardiotoxicity evaluation. Furthermore, the three-dimensional myocardial tissue of the present invention may be placed on a substrate to construct a chip and used for high-throughput testing. Other uses of the three-dimensional myocardial tissue of the present invention include, but are not limited to, tissue engineering, cell culture, biosensors, and the like.

In another aspect, the present invention provides a cultured three-dimensional myocardial tissue comprising cardiomyocytes or a cell mixture comprising cardiomyocytes in a container having an extracellular matrix gel on the inner wall thereof.

The extracellular matrix gel, container, inner wall, cardiomyocytes, cell mixture containing cardiomyocytes, and three-dimensional myocardial tissue are as described above. The cultured three-dimensional myocardial tissue is the cultured three-dimensional myocardial tissue present in the container. Cultivation is also as described above.

The cardiomyocytes that constitute the cultured three-dimensional myocardial tissue are preferably cells that have been pretreated to alternately layer an extracellular matrix. Cells other than myocardial cells constituting the cultured three-dimensional myocardial tissue may also be subjected to the pretreatment.

The cultured three-dimensional myocardial tissue has an average maximum contraction velocity of 50 μm/s or more, preferably 100 μm/s or more, more preferably 150 μm/s or more, and an average maximum relaxation velocity of 30 μm/s or more, preferably 60 μm/s. or more, more preferably 90 μm/s or more.

The meanings of the terms in this specification other than the terms explained above should be understood as commonly understood in the fields of medicine, cell engineering, biology and the like.

EXAMPLES The present invention will be described in more detail and specifically below by way of examples, but the examples are not intended to limit the scope of the present invention.

A three-dimensional myocardial tissue was constructed using a collagen gel culture vessel according to the method shown in FIG. A collagen solution (2.1 mg/ml) was dropped onto a 6-well plate and culture inserts (24-well size) were placed. Further, a collagen solution was added dropwise to the surrounding area, and the mixture was allowed to stand at 37°C for 30 minutes to gel. After gelation, the culture insert was removed, and the formed recessed portion was used as a culture vessel. Here, iPS cell-derived cardiomyocytes (iPS cell line 253G1 (Riken)) in which fibronectin and gelatin were mutually laminated were induced to differentiate into myocardium by the applicant. ) and myocardial fibroblasts (Lonza, catalog number: CC-2904) in which fibronectin and gelatin are mutually laminated, and seeded by dropping a cell suspension (1.0×10 ⁶ cells: 10 layers), A three-dimensional myocardial tissue was constructed by culturing (Fig. 2). The ratio of seeded myocardial cells to myocardial fibroblasts was myocardial cells: myocardial fibroblasts = 75:25. After seeding the cells, a DMEM medium (containing 10% FBS) was added and cultured at 37° C. for 5 days to evaluate the beating contractile behavior of the constructed three-dimensional myocardial tissue.

High-speed camera imaging and particle image velocimetry (PIV method) were used as evaluation methods for the constructed three-dimensional myocardial tissue. As a comparative experiment, a three-dimensional myocardial tissue was constructed by seeding and culturing on a culture insert in the same manner as described above (construction by a conventional method), and the contractile behavior of pulsation was evaluated using the PIV method.

As a result, the three-dimensional myocardial tissue on the collagen gel culture vessel exhibited contraction as large as about 10 times that of the three-dimensional myocardial tissue on the conventional culture insert (FIGS. 3 and 4).

iPS cell-derived cardiomyocytes laminated with fibronectin and gelatin, myocardial fibroblasts laminated with fibronectin and gelatin, and vascular endothelial cells (Lonza, catalog number: CC-7030) were mixed at a ratio of 75:25:10, respectively. The cell suspension was seeded in a collagen gel container (1.0×10 ⁶ cells: 10 layers), and a three-dimensional myocardial tissue was constructed in the same manner as in Example 1.

The resulting three-dimensional tissue was subjected to anti-CD31 antibody staining, anti-actin filament antibody staining and DAPI staining to examine the structures in the three-dimensional tissue. As a result, similar to construction by the conventional method, a vascular network was formed in the three-dimensional tissue, and vascular endothelial cells were seen to infiltrate inside the gel container (Fig. 5).

INDUSTRIAL APPLICABILITY The present invention can be used in fields such as regenerative medicine and drug discovery research. Specifically, the three-dimensional myocardial tissue obtained by the present invention can be used for safety/toxicity, pharmacokinetics, drug efficacy, pharmacological evaluation, drug screening, graft production, tissue engineering, cell culture, and biosensors in drug discovery research. , biochips, etc.

Claims (5)

A method for producing a three-dimensional myocardial tissue, comprising seeding and culturing a cell mixture containing myocardial cells, myocardial fibroblasts and vascular endothelial cells in a container having an extracellular matrix gel on the inner wall thereof, the container comprising: All of the bottom surface and all of the side surfaces of the inner wall are covered with extracellular matrix gel or are made of extracellular matrix gel, and pretreatment is performed to alternately layer the extracellular matrix on cardiomyocytes and myocardial fibroblasts. The ratio of cardiomyocytes, myocardial fibroblasts and vascular endothelial cells to be seeded is cardiomyocyte: myocardial fibroblast: vascular endothelial cell = 2.5-3.5: 1: 0.3-0 .5, wherein a vascular network is formed in the three-dimensional myocardial tissue, and vascular endothelial cells are infiltrating into the extracellular matrix gel surrounding the three-dimensional myocardial tissue. Extracellular matrix is one or more selected from the group consisting of collagen, atelocollagen, gelatin, fibronectin, laminin, cadherin, tenascin, entactin, elastin, fibrin, proteoglycan, hyaluronic acid, chitin, chitosan, and derivatives of the above substances 2. The method of claim 1, wherein: 3. The method of claim 2, wherein the extracellular matrix is collagen. The method according to any one of claims 1 to 3, wherein the three-dimensional myocardial tissue has an average maximum contraction velocity of 150 µm/s or more and an average maximum relaxation velocity of 90 µm/s or more. A method for drug screening or cardiotoxicity evaluation, which comprises using a three-dimensional myocardial tissue obtained by the method according to any one of claims 1 to 4.

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