Construction method of cardiopulmonary organs based on human iPSC (induced pluripotent stem cell) differentiation and culture medium composition

Abstract

The invention discloses a construction method of a cardiopulmonary organ based on human induced pluripotent stem cell (iPSC) differentiation and a special culture medium composition. The method comprises the following steps: firstly, synchronously inducing and differentiating the iPSCs into mesoderm aggregates and anterior intestinal derm, then embedding the mesoderm aggregates and the anterior intestinal derm into matrigel for 3D culture, and adding a culture medium containing factors such as Activin A, CHIR99021, SB431542, Noggin, SAG, FGF4, Y27632 and A8301 in the culture process. After 25 days of culture, the human iPSCs can be successfully differentiated into various cell types of cardiopulmonary organs including cardiac myocardial cells, cardiac vascular endothelial cells, lung near-end and far-end airway epithelial cells, airway secretory cells, alveolar secretory cells, airway basal cells, cilia cells and the like. According to the method, collaborative culture of the heart and lung organs is realized for the first time, the limitation of independent culture of the center organs and the lung organs in the prior art is broken through, and the method has important application value in the fields of cardiopulmonary disease modeling, drug screening, toxicity testing, personalized treatment, organ repair and the like.

Description

Technical Field

[0001] This patent belongs to the field of biotechnology and mainly involves the culture technology and culture medium combination of human pluripotent stem cells to induce cardiopulmonary organoids. Background of the Invention

[0002] Organoids are miniature organ models formed by self-organization of stem cells or specific tissue cells through in vitro three-dimensional culture technology. They can closely simulate the structure and function of human organs. They are of great value in disease mechanism research, drug screening, personalized medicine, organ development research and regenerative medicine. [1,2] Organoids not only provide scientists with an experimental platform that is closer to the human physiological environment, but also reduce dependence on animal experiments and promote the advancement of precision medicine and drug development. [3] In addition, organoid technology has shown great potential in studying complex diseases (such as cancer and infectious diseases) and developing new treatments. [4] , is an indispensable tool in biomedical research and clinical applications.

[0003] Currently, iPSC-induced organoid technology has successfully induced cardiac organoids [5,6] and lung organoids [7,8] , providing important tools for disease research and drug development [9] However, as organs with closely connected functions, the heart and lungs often suffer from cardiopulmonary comorbidities (such as pulmonary hypertension, heart failure with pulmonary insufficiency, etc.)

[10] , but the iPSC-induced cardiopulmonary organoid culture model has not yet been realized. Therefore, the establishment of a co-induced differentiation culture method for cardiopulmonary organoids is of great significance for studying cardiopulmonary interactions, developing treatment plans for related diseases, and evaluating the comprehensive effects of drugs on the cardiopulmonary system. It not only has strong market demand and application potential, but also fills the gaps in existing technologies. The method we created solves the key problem of the lack of co-culture of cardiopulmonary organoids in existing technologies by innovatively integrating the culture conditions of heart and lung organoids, providing a new solution for cardiopulmonary co-disease research, drug screening and personalized medicine, and is significantly innovative and practical.

[0004] Purpose of the Invention

[0005] The present invention aims to address the limitations of the existing technology of separate culture of cardiopulmonary organoids, and to achieve functional integration and collaborative culture of heart and lung organoids by developing an innovative co-culture method of cardiopulmonary organoids. The heart and lungs are closely connected in physiological function, and separate culture cannot fully simulate their interaction. Cardiopulmonary co-culture can more realistically reflect the physiological and pathological states in the body, providing a more accurate research model and drug screening platform for studying cardiopulmonary co-diseases. In addition, this method is of great value in drug research and development, and can simultaneously evaluate the efficacy and toxicity of drugs on the cardiopulmonary system, significantly improving the efficiency and accuracy of drug screening. With the increasing incidence of cardiopulmonary diseases and the growing demand for personalized medical care, cardiopulmonary co-culture technology has strong market demand and application potential. It not only promotes disease mechanism research and drug development, but also provides a new solution for regenerative medicine and clinical treatment, which has important scientific significance and commercial value. Summary of the Invention

[0006] The present invention provides a method for constructing heart-lung symbiotic organoids, wherein the heart-lung symbiotic organoid generation process includes the lung endoderm, lung foregut germ layer, lung organoids, and heart organoid mesoderm, as well as various stages of development. To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A method for constructing a cardiopulmonary symbiotic organoid, comprising:

[0008] S1: iPSCs were differentiated into mesoderm and endoderm: mesoderm differentiation was completed in E8 medium supplemented with Y27632 (10-20 μM), followed by centrifugation to form cell spheres. Endoderm differentiation was then induced for four days in Advanced DMEM / F12 basal medium supplemented with Activin A (100-200 ng / mL), CHIR-99021 (2-5 μM), and FBS (2%).

