JP2020031548A - Method for producing three-dimensional myocardial tissue - Google Patents

Abstract

To provide a three-dimensional myocardial tissue that has a high cell density, significantly great shrinkage force and can sufficiently reproduce a biological tissue and a method of producing the same.SOLUTION: A method for producing a three-dimensional myocardial tissue includes culturing a cardiomyocyte or a cell mixture containing a cardiomyocyte in a container having an extracellular matrix gel in an inner wall. There is also provided a cultured three-dimensional myocardial tissue including a cardiomyocyte or a cell mixture containing a cardiomyocyte in a container having an extracellular matrix gel in an inner wall.SELECTED DRAWING: None

Description

  The present invention relates to an artificial living tissue and a method for producing the same. More specifically, the present invention relates to a three-dimensional myocardial tissue and a method for producing the same.

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

  In order to construct a three-dimensional cardiomyocyte tissue having a higher cell density, a cell sheet technique has been reported (for example, Non-Patent Document 2). By laminating the cell sheets, it is possible to construct a three-dimensional tissue having a high cell density. However, since the cell density in the cell sheet is high, internal necrosis occurs, and the contraction force of the tissue itself is not sufficient.

  On the other hand, by coating the cell membrane with a protein, which is a cell adhesion factor, on the surface of the cell, it promotes cell-cell adhesion and builds a three-dimensional structure with high cell density. Have been reported (Patent Documents 1 and 2, Non-Patent Documents 3 and 4). With these techniques, a three-dimensional myocardial tissue having a high cell density and a vascular network structure has been constructed (Non-Patent Document 5). Such a myocardial tissue exhibits a reaction close to that of a living body, and research for drug discovery application and evaluation of drug response are being advanced. In addition, since such a myocardial tissue has a vascular network, engraftment and maturation have been confirmed when transplanted (Non-Patent Document 6). However, the myocardial tissue obtained by the above technique cannot be said to sufficiently reproduce living tissue from the viewpoint of contraction force.

Japanese Patent No. 4991964 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 having a high cell density, a large contraction force and sufficient reproduction of a living tissue. It is desired to apply such three-dimensional myocardial tissue to regenerative medicine and drug discovery.

  The present inventors have conducted intensive studies and as a result, found that the above-mentioned 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 completed the present invention. Was.

That is, the present invention provides the following.
(1) A method for producing three-dimensional myocardial tissue, comprising culturing cardiomyocytes or a cell mixture containing cardiomyocytes in a container having an extracellular matrix gel on the inner wall.
(2) The group whose extracellular matrix is composed 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 the group consisting of:
(3) The method according to (1) or (2), wherein the cell mixture contains cardiomyocytes 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 a pretreatment for alternately stacking extracellular matrices on the cardiomyocytes is performed.
(6) The method according to any one of (1) to (5), wherein the average maximum contraction speed of the three-dimensional myocardial tissue is 150 μm / s or more, and the average maximum relaxation speed is 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) The group consisting 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 the group consisting of:
(9) The tissue according to (7) or (8), wherein the cell mixture contains cardiomyocytes 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 a pretreatment for alternately laminating an extracellular matrix on the cardiomyocytes is performed.
(12) The tissue according to any one of (7) to (11), wherein the average maximum contraction speed is 150 µm / s or more, and the average maximum relaxation speed is 90 µm / s or more.

  According to the present invention, a three-dimensional myocardial tissue having a high cell density and a large contraction force close to a living body and a method for producing the same are provided. It is considered that the large contraction force of the three-dimensional myocardial tissue of the present invention is because the extracellular matrix gel on the inner wall of the container does not inhibit the pulsatile function of the myocardial tissue and can retain the myocardial tissue. Since the three-dimensional myocardial tissue of the present invention can reproduce the same reaction as in-vivo myocardium in vitro, it is possible to realize more advanced regenerative medicine and drug discovery research.

