A method of constructing a lung organoid that spontaneously generates blood vessels and has a branched morphology

Abstract

This invention discloses a method for constructing lung organoids with spontaneous vascularization and branching morphology. By optimizing the culture protocol and progressively inducing directional mesoderm, vascularized foregut endoderm, vascularized lung bud organoids, and vascularized lung organoids, lung organoids with both vascularization and branching morphology are successfully cultured, thus more realistically simulating the structural characteristics of the human lung. This invention has significant advantages: First, it can effectively induce the formation of vascularization and branching structures in lung organoids, overcoming the shortcomings of traditional lung organoids that lack vascularization and branching structures; second, by adjusting the culture method in the early stages, the uniformity of the lung organoids is significantly improved, enhancing the reliability and reproducibility of experimental results. This invention provides a more precise and efficient experimental platform for preclinical studies such as lung disease research, drug screening, and toxicology testing, and has significant application value.

Description

Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to organoid preparation technology, and in particular to a method for constructing lung organoids with branching morphology that are induced to spontaneously generate blood vessels using embryonic stem cells (ESCs). Background Technology

[0004] Organoid technology has become an important research direction in the biomedical field in recent years, with wide applications, particularly in regenerative medicine, drug screening, and disease models. Organoids are three-dimensional tissue models cultured in vitro that possess structures and functions similar to native organs. They can simulate the physiological and pathological environments of human organs, providing a more realistic experimental platform for related research. Compared to traditional animal models, organoids are human-derived, more accurately reflecting the physiological responses and pathological changes of human tissues, thus overcoming species differences inherent in animal experiments. This characteristic of organoids gives them significant advantages in drug screening, toxicity testing, and disease models, enabling better prediction of drug performance in humans and reducing biases in experimental results caused by species differences. In recent years, the U.S. Food and Drug Administration (FDA) and other regulatory agencies have begun recommending the use of human-derived organ models such as organoids and organ-on-a-chip to replace traditional animal experiments.

[0005] Lung organoids are three-dimensional organoid models that mimic the structure and function of lung tissue through in vitro culture. To date, based on the source of the starting material, lung organoids are mainly classified into three different types: (a) adult lung stem / progenitor cells (AdSCs), (b) fetal lung stem cells (FSCs), and (c) pluripotent stem cells (PSCs). Lung organoids derived from adult stem cells have higher maturity but lower differentiation potential and simpler structures. In contrast, lung organoids derived from embryonic stem cells possess stronger pluripotency and self-renewal capacity, can more broadly mimic the developmental process of lung tissue, and have higher regenerative potential and more complex structures. Currently, lung organoids derived from embryonic stem cells have successfully differentiated into lung-specific airway and alveolar cells, but they still suffer from vascularization and a lack of immune cells, which are core issues in organoid research.

[0006] Several research teams have focused on an "assembly-style" approach that uses multiple cell types to differentiate and then fuse, enabling lung organoids to possess blood vessels and immune cells. However, the blood vessels or immune cells generated by this assembly lack organ-specificity or structural features, thus limiting the spatial patterning and functional maturation of organoids. Miao et al. described the first spontaneously vascularized lung organoid generated through the coordinated development of the mesoderm and endoderm, but it lacked the branching structure of the lung (Miao Y, et al. Cell. 2025;188(16):4295-4313.e27.).

[0007] In summary, there is an urgent need to develop a method for constructing lung organoids that can induce embryonic stem cells (ES) or induced pluripotent stem cells (iPSCs) to spontaneously form blood vessels and have a branching morphology, in order to solve the problems of insufficient vascularization, lack of immune cells and lack of branching structure in existing technologies. Summary of the Invention

[0009] This application proposes a method for constructing lung organoids that spontaneously generate blood vessels and have a branching morphology. By optimizing culture conditions, this method has been successfully developed to use embryonic stem cells (ESCs) as a source to cultivate lung organoids that not only spontaneously generate blood vessels but also have a branching morphology. This allows for a more realistic reproduction of the structural features of the lungs. Unlike traditional methods, this technology can better simulate the structural features of the human lungs, providing a more precise and effective experimental platform for preclinical research on lung diseases (pulmonary fibrosis, pulmonary vascular developmental abnormalities), drug screening, toxicology testing, and other fields.

