An in vitro model of coronary heart disease based on vascularized myocardium and its construction method and application

Abstract

The present invention provides an in vitro model of coronary heart disease based on vascularized myocardium, and a construction method and application thereof. The method is mainly divided into processes such as engineering blood vessel production, myocardial tissue preparation, perfusion culture, and atherosclerosis simulation, which are respectively used to realize perfusable blood vessel construction, in vitro myocardial tissue regeneration, and pathological feature formation. First, a three-dimensional myocardial tissue with a complete vascular structure is prepared by layer-by-layer assembly of matrix gel; and a long-term culture of myocardial tissue is achieved by a perfusion culture method based on a vascular interface; finally, the pathological environment of coronary artery sclerosis is simulated by simulating high fat, inflammatory response, and immune cell enrichment, so as to realize the construction of a coronary heart disease model. The method of the present invention can realize the in vitro regeneration of large-scale myocardial tissue and the construction of an in vitro coronary heart disease model, providing new strategies and methods for the mechanism research and drug evaluation of coronary heart disease.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro construction of organ chip pathological models, and more specifically, to an in vitro model of coronary heart disease based on vascularized myocardium, and a construction method and application thereof. Background Art

[0002] Coronary heart disease is one of the main causes of cardiovascular disease and seriously threatens human life and health. Due to the complexity of the human body and the particularity of human tissue, in recent years, the study of heart disease is shifting from animal models to new in vitro tissue engineering models. Traditional 2D cardiomyocyte culture can provide a simple in vitro humanized platform for drug testing and disease modeling, but due to the randomness of the direction of myotube formation and the lack of specific tissue structure, 2D cells have great limitations in cardiovascular research. In contrast, in vitro 3D reconstruction of myocardial tissue can more closely simulate the structure and microenvironment of myocardial tissue in vivo and reproduce the contraction and other functions of myocardial tissue. In particular, the myocardium is highly vascularized to meet the high energy demand of myocardial activity. Therefore, constructing vascularized myocardial tissue is the key to building a cardiovascular disease model. At the same time, vascularized myocardium can provide better material exchange for 3D tissue samples and achieve long-term in vitro culture. It is also closer to the in vivo tissue microenvironment, providing a tissue-level disease research and drug evaluation model.

[0003] At present, myocardial vascularization methods are all based on organoid technology or 3D printing technology, while vascularized myocardial chips based on organ chip layer-by-layer assembly technology have not been reported. At the same time, in vitro tissue-level coronary heart disease models are a platform that is urgently needed for clinical research. Summary of the invention

[0004] According to the above-mentioned technical problem of the lack of in vitro humanized coronary heart disease models in biological and medical research, an in vitro model of coronary heart disease based on vascularized myocardium and its construction method and application are provided. The method of the present invention is mainly divided into processes such as engineering blood vessel production, myocardial tissue preparation, perfusion culture and atherosclerosis simulation, which are used to achieve perfusable blood vessel construction, in vitro myocardial tissue regeneration and pathological feature formation respectively. First, a three-dimensional myocardial tissue with a complete vascular structure is prepared by layer-by-layer assembly of matrix gel; and long-term culture of myocardial tissue is achieved by perfusion culture based on vascular interface; finally, the pathological environment of coronary artery sclerosis is simulated by simulating high fat, inflammatory response, immune cell enrichment, etc., to achieve the construction of a coronary heart disease model. The method of the present invention can achieve the in vitro regeneration of large-scale myocardial tissue and the construction of an in vitro coronary heart disease model, providing new strategies and methods for the mechanism research and drug evaluation of coronary heart disease.

[0005] The technical means adopted by the present invention are as follows:

[0006] A method for constructing an in vitro model of coronary heart disease based on vascularized myocardium comprises the following steps:

[0007] Step 1: construct a first mold, a second mold, and a third mold respectively by assembling multiple molds layer by layer;

[0008] Step 2: preparing vascular tissue using the first mold, and fixing the vascular tissue on the metal pipe joint using surgical thread or surgical vascular clip;

[0009] Step 3: assembling a second mold with a larger size outside the vascular tissue obtained in step 1, and pouring matrix glue mixed with cells into the second mold, and solidifying the matrix glue on the outer layer of the vascular tissue;

[0010] Step 4: Assemble the third mold into a culture chip with a culture chamber, and connect it to a perfusion tube and a peristaltic pump to perform perfusion culture on the vascular tissue obtained in step 3;

[0011] Step 5: Add free fatty acids, inflammatory factors, monocytes and other stimuli to the perfusion culture medium to simulate the pathological microenvironment of atherosclerosis and construct a coronary heart disease model.