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 (in a broken line) constructed on a collagen gel culture vessel. FIG. 3 is a graph showing the contraction behavior of a three-dimensional myocardial tissue constructed using a collagen gel culture vessel and a new three-dimensional tissue constructed using a culture insert. FIG. 4 is a graph showing the maximum contraction rate and maximum relaxation rate of a three-dimensional myocardial tissue constructed using a collagen gel culture vessel and a new three-dimensional tissue constructed using a culture insert. FIG. 5 is a fluorescent staining image (cross-sectional view from the horizontal direction) 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, which comprises 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 secreted from living cells and accumulated outside the cells, and is involved in regulation of cell adhesion, proliferation and differentiation. The extracellular matrix used for the container in the present invention may be one that exists in the living body, or one that does not exist in the living body but has the same function and characteristics as those present in the living body. . In the present specification, these are collectively referred to as an extracellular matrix. The preferred extracellular matrix used for the container in the present invention is a cell-adhesive and gellable one. Further, those having biocompatibility are also preferable. Examples of preferred extracellular matrix 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 / chitosan, or , Polyethylene glycol, polyvinyl alcohol, and other water-soluble synthetic polymers, and derivatives of the above substances, but are not limited thereto. Collagen, gelatin and fibrin are particularly preferably used for the container of the present invention because they are very excellent in both cell adhesiveness and gelling ability and are also biocompatible. Derivatives of the above substances include all derivatives as long as cell adhesion, gelling ability and biocompatibility are not impaired. Methods for producing derivatives are known to those skilled in the art.

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

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

  The properties 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 rate and size of the three-dimensional myocardial tissue, and the like. It is preferable to determine the properties and physical properties of the extracellular matrix gel so as not to inhibit the pulsatile function of the myocardial tissue and retain the myocardial tissue. It is also preferable to determine the properties and properties of the extracellular matrix gel so that the three-dimensional myocardial tissue does not fall off the gel. Such a determination is within the skill of those 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 inner wall of the container includes a bottom surface, sides, and a top surface, if present, inside the container. For example, the entire bottom and side surfaces of the container may be covered with extracellular matrix gel, or may be made of extracellular matrix. Also, for example, a part of the bottom surface and a part of the side surface of the container may be covered with an extracellular matrix gel, or may be made of an extracellular matrix. Further, for example, the entire bottom surface and a part of the side surface of the container may be covered with an extracellular matrix gel, or may be made of an extracellular matrix. Further, for example, a part of the bottom surface and the entire side surface of the container may be covered with an extracellular matrix gel, or may be made of an extracellular matrix. Preference is given to containers in which the entire bottom surface is covered with extracellular matrix gel or made of extracellular matrix. More preferred are containers in which the entire bottom surface and all of the side surfaces are covered with an extracellular matrix gel or are made of an extracellular matrix.

  In order to prepare such a container, an extracellular matrix gel may be applied to the inner wall of an existing glass or plastic culture container. Alternatively, as shown in Examples of the present application, a container may be produced by solidifying while pressing the mold against the extracellular matrix gel, and removing the mold after solidification. As for the container prepared by forming the concave portion in the extracellular matrix gel as described above, the inner wall of the container indicates the concave portion.

  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, and the like, and are not particularly limited. For example, the bottom surface of the container may be a circle having a diameter of about 100 μm to several cm.

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

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

  In the method for producing 3D myocardial tissue of the present invention, 3D myocardial tissue can be constructed using only cardiomyocytes, but 3D myocardial tissue is constructed using a mixture of myocardial cells and myocardium-derived fibroblasts. Is preferred. A three-dimensional myocardial tissue having a vascular network can be obtained by using a mixture of myocardial cells, myocardial fibroblasts and vascular endothelial cells.

  The culturing of the cells in the method for producing a three-dimensional myocardial tissue of the present invention includes, as a preceding step, seeding of the cells into the container. Seeding of cells 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 cells using a pipette. Alternatively, seeding may be performed using various 3D printers. In the printer system, there are an ink jet printer, a micro extrusion printer, a laser assist printer, and the like. There is another method of sowing using an application needle. The printer method and the method using an application needle are suitable for producing a microchip of three-dimensional myocardial tissue.