[0010] This application is achieved through the following technical solution:

[0011] A method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology, specifically including the following steps: (1) Embryonic stem cell culture and maintenance methods; (2) Directional induction of meso-endoderm; (3) Induction of vascularized foregut endoderm; (5) Induction of vascularized lung bud organoids; (5) Branching morphogenesis and maturation of vascularized lung organoids.

[0012] The maintenance and culture of embryonic stem cells in step (1) includes two methods: ① culturing ES cells in Matrigel; ② culturing ES cells on mouse embryonic fibroblasts (MEF).

[0013] ① The main steps in culturing embryonic stem cells on Matrigel are as follows: a. Mix Matrigel with low growth factor at a certain ratio, spread the diluted Matrigel on a plate, place it in an incubator and use it the next day; b. Digest ES cells with a confluence of 70%-90%, and seed the digested cell clumps into Matrigel-coated microplates with low growth factors. Change the medium daily until the cells reach a confluence of 70%-90% before passage, cryopreservation, or further organoid induction.

[0014] ② The main steps in culturing ES cells on mouse embryonic fibroblasts (MEF) are as follows: a. Prepare gelatin-coated tissue culture plates in advance, and seed thawed MEF cells into the gelatin-coated tissue culture plates for overnight culture; b. On the second day, seed the thawed ES cells into tissue culture plates lined with MEF, and change the culture medium daily using ES cell-specific medium until the cell confluence reaches 70%-90%. c. Digest the ES cells seeded on MEF and transfer them to a gelatin-coated tissue culture plate. After the MEF adheres to the bottom of the plate, remove the ES cells suspended in the supernatant for subsequent organoid induction.

[0015] The induction scheme for the directional mesoderm in step (2) mainly includes the following steps: ① Use digestive enzymes to digest ES cells into single cells and count them. After counting, seed 2000-5000 cells / well into 96-well cell culture plates and culture them in mTeSR1plus medium containing Y-27632. Centrifuge to make the cells aggregate into embryoids. ② Change the medium daily for days 1-3, and culture the embryoids using directional meso-endodermal induction media I, II, and III respectively.

[0016] Step (3), the induction of vascularized foregut endoderm, mainly includes the following steps: The embryoids formed were replaced with a special culture medium for vascularized foregut endoderm on days 4-7.

[0017] The induction of vascularized lung bud organoids in step (4) mainly includes the following steps: ① Thaw the low growth factor substrate in advance and prepare the Transwell plate; ② Prepare a collagen mixture and a collagen-marigel mixture; ③ Select embryoids of well-grown vascularized foregut endoderm, wrap the embryoids with low growth factor matrix gel and then inoculate them into transwell plates. Determine the number of organoids to be wrapped and the volume of the collagen-marigel mixture according to the size of the transwell plate. ④ Place the transwell plate inoculated with embryoids in a cell culture incubator until the collagen-marigel mixture solidifies; ⑤ After the collagen-marigel mixture solidifies, add it to the vascularized lung bud organoid induction medium I containing BMP4 for culturing; ⑥ Replace the medium with BMP4-free vascularized lung bud organoid induction medium II on day 10 and culture until day 21; ⑦ Vascularized lung buds were observed on day 21, and vascular endothelial cells (CD31) and lung progenitor cells (NKX2.1) could be identified.

[0018] Step (5), the formation and maturation of vascularized lung organoid branching morphology, mainly includes the following steps: ① Continue to use vascularized lung organoid branching culture medium for maintenance after day 21, and change the culture medium daily; add collagen-marigel mixture to the upper chamber of the transwell on day 22 and day 31, and place it in an incubator to solidify it; ② From day 21 to 39, the branching morphology and expansion of vascularized lung organoids and vascularized structures can be observed. On day 39, vascular endothelial cells and various lung cells such as lung progenitor cells (NKX2.1), lung epithelial cells (EPCAM), lung proximal cells (SOX2), lung distal cells (SOX9), lung interstitial cells (VIM), basal cells (P63), Club cells (SCGB1A1), ciliated cells (ACCTUB), goblet cells (MUC5AC), alveolar epithelial cells (SPB, HOPX), and lung fibroblasts (α-SMA, PDGFR-α) can be identified.