[0012] Furthermore, the materials of the first mold, the second mold and the third mold are hard plastic materials, and the hard plastic materials are acrylic or polycaprolactone.

[0013] Furthermore, the first mold is a blood vessel production unit, including multiple blood vessel injection holes, multiple cylindrical chambers I, metal joint tubes and metal placeholder tubes. Multiple cylindrical chambers I are opened inside the first mold, the metal placeholder tubes are placed inside the cylindrical chamber I and are respectively connected to the metal joint tubes on both sides. The metal joint tubes on both sides extend to the outside of the first mold, and a gap is left between the metal placeholder tubes and the cylindrical chamber I. The blood vessel injection hole is opened on the first mold and is connected to the cylindrical chamber I, which is used to inject matrix glue into the cylindrical chamber I to generate vascular tissue.

[0014] Furthermore, the second mold is a myocardial bundle production unit, which includes multiple myocardial injection holes and multiple cylindrical chambers II. The myocardial injection holes are opened on the second mold and connected to the cylindrical chamber II, and are used to inject matrix glue into the cylindrical chamber II to generate a myocardial bundle structure.

[0015] Furthermore, the third mold is a perfusion unit, including a culture chamber, a perfusion inlet and a perfusion outlet. The culture chamber is opened inside the third mold, and the perfusion inlet and the perfusion outlet are arranged on both sides of the third mold. The perfusion culture medium of the culture chamber is dynamically perfused by connecting the perfusion inlet and the perfusion outlet through a perfusion tube.

[0016] Furthermore, in the step 2, vascular tissue is generated by perfusing matrix glue into the first mold, wherein the matrix glue is a collagen solution, the concentration of the collagen solution is 6-8 mg / ml, and 90% of its water is removed by in situ dehydration using dust-free paper to improve the mechanical strength; wherein the matrix of the collagen solution is mixed with smooth muscle or fibroblasts to form a vascular wall tube structure, which is a collagen tube with a cavity inside; endothelial cells are perfused into the collagen tube cavity and rotated and cultured to form a vascular endothelial structure.

[0017] Furthermore, in step three, the outer layer of vascular tissue formed is a myocardial bundle structure, the matrix gel used to generate the myocardial bundle structure is one or a mixture of collagen, Matrigel or Fibrinogen, and the cells are myocardial cells.

[0018] The present invention also provides an in vitro model of coronary heart disease based on vascularized myocardium constructed by the construction method.

[0019] The present invention also provides an application of the in vitro model of coronary heart disease based on vascularized myocardium in in vitro vascularized myocardial tissue regeneration, construction of a coronary heart disease model, and research on the mechanism of coronary heart disease and drug screening.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The in vitro model of coronary heart disease based on vascularized myocardium provided by the present invention, and its construction method and application, use a multi-module assembly system to construct a vascularized myocardium model, and establish a large-sized perfusable blood vessel and a three-dimensional myocardial model with good functions. Cultivate through the perfusion system to establish a three-dimensional vascularized myocardial tissue model. The system is mainly divided into a blood vessel production unit, a myocardial bundle production unit and a perfusion unit. By forming blood vessels, sufficient nutrients and oxygen are provided to large-sized myocardial bundles, simulating the myocardial tissue microenvironment in vivo, and providing physical support to the myocardial bundles, achieving a mechanical stretching effect, which can be used to observe cell activity and characterize functional changes, as well as for subsequent drug screening.

[0022] 2. The in vitro model of coronary heart disease based on vascularized myocardium provided by the present invention, and its construction method and application, use a multi-module assembly method to construct vascularized myocardial tissue, which can be used for myocardial tissue research and drug evaluation. In particular, additional equipment can be added to evaluate the effects of electrical or mechanical stimulation on myocardial maturation during myocardial formation and to establish a myocardial model of disease origin for characteristic drug screening.