[0009] S2: Use RPMI1640 medium containing FGF2 (30-50 ng / mL), Activin A (100-500 ng / ml), BMP4 (10-20 ng / mL), Y-27632 (5-10 μM), and Insulin (1-5 μg / mL) to direct the mesodermal cell spheres into embryoid bodies. Advanced DMEM / F12, SB431542 (10-50 μM), Noggin (200-500 ng / mL), SAG (1-5 μM), FGF4 (200-500 ng / mL), CHIR99021 (1-5 μM), N-2 (1×), B27+ (50×), L-glutamine (1×), BSA (0.05%), monothinoglycerol (0.4-1 μM), ascorbic acid (0.1%), and β-glutamic acid (0.1%) were added to the endoderm induced in S1. acid (50-100 μg / mL), FGF7 (10-50 ng / mL), and ATRA (50 nM) for six days to complete the induction of the foregut germ layer;

[0010] S3: Obtain oriented embryoid bodies and foregut in step S2. Further, the foregut and embryoid bodies are simultaneously transferred into Matrigel. This step begins on the tenth day after the endoderm begins to be cultured.

[0011] S4: Further, the culture medium in step S3 is changed according to different time periods, and first E8, N-2 (1×), B27+ (50×), L-glutamine (1×), penicillin-streptomycin (1×), BSA (2%), monothinoglycerol (0.4-1 μM), ascorbic acid (50-100 μg / mL), FGF7 (10-50 ng / mL), ATRA (50-100 nM), CHIR-99021 (3-5 μM), Y-27632 (2-5 μM), and Noggin (100-300 ng / mL) are added;

[0012] Furthermore, five days later, the medium was replaced with Advanced DMEM / F12, N-2 (1×), B27- (50×), L-glutamine (1×), penicillin-streptomycin (1×), BSA (2%), monothinoglycerol (0.4-1 μM), FGF7 (10-50 ng / mL), ATRA (50-100 nM), CHIR-99021 (5-10 μM), BMP4 (10-20 ng / mL), SAG (2-10 μM), and FGF4 (100-300 ng / mL);

[0013] Furthermore, seven days later, the medium was replaced with Advanced DMEM / F12, N-2 (1×), B27- (50×), L-glutamine (1×), BSA (2%), monothinoglycerol (0.4-1 μM), ascorbic acid (50-100 μg / mL), FGF7 (10-50 ng / mL), ATRA (50-100 nM), IWP2 (5-10 μM), SB431542 (50 μM), SAG (2-5 μM), and A8301 (200-500 nM);

[0014] Furthermore, eight days later, the medium was replaced with Advanced DMEM / F12, N-2 (1×), B27- (50×), L-glutamine (1×), penicillin-streptomycin (1×), BSA (2%), ascorbic acid (50-100 μg / mL), FGF7 (10-50 ng / mL), ATRA (50-100 nM), FGF2 (20-50 ng / mL), and A8301 (200-500 nM);

[0015] S5: Differentiate the foregut germ layers into lung organoids and then convert the culture into mature cardiopulmonary symbiotic organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings used in the embodiments of the present invention. The drawings in the following description are only used to explain some embodiments of the present invention. Figure 1 This is a flow chart of the method for constructing a cardiopulmonary symbiotic organoid provided by the present invention;

[0017] Figure 2 The cell morphology and immunofluorescence staining identification images (Nanog) of iPSCs provided by the present invention are shown. Scale bar: 50 μm.