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

  The number of cells to be seeded may be any number as long as a three-dimensional myocardial tissue can be obtained. The number of cells can be determined according to the size, shape, and use of the three-dimensional myocardial tissue, and the size and shape of the container. Generally, the number may be such that several to several tens of cells accumulate on the bottom surface of the container, or may be such that the thickness of the seeded cell layer is several tens μm to several hundred μm.

  In the method for producing a three-dimensional myocardial tissue of the present invention, it is preferable to perform a pretreatment for alternately laminating an extracellular matrix on cells constituting the tissue. Cells subjected to such pretreatment are referred to as LbL cells. By performing such a pretreatment, adhesion between cells is induced, and a three-dimensional tissue can be constructed in a short time. It is preferable to perform such a pretreatment on cardiomyocytes. Cells other than cardiomyocytes may be subjected to the above pretreatment. For example, when producing a three-dimensional myocardial tissue using cardiomyocytes, cardiac fibroblasts and vascular endothelial cells, the above-mentioned pretreatment may be applied to myocardial cells and cardiac fibroblasts.

  In the above pretreatment, the first and second extracellular matrices are alternately stacked on cells. 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 a first cell. A protein that interacts with the outer matrix. Here, the interaction means, for example, chemical interaction by electrostatic interaction, hydrophobic interaction, hydrogen bond, charge transfer interaction, covalent bond formation, specific interaction between proteins, van der Waals force, etc. Physically, it is close enough to bond, adhere, adsorb, or transfer electrons with a polymer containing an RGD sequence. Note that the first and second extracellular matrices are preferably biodegradable.

  Specific examples of the combination 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, elastin. And polylysine, fibronectin and collagen, laminin and collagen, vitronectin and collagen, RGD-bound collagen or RGD-bound gelatin and collagen or gelatin, and the like. 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 each be of one type, or two or more types may be used in combination as long as they exhibit an interaction.

  The method for alternately laminating the extracellular matrix on the cells is not particularly limited. For example, the cells are immersed in an aqueous solution containing the first extracellular matrix, and then the aqueous solution containing the second extracellular matrix is used. May be immersed in the cells. Further, for example, an aqueous solution containing the first extracellular matrix and an aqueous solution containing the second extracellular matrix may be alternately contacted on the cell surface of the monolayer. For these methods, see, for example, Akagi et al., Surface, Vol. 51, No. 4, p. 1-11 (2013), 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 above-mentioned container. Culture of cardiomyocytes or a cell mixture containing cardiomyocytes can be carried out by using a usual animal cell culture method. For example, the cells may be cultured at 20 to 40 ° C, preferably 35 to 39 ° C, more preferably 37 ° C, for example, for 1 to 14 days, preferably several days to 10 days. Known media can also be used. Examples of the medium 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 can be determined according to 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 are not particularly limited.

  The thickness of the three-dimensional myocardial tissue of the present invention obtained by the above manufacturing method can be several tens 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 may be about 10 times or more that of the myocardial tissue obtained by the conventional method. The average maximum contraction rate of the three-dimensional myocardial tissue of the present invention can be 50 μm / s or more, preferably 100 μm / s or more, more preferably 150 μm / s or more, and the average maximum relaxation rate is 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 contraction rate and a high relaxation rate, a reaction can be observed even when a small amount of a test drug is given, for example, during drug screening. 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 is possible to reproduce the same reaction as in vivo in vitro.

  The three-dimensional myocardial tissue obtained by the production method of the present invention may be used separately 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 graft applied to the heart. Further, the three-dimensional myocardial tissue of the present invention may be used for tests such as drug screening and cardiotoxicity evaluation. Further, the three-dimensional myocardial tissue of the present invention may be arranged on a substrate to form a chip and used for a high-throughput test. 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.

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

  The cardiomyocytes constituting the cultured three-dimensional myocardial tissue are preferably cells that have been subjected to a pretreatment for alternately laminating extracellular matrices. The pretreatment may be applied to cells other than cardiomyocytes constituting the cultured three-dimensional myocardial tissue.

  The average maximum contraction rate of the cultured three-dimensional myocardial tissue can be 50 μm / s or more, preferably 100 μm / s or more, more preferably 150 μm / s or more, and the average maximum relaxation rate is 30 μm / s or more, preferably 60 μm / s. Or more, more preferably 90 μm / s or more.