[0023] In summary, the application of the technical solution of the present invention can solve the problem of lack of in vitro human coronary heart disease models in biological and medical research in the prior art.

[0024] Based on the above reasons, the present invention can be widely promoted in the field of in vitro construction of organ chip pathological models and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0026] Figure 1 Schematic diagram of the multi-unit structure of the present invention, wherein (a) is a blood vessel production mold, (b) is a muscle bundle production mold, and (c) is a perfusion mold.

[0027] Figure 2 The perfusion operation diagram and schematic diagram of the present invention, wherein (a) is a perfusion operation structure diagram, and (b) is a perfusion operation schematic diagram.

[0028] Figure 3 This is a physical picture of the vascularized myocardial model constructed in Example 1 of the present invention.

[0029] Figure 4 This is a diagram for identifying the expression of functional proteins of muscle and HUVEC in the mold in Example 2 of the present invention.

[0030] In the figure: 1. Blood vessel injection hole; 2. Cylindrical chamber I; 3. Metal joint tube; 4. Metal placeholder tube; 5. Myocardial injection hole; 6. Cylindrical chamber II; 7. Culture chamber; 8. Perfusion inlet; 9. Perfusion outlet. DETAILED DESCRIPTION

[0031] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0034] Unless otherwise specifically stated, the relative arrangement of the parts and steps described in these embodiments, the numerical expressions and numerical values ​​do not limit the scope of the present invention. At the same time, it should be clear that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The technology, methods and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but in appropriate cases, the technology, methods and equipment should be regarded as part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0035] In the description of the present invention, it is necessary to understand that the directions or positional relationships indicated by directional words such as "front, back, up, down, left, right", "lateral, vertical, perpendicular, horizontal" and "top, bottom" are usually based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description. Unless otherwise specified, these directional words do not indicate or imply that the device or element referred to must have a specific direction or be constructed and operated in a specific direction. Therefore, they cannot be understood as limiting the scope of protection of the present invention: the directional words "inside and outside" refer to the inside and outside relative to the contours of each component itself.

[0036] For ease of description, spatially relative terms such as "above", "above", "on the upper surface of", "above", etc. may be used here to describe the spatial positional relationship between a device or feature and other devices or features as shown in the figure. It should be understood that spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation described in the figure. For example, if the device in the accompanying drawings is inverted, the device described as "above other devices or structures" or "above other devices or structures" will be positioned as "below other devices or structures" or "below their position devices or structures". Thus, the exemplary term "above" can include both "above" and "below". The device can also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatially relative descriptions used here are interpreted accordingly.

[0037] In addition, it should be noted that the use of terms such as "first" and "second" to limit components is only for the convenience of distinguishing the corresponding components. If not otherwise stated, the above terms have no special meaning and therefore cannot be understood as limiting the scope of protection of the present invention.

[0038] As shown in the figure, the present invention provides a method for constructing an in vitro model of coronary heart disease based on vascularized myocardium, and the specific steps of the method are as follows:

[0039] Step 1: Prepare vascular tissue by assembling multiple molds layer by layer, and fix it on the metal pipe joint of the chip by surgical thread fixation or surgical vascular clips; wherein, the vascular tissue is generated by perfusing matrix glue into the mold, the matrix glue is a collagen solution, the concentration of the collagen solution is 6-8 mg / ml, and 90% of its water is removed by in-situ dehydration with dust-free paper to improve the mechanical strength; the matrix of the collagen solution is mixed with smooth muscle or fibroblasts, which is used to form a vascular wall tube structure, which is a collagen tube with a cavity inside; endothelial cells are perfused into the collagen tube cavity and rotated and cultured to form a vascular endothelial structure;

[0040] Step 2: Assemble the above-mentioned vascular tissue with a larger mold, perfuse the mold with matrix glue mixed with cells and solidify the matrix glue on the outer layer of the vascular tissue; wherein the outer layer of the vascular tissue formed is a myocardial bundle structure, the matrix glue used to generate the myocardial bundle structure is one or a mixture of collagen, Matrigel or Fibrinogen, and the cells are myocardial cells;

[0041] Step 3: Assemble the mold into a culture chip with a culture chamber, and connect it to a perfusion tube and a peristaltic pump to perform perfusion culture on the vascularized tissue.