[0018] Figure 3 The figure shows the cell morphology of the endoderm in the embodiment of the present invention, with a scale bar of 300 μm, and immunofluorescence staining identification (FOXA2, SOX17), with a scale bar of 30 μm;

[0019] Figure 4 This is a picture of the foregut germ layer cell morphology taken with an optical microscope in an embodiment of the present invention, scale bar: 100 μm;

[0020] Figure 5 Figures 1 and 2 are images of embryoid body formation and error examples in the examples of the present invention, as well as morphological images taken by light microscopy during cardiac organoid development. Scale bar: 100 μm.

[0021] Figure 6 This is an example diagram of the cardiopulmonary culture process in an embodiment of the present invention, scale: 500 μm;

[0022] Figure 7 This is an immunofluorescence staining image of the cardiopulmonary organoids produced in this example. The images show staining for vascular endothelial cells (CD31), cardiomyocytes (CTNT), proximal lung epithelial cells (SOX2), alveolar epithelial cells (SPC), distal lung epithelial cells (SOX9), basal cells (KRT5), ciliated cells (AC-TUBULIN), and airway secretory cells (SCGB3A1). Scale bar: 20 μm. DETAILED DESCRIPTION

[0023] In order to explain the specific solutions of the present invention more clearly, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0024] An embodiment of the present invention provides a method for constructing a heart-lung symbiotic organoid, which effectively regulates the signal pathway for directed differentiation of iPSCs, so that iPSCs can differentiate into the endoderm and foregut germ layer, and then the embryoid bodies and foregut germ layer are transferred to Matrigel together to realize the co-culture of heart-lung organoids. According to the construction method of the present invention, the differentiation of lung organoids derived from the foregut germ layer, embryoid bodies, mesoderm, cardiac progenitor cells and cardiomyocytes can be cultured simultaneously. This heart-lung symbiotic organoid differentiation scheme realizes for the first time the simultaneous differentiation of symbiotic heart organoids and lung organoids in the same differentiation system.

[0025] An embodiment of the present invention provides a method for constructing cardiopulmonary symbiotic organoids, which includes: in the early stage of culture, adding ActivinA and CHIR-99021 culture additives to the culture medium to direct the differentiation of iPSCs to form the endoderm, and then adding Noggin, SAG, FGF4, N-2, B27, etc. to direct their differentiation into the foregut germ layer, and inducing their differentiation into cardiopulmonary organoids by adding CHIR99021, IWP2, SB431542, SAG, A8301, etc.

[0026] The differentiation of heart-lung symbiotic organoids requires multiple stages, including the process from iPSCs to directed endoderm, foregut, co-culture of embryoid bodies and foregut, and finally mature heart-lung organoids. To achieve heart-lung co-culture, the induction of the endoderm and foregut is crucial, followed by the formation of lung organoids. Simultaneously, the centrifuged embryoid bodies are induced to form heart organoids.

[0027] The construction method of the present invention can be divided into the following stages

[0028] In the following examples, unless otherwise specified, all experiments were carried out under sterile conditions, and the culture conditions were all at 37° C. and 5% CO 2 .

[0029] The present invention relates to a technology and culture medium combination for inducing co-culture of cardiopulmonary organoids using human iPSCs.

[0030] (1) iPSC culture

[0031] iPSCs were cultured on a culture plate pre-coated with Matrigel (Matrigel was diluted with DMEM / F12 at a ratio of about 1:80) for at least one hour, and the culture plate was placed in a cell culture incubator for culture. The mTeSR was replaced every day. TM When the cell density reaches 75%-85%, the cells need to be passaged. TM Digestion and passage were performed according to the instructions of the manufacturer.

[0032] (2) Establishing a protocol for the iPSC-to-cardiac embryoid body formation stage

[0033] In order to form embryoid bodies, the present application uses A1 culture additives in the culture system at this stage to direct the differentiation of iPSCs. Take an appropriate amount of cells (prepare a cell suspension with a density of 5000-8000 cells / 100 μL culture medium) and seed 100 μL of cell suspension per well on a U-shaped ultra-low attachment 96-well plate. Centrifuge at 300 × g and 4°C for 3 minutes, and place the cells at the bottom of the culture plate to obtain cardiac spheres. Then place them in an incubator and direct them into mesoderm in A2 culture medium. Among them, A1 culture medium is E8 and Y27632 (10-20 μM); A2 culture medium components are RPMI1640, FGF2 (30-50 ng / mL), ActivinA (100-500 ng / ml), BMP4 (10-20 ng / mL), Y-27632 (5-10 μM), Insulin (1-5 μg / mL).