[0019] Beneficial effects: The culture protocol provided by this invention further improves and optimizes the culture of lung organoids, with specific advantages and effects including: (1) This culture protocol can effectively induce ES cells to form lung organoids with both vascularization and branching morphology. This makes the formed lung organoids more consistent with the real morphological structure of human lungs, thus solving the problem of the lack of vascularization and branching structure in existing lung organoids and providing a more accurate experimental model for simulating lung function. (2) Achieving homogeneity of lung organoids: The embryoids were cultured in 96-well plates in the early stages. This research protocol effectively improves the homogeneity of lung organoids, ensuring consistency in the developmental process and functional characteristics of each organoid. This improvement in homogeneity is of great significance for drug screening, disease modeling, and other research, and can significantly enhance the reliability and reproducibility of experimental results. (3) Simplify the culture process and improve the ease of operation: This research scheme optimizes the culture scheme and simplifies the operation process. This scheme can not only efficiently culture lung organoids, but also minimize human error in the experiment, thus improving the operability and feasibility of the experiment. Attached Figure Description

[0020] Figure 1 This is a flowchart of the induction protocol for inducing spontaneous blood vessel formation and branching morphology of lung organoids from human embryonic stem cells (ESc) in this application. Figure 2 This is a morphological diagram of the human embryonic stem cells (ESc) cultured on Matrigel used in this application. Figure 3 This is a morphological diagram of the human embryonic stem cells (ESc) cultured on MEF used in this application. Figure 4 A comparison image of well-growing ES cells in the application and differentiated embryonic stem cells (ESc) in poor growth condition; Figure 5 This is an example diagram illustrating the formation process of the vascularized foregut endoderm in this application; Figure 6 This is a bright-field photograph of the vascularized lung bud organoid in this application; Figure 7 This is a photograph showing the branching morphology and maturity of the vascularized lung organoids in this application; Figure 8 This is an immunofluorescence identification image of the vascularized lung organoids in this application. Detailed Implementation

[0022] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0023] like Figure 1 As shown, this invention provides a culture protocol for lung organoids that can induce spontaneous angiogenesis and branching morphology in embryonic stem cells (ES) or induced pluripotent stem cells (iPSCs), specifically including the following steps: (1) Maintenance and culture of embryonic stem cells: ① Culture ES cells on MATLAB: a. 1% (v / v) low growth factor Matrigel-coated tissue culture plates: Thaw the low growth factor Matrigel and mix it with DMEM / F12 medium at 4°C at a v / v ratio of 1:100. After mixing thoroughly, add 1 ml of the diluted Matrigel mixture to each well of a 6-well culture plate. Place the plate in a 37°C incubator for 1 hour, then remove it, seal the plate with sealing film, and store it in a 4°C refrigerator. It can be stored for up to one week. Before use, the diluted Matrigel mixture needs to be aspirated. b. Seed the ES cells to be passaged or thawed into 6-well plates coated with low-growth factor Matrigel. For passage, ES cells were seeded at a 1:10 ratio, and frozen cells were seeded at 2 × 10⁶ cells / well. 5 -2.5×10 5 Seed cells were placed in each well, and ES cells were subsequently cultured in Stemcell's mTeSR1 plus medium. For cryopreserved and thawed cells, a final concentration of 10-50 μM of ROCK inhibitor (Y-27632) was added to the medium on day 1 of thaw. The medium for ES cells needed to be changed daily. After approximately 3-4 days, the cells reached 70%-90% confluence (e.g., ...). Figure 2 (as shown), so as to carry out subsequent passage / cryopreservation or organoid induction experiments.

[0024] ②Culturing ES cells on MEF: a. Preparation of 0.1% (wt / vol) gelatin solution: Prepare a 0.1% (wt / vol) gelatin solution in 1×PBS by dissolving 2 g of bovine gelatin powder in 1 L of tissue culture-grade water and boiling for 10 minutes. Add 200 mL of cooled 10×PBS, followed by an additional 800 mL of cooled tissue culture-grade water. Filter the solution through a 0.22 μm pore filter into a 500 mL or 1 L sterile bottle. Store at 4°C and use within 1 month. b. Thaw mouse embryonic fibroblasts that had been arrested after gamma-ray irradiation in a 37°C water bath, and then fill each well with 1.5 × 10⁻⁶ cells. 5 -2.0×10 5 Cells were seeded at a density of 1,000 cells into gelatin-coated 6-well culture plates and then incubated overnight at 37 degrees Celsius with 5% CO2 to allow cell attachment. c. Seed the ES cells that need to be passaged or thawed into MEF-coated cell culture plates at the same seeding density as described above. Culture the ES cells in Stemcell's mTeSR1 plus medium, changing the medium daily. They should reach 70%-90% confluence in approximately 3-4 days (e.g., ...). Figure 3 (As shown) for subsequent cell passage / cryopreservation or organoid induction.