[0042] Step 4: Add free fatty acids, inflammatory factors, monocytes and other stimuli to the perfusion culture medium to simulate the pathological microenvironment of atherosclerosis and construct a coronary heart disease model.

[0043] The mold (chip) used in the present invention is prepared by mechanical processing, and the mold includes 7 parts ( Figure 1 ). The core component A is an acrylic module with symmetrical through holes at both ends and a hollow middle. The other six components (B, C, D, E, F) can be assembled with the core component A in pairs to form angiogenesis molds (A+B\C), myocardium preparation molds (A+D / E) and perfusion culture chips (A+F / G). The preparation of vascularized myocardium is achieved by assembling the matrix gel layer by layer using molds of different sizes.

[0044] In a method for constructing an in vitro model of coronary heart disease based on vascularized myocardium provided by the present invention, myocardial tissue can be perfused and cultured through the vascular structure in the myocardial tissue. Adding free fatty acids, IL-1β, etc. to the culture medium can simulate the pathological microenvironment of atherosclerosis, leading to the NO synthesis dysfunction of myocardial blood vessels, decreased barrier function, and activation of endothelial cell adhesion factors. Further, monocytes are added to the perfusion culture system to simulate the adhesion and infiltration of monocytes in blood vessels in vitro to construct an early plaque model of atherosclerosis. Then, the influence of atherosclerosis formation on myocardial function is studied.

[0045] The present invention also provides a multi-module assembly mold system for constructing an in vitro model of coronary heart disease of the present invention, which is divided into three units. The designed acrylic molds are assembled to form a blood vessel production unit, a myocardial bundle production unit and a perfusion unit. The blood vessel production unit and the myocardial bundle production unit are used to prepare vascularized myocardial tissue by assembling layer by layer.

[0046] In the system, the blood vessel production unit mainly includes a plurality of blood vessel injection holes 1, a plurality of cylindrical chambers 2, a metal support tube 3, and a metal placeholder tube 4. A plurality of cylindrical chambers Ⅰ2 are opened inside the blood vessel production unit. The metal placeholder tube 4 is placed inside the cylindrical chamber Ⅰ2 and is respectively connected to the metal joint tube 3 on both sides. The metal joint tubes 3 on both sides extend to the outside of the blood vessel production unit. A gap is left between the metal placeholder tube 4 and the cylindrical chamber Ⅰ2. The blood vessel injection hole 1 is opened on the blood vessel production unit and is connected to the cylindrical chamber Ⅰ2 (each cylindrical chamber Ⅰ2 is connected to at least one blood vessel injection hole 1), which is used to perfuse matrix glue into the cylindrical chamber Ⅰ2 to generate vascular tissue. The myocardial bundle production unit mainly includes a plurality of myocardial injection holes 5 and a plurality of cylindrical chambers 6. The myocardial injection holes 5 are provided on the myocardial bundle production unit and are connected to the cylindrical chamber II 6 (each cylindrical chamber II 6 is connected to at least one myocardial injection hole 5), and are used to inject matrix glue into the cylindrical chamber II 6 to generate a myocardial bundle structure. The perfusion unit includes: a perfusion chamber 7, a perfusion inlet 8, and a perfusion outlet 9. The culture chamber 7 is provided inside the perfusion unit, and the perfusion inlet 8 and the perfusion outlet 9 are arranged on both sides of the perfusion unit. The perfusion culture medium of the culture chamber 7 is connected to the perfusion inlet 8 and the perfusion outlet 9 through a perfusion tube for dynamic perfusion. The structure is as follows Figure 1 shown.

[0047] The method of the present invention can be used for in vitro vascularized myocardial tissue regeneration and coronary heart disease model construction, and has transformation potential in coronary heart disease mechanism research and drug screening.

[0048] The present invention uses a multi-module assembly method to construct vascularized myocardial tissue, which can be used for myocardial tissue research and drug evaluation. In particular, additional equipment can be added to evaluate the impact of electrical stimulation or mechanical stimulation on myocardial maturation during myocardial formation and to establish a myocardial model of disease origin for characteristic drug screening.