[0034] (3) Establish a differentiation protocol for the “iPSC→directed endoderm” stage of endoderm differentiation.

[0035] In order to enable iPSC to achieve endoderm differentiation, the present application uses Activin A and CHIR-99021 culture additives in the culture system at this stage to carry out directed differentiation of iPSC.

[0036] Endoderm construction is completed in 4 days:

[0037] Prepare the culture medium B1 corresponding to different stages according to the contents shown in Table 1

[0038] Table 1: Details of B1 culture medium at different stages

[0039]

[0040] (4) Establish a differentiation scheme for the "endoderm→foregut" stage of foregut germ differentiation.

[0041] In order to enable the endoderm to achieve foregut differentiation, the present application uses SB431542, Noggin, SAG, FGF4, CHIR-99021, ascorbic acid, FGF7, and ATRA culture additives in the culture system at this stage to carry out directed differentiation of iPSCs.

[0042] The foregut germ layer is constructed in six days:

[0043] Prepare medium B2 according to the contents shown in Table 2

[0044] Table 2: Medium B 2 Details

[0045]

[0046]

[0047] (5) Differentiation plan for establishing a co-culture model of heart and lung organoids

[0048] In order to co-culture heart and lung organoids, the present application uses culture additives such as IWP2, ascorbic acid, FGF7, CHIR-99021 in the culture system at this stage to direct the differentiation of the foregut germ layer and embryoid bodies.

[0049] The specific implementation plan is as follows: Collect all floating foregut spheroids into a single centrifuge tube and allow gravity to settle to the bottom of the tube for 15 minutes. Discard as much supernatant culture medium as possible, then add Matrigel and pipette to mix thoroughly (avoiding bubbles). Plate 30 μL / well.

[0050] Next, take a 2-20 μL pipette, hold the scissors at a 90° angle to the pipette tip, cut a small section, and set the pipette volume to 3 μL (this pipette will be used to extract the embryoid bodies). Embryoid bodies will be embedded one by one in the Matrigel droplet. Place in a 37°C incubator to solidify for 30 minutes. After solidification, add 100 μL of medium C and return to the 37°C incubator for continued incubation.

[0051] After five days, the embryoid bodies will begin to differentiate. Remove the culture medium from the wells without aspirating or disrupting the matrix droplets containing aggregates. Turn off the lights and prepare to add 100 μL of culture medium D per well. Return the dish to the incubator.

[0052] After seven days, carefully remove the culture medium and add 200 μL of culture medium E per well by pressing the plunger of the pipette until the first stop.

[0053] Eight days later, medium E was removed and 100 μL of medium F was added to each well.

[0054] The construction of the cardiopulmonary symbiotic organoids is completed in about 25 days. The corresponding culture medium needs to be replenished every 2-3 days. The specific method of changing the medium is: aspirate all the culture medium as much as possible, being careful not to destroy the cardiopulmonary cell clusters, and then add 100μL of the required culture medium.

[0055] Prepare the culture medium corresponding to different stages according to the contents shown in Table 3.

[0056] Table 3: Culture medium details at different stages of heart-lung co-culture

[0057]

[0058]

[0059] The suppliers and product numbers of the relevant reagents used in this example are shown in Table 4.

[0060] Table 4: Reagent information list

[0061]

[0062]