[0025] (2) Directional meso-endoderm induction protocol: a. When ES cells reach 70-90% confluence, normal cell morphology, high nucleoplasm-to-cytoplasm ratio, and tight cell arrangement indicate that the ES cells are in an undifferentiated primitive state. If the ES cell clones have spiky edges or are clumped together in the middle, it suggests that the cells may have already differentiated and cannot be used for subsequent organoid induction (specifically, as shown in...). Figure 4 (as shown) b. Digest ES cells into single cells: Aspirate the culture medium from the ES cells in the wells and use a calcium-free medium. 2+ Mg 2+ Wash cells with DPBS, then add 1 ml of pre-warmed Accutase digestive enzyme to each well and incubate at 37°C for 10-20 minutes. During digestion, tap the cell culture plate to observe the cell digestion status. Stop digestion when 80% of the cells are suspended. Add 1 ml of the same volume of DMEM / F12 to terminate the digestion. c. Collect the digested cell suspension and centrifuge at 400g for 3min. After discarding the supernatant, resuspend the cells in mTeSR1 medium containing Y27632 at a final concentration of 10-50µm. Count the cells and then seed 2000-5000 cells per well in a U-bottom, low-adsorption 96-well plate. Add 100-200µl of mTeSR1 medium containing Y27632 at a final concentration of 10-50µm to each well. d. Centrifuge the 96-well plate inoculated with ES cells at 400g for 3min to allow the ES cells to aggregate at the bottom of the U-shaped 96 wells, so that they can better aggregate to form embryoids; then place the 96-well plate in a cell culture incubator at 37 degrees and 5% CO2 for culture.

[0026] e. On day 1, carefully aspirate the culture medium from the wells and add 100-200 μl of directional meso-endodermal I medium to each well: 50 ml medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, of which 100-500 ng / ml Activin A and 12-50 μM CHIR99021 need to be added.

[0027] d. On day 2, carefully aspirate the culture medium from the wells and add 100-200 μl of the following directional meso-endodermal II medium to each well: 50 ml medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which should be supplemented with 100-500 ng / ml Acticin A and 0.2%-2% FBS (Fetal Bovine Serum, FBS).

[0028] e. On day 3, carefully aspirate the culture medium from the wells and add 100-200 μl of the following directional meso-endoderm III medium to each well: 50 ml medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which should be supplemented with 100-500 ng / ml Acticin A and 2%-5% FBS.

[0029] (3) Induction protocol for vascularized foregut endoderm: a. On day 4, the vascularized foregut endoderm induction protocol is performed. All culture medium in the wells needs to be aspirated. Subsequently, 100-200 μl of the following vascularized foregut endoderm induction medium is added to each well: 50 ml medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which needs to be supplemented with 50-500 ng / ml Noggin and 2%-5% FBS.

[0030] b. Use the following culture medium from days 5 to 7: 50ml medium containing 25ml DMEM / F12 medium, 25ml Neurobasal Medium, 1ml B27 supplement, 0.5ml N2 supplement, 0.25ml GlutaMAX, 35-50μl β-Mercaptoethanol, 0.5ml Antibiotic-Antimycotic, with 50-500ng / ml Noggin and 2%-5% FBS added. Note that a half-medium change is required from days 5 to 7. If there is still 200ul of culture medium in the original well, aspirate 100ul of the medium from the well on day 2, then add 100ul of freshly prepared culture medium. The initial culture flow chart for vascularized lung organoids is shown below. Figure 5 As shown.

[0031] (4) Induction protocol for vascularized lung bud organoids: a. Thaw the low-growth-factor Matrigel in advance and prepare 6-well, 12-well, or 24-well culture plates containing Transwell chambers.

[0032] b. Prepare the Collagen solution: Slowly add one part of 4°C 10X PBS or 10X culture medium to eight parts of 4°C collagen solution, and stir gently; adjust the pH of the mixture to 7.0–7.5 using sterile 0.1 M NaOH. Carefully monitor pH adjustments (using a pH meter, phenol red, or pH test strips); adjust the final volume to a total of 10 parts using sterile water; subsequently, to prevent gelation, maintain the temperature of the mixture between 2–10°C.