[0049] The present invention uses a multi-module assembly system to construct a vascularized myocardial model, establishes a large-sized perfusable blood vessel and a well-functioning three-dimensional myocardial model. Cultivation is carried out through a perfusion system to establish a three-dimensional vascularized myocardial tissue model. The system is mainly divided into a blood vessel production unit, a myocardial bundle production unit and a perfusion unit. By forming blood vessels, sufficient nutrients and oxygen are provided to large-sized myocardial bundles, simulating the myocardial tissue microenvironment in the body, and having a physical support effect on the myocardial bundle, achieving a mechanical stretching effect, which can be used to observe cell activity and characterize functional changes and subsequently for drug screening.

[0050] Example 1

[0051] The present invention also provides a method for constructing a myocardial model based on a multi-module assembly mold system, which is performed according to the following steps:

[0052] (1) Two-dimensional cell culture

[0053] Human cardiomyocytes were cultured in myocardial medium (RPMI 1640 plus B27 Supplement) at 37°C and 5% CO2. When the cell density reached 70-80%, they were digested with 0.2% trypsin and set aside. Human umbilical vein endothelial cells (HUVEC) were cultured in endothelial cell medium (ECM) supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement and 1% penicillin / streptomycin solution at 37°C and 5% CO2. When the cell density reached 70-80%, they were digested with 0.2% trypsin and set aside.

[0054] (2) Preparation of engineered blood vessels

[0055] The blood vessel production unit mold was sterilized, and a 7 mg / mL collagen solution was prepared and injected into the cylindrical channel I 2 through the blood vessel injection hole 1, and solidified at 37°C for 30 minutes to form a collagen tube. After that, it was dehydrated and the port was fixed to the metal support tube 3.

[0056] (3) Preparation of myocardial bundles

[0057] The myocardial bundle production unit mold was sterilized, and myocardial cells and human umbilical vein endothelial cells (HUVEC) were distributed (resuspended) in type I rat tail collagen at a certain cell density, and injected into cylindrical channel II 6 from myocardial injection hole 5, respectively, and solidified at 37°C for 30 minutes.

[0058] (4) Inoculation of endothelial cells

[0059] The metal placeholder tube 4 was removed, and a cavity was formed in the collagen tube. Human umbilical vein endothelial cells (HUVECs) were seeded into the cavity, and after being allowed to stand in an incubator for 30 minutes, the tube was turned upside down and allowed to stand for 24 hours.

[0060] (5) Perfusion co-culture

[0061] The vascular perfusion medium is connected to both ends of the metal support tube 3 through a perfusion tube for dynamic perfusion culture. The culture chamber perfusion medium is connected to the perfusion inlet 8 and the perfusion outlet 9 through a perfusion tube for dynamic perfusion. Cardiomyocyte basal medium (RPMI 1640 plus B27 Supplement) and endothelial cell medium (1:1) are used for perfusion for 3 days, and stimulation conditions are added. Figure 2 shown.

[0062] Example 2

[0063] Identification of Myocardial and Vascular Cells:

[0064] After constructing vascularized myocardium using a mold designed and made in the laboratory, the structure is as follows Figure 3As shown. After the perfusion culture, the cardiomyocytes were functionally identified. The method is as follows: the myocardial bundle mold was taken out, and the cells were fixed with 4% paraformaldehyde for 90 minutes, and rinsed with PBS buffer three times, each time for 10-15 minutes; 0.1% triton X-100 porogen was used for 40 minutes, and the cells were rinsed with PBS buffer three times, each time for 10-15 minutes; goat blocking serum was used at 4°C overnight, the primary antibody was incubated overnight, and the cells were rinsed with PBS buffer three times, each time for 10-15 minutes; the secondary antibody was incubated at room temperature and away from light for 1 hour, and the cells were rinsed with PBS buffer three times, each time for 10-15 minutes; after rinsing, a 1:5000 diluted DAPI working solution was added and incubated for 15 minutes; the cells were rinsed with PBS buffer twice; the endothelial cells were functionally identified, the primary antibody was incubated overnight, and the cells were rinsed with PBS buffer three times, each time for 10-15 minutes; the secondary antibody was incubated at room temperature and away from light for 1 hour, and the cells were rinsed with PBS buffer three times. The other steps were consistent with the above, and pictures were taken under a fluorescence microscope to record the expression of the corresponding proteins. The results are shown as follows Figure 4 shown.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an in vitro model of coronary heart disease based on vascularized myocardium, characterized in that: The steps include: Step 1: construct a first mold, a second mold, and a third mold respectively by assembling multiple molds layer by layer; Step 2: preparing vascular tissue using the first mold, and fixing the vascular tissue on the metal pipe joint using surgical thread or surgical vascular clip; Step 3: assembling a second mold with a larger size outside the vascular tissue obtained in step 1, and pouring matrix glue mixed with cells into the second mold, and solidifying the matrix glue on the outer layer of the vascular tissue; Step 4: Assemble the third mold into a culture chip with a culture chamber, and connect it to a perfusion tube and a peristaltic pump to perform perfusion culture on the vascular tissue obtained in step 3; Step 5: Add free fatty acids, inflammatory factors and monocyte stimulation to the perfusion medium to simulate the pathological microenvironment of atherosclerosis and construct a coronary heart disease model; The first mold is a blood vessel production unit, comprising a plurality of blood vessel injection holes (1), a plurality of cylindrical chambers I (2), a metal joint tube (3) and a metal placeholder tube (4). The plurality of cylindrical chambers I (2) are opened inside the first mold. The metal placeholder tube (4) is placed inside the cylindrical chamber I (2) and is connected to the metal joint tube (3) on both sides. The metal joint tubes (3) on both sides extend to the outside of the first mold. A gap is left between the metal placeholder tube (4) and the cylindrical chamber I (2). The blood vessel injection hole (1) is opened on the first mold and is connected to the cylindrical chamber I (2) for injecting matrix glue into the cylindrical chamber I (2) to generate blood vessel tissue. The second mold is a myocardial bundle production unit, comprising a plurality of myocardial glue injection holes (5) and a plurality of cylindrical chambers II (6). The myocardial glue injection holes (5) are provided on the second mold and are connected to the cylindrical chambers II (6) for injecting matrix glue into the cylindrical chambers II (6) to generate a myocardial bundle structure. The third mold is a perfusion unit, comprising a culture chamber (7), a perfusion inlet (8) and a perfusion outlet (9); the culture chamber (7) is opened inside the third mold, the perfusion inlet (8) and the perfusion outlet (9) are arranged on both sides of the third mold, and the perfusion culture medium of the culture chamber (7) is connected to the perfusion inlet (8) and the perfusion outlet (9) through a perfusion tube for dynamic perfusion; In the step 2, vascular tissue is generated by perfusing matrix glue into the first mold, where the matrix glue is a collagen solution with a concentration of 6-8 mg / ml, and 90% of its water is removed by in situ dehydration with dust-free paper to improve the mechanical strength; wherein the matrix of the collagen solution is mixed with smooth muscle or fibroblasts to form a vascular wall tube structure, which is a collagen tube with a cavity inside; endothelial cells are perfused into the collagen tube cavity and rotated and cultured to form a vascular endothelial structure.

2. The method for constructing an in vitro model of coronary heart disease based on vascularized myocardium according to claim 1, characterized in that: The materials of the first mold, the second mold and the third mold are hard plastic materials, and the hard plastic materials are acrylic or polycaprolactone.

3. The method for constructing an in vitro model of coronary heart disease based on vascularized myocardium according to claim 1, characterized in that: In the step three, the outer layer of the vascular tissue formed is a myocardial bundle structure, the matrix gel used to generate the myocardial bundle structure is one or a mixture of collagen, Matrigel or Fibrinogen, and the cells are myocardial cells.

4. An in vitro model of coronary heart disease based on vascularized myocardium constructed by the construction method described in any one of claims 1 to 3.

5. Use of the in vitro model of coronary heart disease based on vascularized myocardium according to claim 4 in in vitro vascularized myocardial tissue regeneration, construction of a coronary heart disease model, and research on the mechanism of coronary heart disease and drug screening.

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