[0063] The inventors also verified the cardiopulmonary symbiotic organoids obtained in the above steps, and the verification content is as follows: Figure 2 The typical clone morphology of iPSCs observed by optical microscopy in the embodiment of the present invention (clear edges, large nuclei and scant cytoplasm) was confirmed by immunofluorescence staining with NANOG antibodies, demonstrating that iPSCs expressed NANOG, indicating that iPSCs were in an undifferentiated state and had multidirectional differentiation potential. Figure 3 In the examples of the present invention, light microscopic observation revealed that the cell morphology transformed from compact colonies to flat epithelial-like structures. Immunofluorescence staining also revealed that the cells were positive for FOXA2 (a marker for foregut endoderm) and SOX17 (a marker for definitive endoderm), indicating that iPSCs were successfully induced into foregut endoderm cells and functional endoderm cells. Figure 4 In the embodiment of the present invention, the cells self-organize to form a tubular or sac-like three-dimensional structure under an optical microscope. Under the microscope, the characteristic polar arrangement and cavity formation of the foregut can be observed, indicating that the induction of the foregut germ layer is complete; Figure 5 In the embodiment of the present invention, normal embryoid bodies (uniform spherical structures) are compared with abnormal samples (such as fragmented or size deviations), and embryoid bodies that meet the requirements of subsequent co-culture are screened according to morphological standards; Figure 6 This is the morphology of organoids at various stages of heart-lung co-culture in the embodiment of the present invention. In the early stage, the foregut germ layer and the cardiac embryoid body are co-cultured, and in the middle and late stages, the lung epithelial branches and the vascular network are intertwined to form a certain complex structure; Figure 7 These are staining identification images of vascular endothelial cells (CD31) and cardiomyocytes (CTNT) in the cardiovascular system of the organoids in the embodiment of the present invention, as well as staining identification images of proximal lung epithelial cells (SOX2), alveolar epithelial cells (SPC), distal lung epithelial cells (SOX9), lung basal cells (KRT5), ciliated cells (AC-TUBULIN), and airway secretory cells (SCGB3A1). The results confirm that the organoids have both cardiopulmonary tissue characteristics and cell diversity.

[0064] The relevant abbreviations used in this embodiment are shown in Table 5:

[0065] Table 5: List of related abbreviations

[0066]

[0067] In summary, this patent has the following beneficial effects:

[0068] 1. Improved accuracy of toxicity prediction: Simultaneous assessment of drug effects on both the cardiopulmonary system (e.g., cardiotoxicity and pulmonary fibrosis risk) avoids the limitations of traditional single-organ models and reduces clinical trial failure rates;

[0069] 2. Personalized disease modeling: Organoids constructed from patient-derived stem cells can simulate cardiopulmonary diseases with specific genetic backgrounds or pathological characteristics (such as congenital cardiopulmonary syndrome and acute lung injury), providing an in vitro validation platform for personalized treatment plans;

[0070] The present invention provides an efficient co-culture method of heart and lung organoids and a culture medium construction method, which can achieve symbiotic differentiation of heart and lung organoids. The heart and lung symbiotic organoids differentiated by this differentiation scheme do not require specific culture medium and culture conditions, thereby achieving an efficient and controllable heart and lung organoid co-culture model, providing an important tool for regenerative medicine and disease model research.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the scope of protection of the present invention.

[0072] References:

[0073] 1. Liu Pan, Kai Jiejing, and Zhao Qingwei. Establishment of a colorectal cancer organoid model and preliminary study of its chemotherapeutic drug sensitivity. Chinese Journal of Pharmaceutical Sciences: p. 1-17.

[0074] 2. Mo Shaobo, et al., Application and research progress of organoids in clinical medicine. Science China: Life Sciences, 2023.53(02):p.221-237.

[0075] 3. Li Yanjiao, et al., Application of patient-derived tumor organoids in the development of anti-tumor drugs. Journal of Dalian Medical University, 2024.46(03):p.236-241.

[0076] 4. Deguchi, S., et al., SARS-CoV-2 research using human pluripotent stemcellsandorganoids. Stem Cells Transl Med, 2021.10(11):p.1491-1499.

[0077] 5. Zhang Yuqing, et al., Research on the construction of cardiac organoid model based on human induced pluripotent stem cells. Chinese Journal of Pharmaceutical Industry, 2024.55(10):p.1381-1389.

[0078] 6. Lee, SG, et al., Generation of human iPSCs derived heartorganoids structurally and functionally similar to heart. Biomaterials, 2022.290: p.121860.

[0079] 7. Suezawa, T., et al., Disease modeling of pulmonary fibrosis using humanpluripotent stem cell-derived alveolar organoids. Stem Cell Reports, 2021.

[0080] 16(12):p.2973-2987.

[0081] 8. Yamamoto, Y., et al., Long-term expansion of alveolar stem cells derived from human iPS cells in organoids. Nat Methods, 2017.14(11): p.1097-1106.