[0033] c. Prepare the Collagen-Matrigel mixture: Mix the collagen solution with low growth factor matrigel at a 1:1 ratio. Note that the entire process should be carried out on ice. The pipette tip that comes into contact with the collagen or matrigel should be pre-cooled in the refrigerator to prevent the collagen or matrigel gel from solidifying prematurely due to temperature rise. The resulting Collagen-Matrigel mixture should be kept in a 4-degree refrigerator or on ice for later use.

[0034] d. Embedding embryoids from the vascularized foregut endoderm into Collagen-Matrigel mixture: Remove the 96-well plate containing the embryoids from the incubator. Use a Pasteur pipette to aspirate the embryoids into a 1.5 ml centrifuge tube. Let stand for 1 minute to allow the embryoids to settle, then carefully aspirate the supernatant. Add 100 μL of Collagen-Matrigel mixture to the centrifuge tube and mix with the embryoids. The centrifuge tube containing the Collagen-Matrigel mixture should be kept on ice throughout the process. Once the Collagen-Matrigel mixture and embryoids are thoroughly mixed, transfer the mixture to a 24-well Transwell plate, positioning the embryoids in the center of each well (12 wells). Then, incubate at 37°C for 30-60 minutes to allow the Collagen-Matrigel mixture to solidify. If using a 6-well plate, a total of 400 μL of Collagen-Matrigel mixture is required; if using a 12-well plate, 200 μL of Collagen-Matrigel mixture is required.

[0035] e. After the Collagen-Matrigel mixture has solidified, add 100 μL of the following vascularized lung bud organoid induction medium I to each well of the lower chamber of a 24-well transwell plate: 50 ml medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μL β-Mercaptoethanol, and 0.5 ml Antibiotic-Antimycotic, which should include 5-50 ng / ml FGF7, 5-50 ng / ml FGF10, 1-10 μM CHIR99021, 50-500 nM Retinoic Acid, 50-500 ng / ml VEGF, 50-500 ng / ml Ang-1, and 5-50 ng / ml BMP4. Add the above medium only to the lower chamber; do not add it to the upper chamber for gas-liquid interface culture.

[0036] f. On days 9-10, perform a half-medium change, i.e., aspirate 500 μL of culture medium and add 500 μL of fresh vascularized lung bud organoid induction medium II as follows: 50 ml of medium contains 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μL β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which should be supplemented with 5-50 ng / ml FGF7, 5-50 ng / ml FGF10, 1-10 μM CHIR99021, 50-500 nM Retinoic Acid, 50-500 ng / ml VEGF, 50-500 ng / ml Ang-1, and 5-50 ng / ml BMP4.

[0037] g. From day 11 to 21, the medium needs to be changed daily. This involves aspirating all the culture medium and adding less than 1 ml of freshly prepared vascularized lung bud organoid induction medium II: 50 ml medium contains 25 ml DMEM / F12 medium, 25 ml Neurobasal medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which should be supplemented with 5-50 ng / ml FGF7, 5-50 ng / ml FGF10, 1-10 μM CHIR99021, 50-500 nM Retinoic Acid, 50-500 ng / ml VEGF, and 50-500 ng / ml Ang-1.

[0038] h. On day 21, the formed vascularized lung bud organoids can be embedded and identified, such as... Figure 6 As shown, the following markers can be identified: vascular endothelial cells (CD31), lung progenitor cells (NKX2.1), and lung mesenchymal cells (FOXF1).

[0039] (5) Branching morphogenesis and maturation of vascularized lung organoids: a. On days 22 / 30, Collagen-Matrigel mixture needs to be added to the culture: The preparation method of Collagen-Matrigel mixture is as described above. First, remove the old culture medium from the wells, then add 100 μL of Collagen-Matrigel mixture to the upper transwell chamber and incubate at 37°C for 30-60 minutes until solidification. Then, add 1 ml of the following vascularized lung branch organoid culture medium to the lower chamber: 50 ml medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μL β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which should contain 2-20 ng / ml FGF7, 2-20 ng / ml FGF10, and 1-10 μM CHIR99021. 50-500nM RetinoicAcid, 20-200ng / ml VEGF, 20-200ng / ml Ang-1.