  The meanings of the terms herein other than those described above are to be understood as commonly understood in the fields of medicine, cell engineering, biology, and the like.

  Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the examples do not limit the scope of the present invention.

According to the method shown in FIG. 1, a three-dimensional myocardial tissue was constructed using a collagen gel culture vessel. A collagen solution (2.1 mg / ml) was dropped on a 6-well plate, and a culture insert (24-well size) was provided. Further, a collagen solution was dropped on the periphery and left at 37 ° C. for 30 minutes to gel. After gelation, the culture insert was removed, and the formed concavity was used as a culture vessel, and the applicant induced iPS cell-derived cardiomyocytes (iPS cell line 253G1 (RIKEN) into myocardium by laminating fibronectin and gelatin. ) And myocardial fibroblasts (Lonza, catalog number: CC-2904) in which fibronectin and gelatin were layered on each other, and the cells were seeded by dropping (1.0 × 10 ⁶ cells: 10 layers), By culturing, a three-dimensional myocardial tissue was constructed (FIG. 2). The ratio of seeded cardiomyocytes to cardiomyocyte fibroblasts was cardiomyocyte: cardiomyocyte fibroblast = 75: 25. After seeding the cells, a DMEM medium (containing 10% FBS) was added and the cells were cultured at 37 ° C. for 5 days, and the beating contraction behavior of the constructed three-dimensional myocardial tissue was evaluated.

  As a method for evaluating the constructed three-dimensional myocardial tissue, imaging by a high-speed camera and a particle image flow velocity measurement method (PIV method) were used. As a comparative experiment, 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 contraction behavior of pulsation was evaluated using the PIV method.

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

IPS cell-derived cardiomyocytes in which fibronectin and gelatin were layered, cardiomyocyte fibroblasts in which fibronectin and gelatin were layered, and vascular endothelial cells (Lonza, catalog number: CC-7030) were mixed at 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 obtained three-dimensional tissue was subjected to anti-CD31 antibody staining, anti-actin filament antibody staining and DAPI staining to examine the structure in the three-dimensional tissue. As a result, a vascular network was formed in the three-dimensional tissue as in the case of the construction according to the conventional method, and it was recognized that vascular endothelial cells had infiltrated inside the gel container (FIG. 5).

  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 to evaluate safety / toxicity, pharmacokinetics, pharmacodynamics, pharmacology, drug screening, graft production, tissue engineering, cell culture, biosensors in drug discovery research , Biochips and the like.

Claims (12)

  A method for producing three-dimensional myocardial tissue, comprising culturing cardiomyocytes or a cell mixture containing cardiomyocytes in a container having an extracellular matrix gel on the inner wall.   The extracellular matrix is selected from the group consisting 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. 2. The method of claim 1, wherein the method is one or more.   The method according to claim 1 or 2, wherein the cell mixture contains cardiomyocytes and cardiomyocyte fibroblasts.   4. The method according to claim 3, wherein the cell mixture further comprises vascular endothelial cells.   The method according to any one of claims 1 to 4, wherein a pretreatment for alternately laminating an extracellular matrix is performed on the cardiomyocytes.   The method according to any one of claims 1 to 5, wherein the average maximum contraction speed of the three-dimensional myocardial tissue is 150 µm / s or more, and the average maximum relaxation speed is 90 µm / s or more.   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.   The extracellular matrix is selected from the group consisting 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. 8. The tissue of claim 7, wherein the tissue is one or more.   9. The tissue according to claim 7, wherein the cell mixture contains cardiomyocytes and cardiomyofibroblasts.   The tissue according to claim 9, wherein the cell mixture further contains vascular endothelial cells.   The tissue according to any one of claims 7 to 10, wherein a pretreatment for alternately laminating an extracellular matrix is performed on the cardiomyocytes.   The tissue according to any one of claims 7 to 11, wherein the average maximum contraction speed is 150 µm / s or more, and the average maximum relaxation speed is 90 µm / s or more.