[0082] 9. Shi Jin, Liu Ke, and Ding Junying. Application and prospects of lung organoid models in the study of infectious lung diseases. China Biotechnology Journal, 2023.43(08):p.30-37.

[0083] 10.Peng,LC,et al.,Corrigendum to "Latentprofile analysis ofdyspnea-relatedkinesiophobia in older adults with chronic obstructive pulmonarydisease"

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[0085] 39522284].Heart Lung,2025.70:p.213.

Claims

1. A method and culture medium for preparing cardiopulmonary organoids with cardiopulmonary function cells, characterized in that: The following steps are involved: Step 1: Culture iPSCs with A1 and B1 culture media, respectively, to induce cell differentiation into mesoderm and endoderm; Step 2: The cells induced by A1 in step 1 are centrifuged into spheres and induced to form embryoid bodies in A2 medium. The cells induced by B1 are induced to form foregut germ under B2 culture conditions; Step 3: Transfer the embryoid bodies and foregut protrusions formed in step 2 into Matrigel, replace the C, D, E, and F culture media at different stages, and finally differentiate into cardiopulmonary organoids with cardiomyocytes and various lung epithelial cells.

2. The method according to claim 1, characterized in that In step 2, the conditions for centrifugation into spheroids are as follows: 5,000-8,000 iPSCs are centrifuged in an ultra-low attachment U-shaped plate at 300×g and 4° C. for 3 minutes.

3. The method according to claim 1, wherein The components of the induction medium A1 include: E8 and Y27632 (10-20 μM); the components of the induction medium A2 include: RPMI1640, FGF2 (30-50 ng / mL), ActivinA (100-500 ng / ml), BMP4 (10-20 ng / mL), Y-27632 (5-10 μM), and Insulin (1-5 μg / mL).

4. The method according to claim 1, wherein The components of the induction medium B1 include: RPMI 1640, ActivinA (100-200 ng / mL), CHIR-99021 (2-5 μM), and FBS (2%); the components of the induction medium B2 include: Advanced DMEM / F12, SB431542 (10-50 μM), Noggin (200-500 ng / mL), SAG (1-5 μM), FGF4 (200-500 ng / mL), CHIR99021 (1-5 μM), N-2 (1×), B27+ (50×), L-glutamine (1×), BSA (0.05%), monothinoglycerol (0.4-1 μM), ascorbic acid (50-100 μg / mL), FGF7 (10-50 ng / mL), and ATRA (50 nM).

5. The method according to claim 1, wherein The induction medium C components include: E8, N-2 (1×), B27+ (50×), L-glutamine (1×), penicillin-streptomycin (1×), BSA (2%), monothinoglycerol (0.4-1μM), ascorbic acid (50-100μg / mL), FGF7 (10-50ng / mL), ATRA (50-100nM), CHIR-99021 (3-5μM), Y-27632 (2-5μM), and Noggin (100-300ng / mL).

6. The method according to claim 1, wherein The induction medium D components include: Advanced DMEM / F12, N-2 (1×), B27-(50×), L-glutamine (1×), penicillin-streptomycin (1×), BSA (2%), monothinoglycerol (0.4-1μM), FGF7 (10-50ng / mL), ATRA (50-100nM), CHIR-99021 (5-10μM), BMP4 (10-20ng / mL), SAG (2-10μM), and FGF4 (100-300ng / mL).

7. The method according to claim 1, wherein The induction medium E components include: Advanced DMEM / F12, N-2 (1×), B27-(50×), L-glutamine (1×), BSA (2%), monothinoglycerol (0.4-1μM), ascorbic acid (50-100μg / mL), FGF7 (10-50ng / mL), ATRA (50-100nM), IWP2 (5-10μM), SB431542 (50μM), SAG (2-5μM), and A8301 (200-500nM).

8. The method according to claim 1, wherein The induction medium F components include: Advanced DMEM / F12, N-2 (1×), B27-(50×), L-glutamine (1×), penicillin-streptomycin (1×), BSA (2%), ascorbic acid (50-100 μg / mL), FGF7 (10-50 ng / mL), ATRA (50-100 nM), FGF2 (20-50 ng / mL), and A8301 (200-500 nM).

9. Use of the cardiopulmonary organoids constructed by the method according to any one of claims 1 to 8 in preparing disease models, drug screening platforms or toxicity detection platforms.

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