[0040] b. From day 22 to 39, the medium needs to be changed daily. This involves aspirating all the original culture medium and adding less than 1 ml of freshly prepared vascularized lung organoid culture medium to each well: 50 ml of medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which should include 2-20 ng / ml FGF7, 2-20 ng / ml FGF10, 1-10 μM CHIR99021, 50-500 nM Retinoic Acid, 20-200 ng / ml VEGF, and 20-200 ng / ml Ang-1.

[0041] c. By day 39, the vascularized lung organoids had essentially completed the development of branching morphology, as shown in the following example. Figure 7As shown, samples can be collected at this time point, and the following markers can be identified, such as lung progenitor cells (NKX2.1), lung epithelial cells (EPCAM), proximal lung cells (SOX2), distal lung cells (SOX9), lung interstitial cells (VIM), basal cells (P63), Club cells (SCGB1A1), ciliated cells (ACCTUB), goblet cells (MUC5AC), alveolar epithelial cells (SPB, HOPX), and lung fibroblasts (α-SMA, PDGFR-α), etc. Figure 8 As shown.

[0042] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology, characterized in that, Includes the following steps: a. Embryonic stem cell culture and maintenance methods: ES cells were cultured on matrigel and on mouse embryonic fibroblasts (MEF); b. Directional induction of mesoderm; c. Induction of vascularized foregut endoderm: On days 4-7, the embryoids formed were partially replaced with a special culture medium for vascularized foregut endoderm. d. Induction of vascularized lung bud organoids; e. Branching morphogenesis and maturation of vascularized lung organoids.

2. The method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, Step b includes the following operational steps: ① Use acutase digestive enzyme to digest ES cells into single cells and count them. After counting, seed 2000-5000 cells / well into 96-well cell culture plates and culture them in mTeSR1plus medium containing Y-27632. Centrifuge to make the cells aggregate into embryoids. ② Change the medium daily for days 1-3, and culture the embryoids using directional meso-endodermal induction media I, II, and III respectively.

3. The method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, Step d includes the following operational steps: ① Thaw the low-growth-factor matrigel in advance and prepare a collagen-marigel mixture; ② Select embryoids of well-grown vascularized foregut endoderm, wrap the embryoids with a collagen-marigel mixture, and then inoculate them into Transwell plates; ③ Place the transwell plate inoculated with embryoids in a cell culture incubator for 30-60 minutes to allow the collagen-marigel mixture to solidify; ④ After the collagen-marigel mixture solidifies, add it to the vascularized lung bud organoid induction medium I containing BMP4 for culturing; ⑤ On day 10, the culture medium was changed to BMP4-free vascularized lung induction medium II and cultured until day 21; ⑥ Vascularized lung bud organoids were observed on day 21, and CD31 vascular endothelial cells and NKX2.1 lung progenitor cells could be identified.

4. The method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, Step e includes the following operational steps: ① Continue to use vascularized lung branch culture medium for maintenance from day 22 to 39, and change the culture medium daily; ② The collagen-marigel mixture needs to be added back into the upper chamber of the transwell culture on days 22 and 31 to allow it to solidify; ③ On day 39, markers such as lung cells, vascular endothelial cells, and lung fibroblasts can be detected.

5. The method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, In step a, ES cells are cultured in Matrigel using a 1% (v / v) low-growth-factor Matrigel-coated tissue culture plate, i.e., thawed low-growth-factor Matrigel, with Matrigel mixed with 4°C DMEM / F12 medium at a 1:100 (v / v) ratio. ES cells are then cultured on MEF plates, thawed in a 37°C water bath after growth arrest due to gamma ray irradiation, and cultured at 1.5 × 10⁶ cells per well. 5 -2.0×10 5 Cells were seeded at a density of 1,000 cells into gelatin-coated 6-well culture plates and then incubated overnight at 37°C with 5% CO2 to allow cell attachment.

6. The method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, Meet at least one of the following conditions: In step b, 1 ml of preheated Accutase digestive enzyme is used and the cells are placed in a 37-degree incubator for 10-20 minutes to digest the ES cells into single cells. Alternatively, after centrifuging the digested cell suspension collected in step b, discard the supernatant, add mTeSR1 medium containing Y27632 at a final concentration of 10-50 μm for resuspending, count the cells, and then seed 2000-5000 cells per well in a U-bottom low-adsorption 96-well plate. Subsequently, add 100-200 μL of mTeSR1 medium containing Y27632 at a final concentration of 10-50 μm to each well. Alternatively, the 96-well plate in which ES cells were seeded in step b can be centrifuged at 400g for 3 minutes to allow the ES cells to aggregate at the bottom of the U-shaped 96-well plate. Alternatively, the directional meso-endodermal I culture medium in step b: 50ml of culture medium containing 25ml DMEM / F12medium, 25ml Neurobasal Medium, 1ml B27 supplement, 0.5ml N2 supplement, 0.25ml GlutaMAX, 35-50μl β-Mercaptoethanol, 0.5ml Antibiotic-Antimycotic, of which 100-500ng / ml Activin A and 12-50uM CHIR99021 need to be added; Alternatively, the directional meso-endodermal II culture medium in step b: 50 ml of medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, of which 100-500 ng / ml Actin A and 0.2%-2% FBS (Fetal Bovine Serum, FBS) need to be added. Alternatively, the directional meso-endodermal III medium in step b: 50ml medium containing 25ml DMEM / F12 medium, 25ml Neurobasal Medium, 1ml B27 supplement, 0.5ml N2 supplement, 0.25ml GlutaMAX, 35-50μl β-Mercaptoethanol, 0.5ml Antibiotic-Antimycotic, of which 100-500ng / ml Actin A and 2%-5% FBS need to be added.

7. The method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, The culture medium for inducing vascularized foregut endoderm in step c is as follows: 50 ml of culture medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, of which 50-500 ng / ml Noggin and 2%-5% FBS need to be added.

8. The method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, Meet at least one of the following conditions: In step d, prepare the collagen solution: Slowly add 1 part of 4°C 10X PBS or 10X culture medium to 8 parts of 4°C collagen solution and stir gently; adjust the pH of the mixture to 7.0–7.5 using sterile 0.1 M NaOH. Carefully monitor pH adjustments; adjust the final volume to a total of 10 parts using sterile water; subsequently, maintain the temperature of the mixture between 2–10°C to prevent gelation; then, mix the low-growth factor matrigel with the collagen mixture at a 1:1 ratio on ice until homogeneous. Or, in step d, the vascularized lung bud organoid induction medium I: 50ml medium containing 25ml DMEM / F12 medium, 25ml Neurobasal Medium, 1ml B27 supplement, 0.5ml N2 supplement, 0.25ml GlutaMAX, 35-50μl β-Mercaptoethanol, 0.5ml Antibiotic-Antimycotic, which requires the addition of 5-50ng / ml FGF7, 5-50ng / ml FGF10, 1-10uM CHIR99021, 50-500nM Retinoic Acid, 50-500ng / ml VEGF, 50-500ng / ml Ang-1, and 5-50ng / ml BMP4; Alternatively, the vascularized lung bud organoid induction medium II in step d: 50 ml of medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which requires the addition of 5-50 ng / ml FGF7, 5-50 ng / ml FGF10, 1-10 μM CHIR99021, 50-500 nM Retinoic Acid, 50-500 ng / ml VEGF, and 50-500 ng / ml Ang-1.

9. A method for constructing a lung organoid with spontaneously generated blood vessels and a branching morphology according to claim 1, characterized in that, Meet at least one of the following conditions: In step e, on days 22 / 30, Collagen-Matrigel mixture needs to be added to the culture. The preparation method of Collagen-Matrigel mixture is as described above. First, the old culture medium in the well needs to be aspirated, and then 100 μL of Collagen-Matrigel mixture is added to the upper chamber of the transwell and placed in an incubator at 37 degrees Celsius for 30-60 minutes to allow it to solidify. Alternatively, the vascularized lung organoid branch culture medium in step e is: 50 ml of culture medium containing 25 ml DMEM / F12 medium, 25 ml Neurobasal Medium, 1 ml B27 supplement, 0.5 ml N2 supplement, 0.25 ml GlutaMAX, 35-50 μl β-Mercaptoethanol, 0.5 ml Antibiotic-Antimycotic, which requires the addition of 2-20 ng / ml FGF7, 2-20 ng / ml FGF10, 1-10 μM CHIR99021, 50-500 nM Retinoic Acid, 20-200 ng / ml VEGF, and 20-200 ng / ml Ang-1.

10. The application of lung organoids based on spontaneous blood vessel formation and branching morphology from induced embryonic stem cells (ES) or induced pluripotent stem cells (iPSC) as described in any one of claims 1-9 in disease simulation, drug screening, and toxicology testing.

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