Mouse airway-like and fibroblast co-culture method

Abstract

The invention discloses a mouse airway-like and fibroblast co-culture method, which comprises the following steps: carrying out in-vitro mouse airway-like culture on lung epithelial stem cells dissociated from mouse lung tissue primary epithelial stem cell spheres (pneumospheroids) in a Transwell upper chamber, after airway-like differentiation of the Transwell upper chamber is completed, replacing the Transwell upper chamber with a lower chamber paved with mouse lung fibroblasts, and carrying out in-vitro mouse airway-like culture. During co-culture, only a culture medium is added into the lower chamber, and the culture medium adopts the same culture medium (gas-liquid interface differential culture medium) as the culture medium in the airway-like culture process, so that a gas-liquid interface co-culture system of the mouse airway-like (upper chamber) and lung fibroblasts (lower chamber) is formed, and the hierarchical structure, morphological characteristics and regeneration function of the airway-like of the upper chamber can be dynamically researched; and lower ventricle fibroblast phenotypes, functions, and related signaling pathway activation levels.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a method for co-culturing mouse airway cells and fibroblasts. Background Technology

[0002] The airway epithelium is a physical barrier covering the surface of the respiratory tract. It is mainly composed of ciliated cells, goblet cells, basal cells, Club cells, and neuroendocrine cells, forming a pseudostratified ciliated columnar epithelium. It can mediate innate immunity by secreting antimicrobial peptides and cytokines, and collaborate with dendritic cells and macrophages to initiate adaptive immunity. Damage to the airway epithelium can induce chronic inflammation, mucinous metaplasia, airway hyperresponsiveness, and tissue fibrosis, forming an important pathological basis for lung-related diseases. Therefore, studying the function and regulatory mechanisms of the airway epithelium is of great significance for the prevention and treatment of related respiratory diseases.

[0003] Airway-like structures (ALIs) are in vitro airway organoids established based on a 2D air-liquid interface (ALI) model. In this culture system, primary bronchial epithelial cells are seeded in the upper chamber of a Transwell chamber, with the upper and lower chambers separated by a permeable membrane with a pore size of 0.4 μm. In the initial stage of cell culture, expansion medium is added to both the upper and lower chambers. After the cells have expanded and covered the bottom surface of the upper chamber, the medium from both chambers is aspirated, and differentiation medium is added only to the lower chamber. This creates a 2D-ALI culture system where the basal surface of the cells is in contact with the medium in the lower chamber, while the apical surface is exposed to air. After ALI-induced differentiation, pseudostratified ciliated columnar epithelium is formed, containing various types of airway epithelial cells. Cell stacking, rhythmic ciliary movement, surface mucus distribution, and tight junction formation are observed. These phenotypes are highly similar to the morphological characteristics, physiological features, and barrier functions of in vivo respiratory epithelium. Currently, the 2D-ALI model is widely used in disease modeling, drug screening, and inhalation toxicology research. 2D-ALI culture of primary cells from donors of certain respiratory diseases (such as bronchial asthma, cystic fibrosis, and chronic obstructive pulmonary disease) can reproduce the pathological features of these diseases in vitro and explore their pathogenic mechanisms. In COVID-19 research, the 2D-ALI system has also been used to simulate SARS-CoV-2 infection in vitro and for rapid drug screening due to its unique advantages. Furthermore, drugs and suspended particles can be directly added to the surface of the apical cells to simulate in vivo airway inhalation, providing an excellent platform for studying the efficacy of related drugs and the toxicity of inhaled substances.

[0004] Airway-like models consist entirely of epithelial cells, thus limiting their application to research on the lung epithelial system. However, the lungs are composed of various cell types, including epithelial cells, fibroblasts, and macrophages. The development and progression of respiratory diseases such as pulmonary fibrosis, bronchial asthma, and chronic obstructive pulmonary disease involve interactions and regulation between epithelial and interstitial cells. Therefore, airway-like models based on a single primary epithelial cell type significantly restrict the study of the complex pathological mechanisms of these related diseases. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a method for co-culturing mouse airways and fibroblasts.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] A method for co-culturing mouse airway cells and fibroblasts, comprising:

[0008] In the upper chamber of a Transwell chamber, airway-like cultures were performed using mouse lung glomeruli and air-liquid interface differentiation medium.

[0009] After the airway-like differentiation is completed, lung fibroblasts are seeded in a multi-well plate. The lower chamber of the Transwell chamber is replaced with the multi-well plate seeded with lung fibroblasts, and gas-liquid interface differentiation medium is added to the new lower chamber for co-culture of airway-like cells and lung fibroblasts.

[0010] The lung spheres are primary epithelial stem cell spheres from mouse lung tissue;

[0011] The gas-liquid interface differentiation culture medium consisted of: PneumaCult supplemented with 0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, 4 μg / mL heparin, and 20 ng / mL EGF. TM -ALI culture medium. In a preferred embodiment, the pore size of the Transwell chamber is 0.4 μm. In the prior art, the upper chamber of the Transwell in a typical co-culture system of tumor organoids and cancer-associated fibroblasts contains cancer-associated fibroblasts, and the lower chamber contains tumor organoids; however, the pore size of the Transwell chamber in this co-culture system is 1.0 μm. In this invention, the upper chamber of the Transwell contains airway-like structures, and the lower chamber contains fibroblasts. The 0.4 μm pore size of the Transwell permeable membrane ensures that lung epithelial stem cells adhere firmly to the upper chamber, facilitating complete differentiation, while also ensuring sufficient nutrient supply to the upper chamber airway-like structures.

[0012] In a preferred embodiment, the lung fibroblasts are third-generation mouse lung fibroblasts.

[0013] In a preferred embodiment, the lung fibroblasts are obtained in the following manner:

[0014] Mouse lung tissue was dissociated using digestive enzymes. The resulting single-cell suspension was filtered and induced to adhere and expand using DMEM / F12 complete culture medium to obtain lung fibroblasts. In this invention, collagenase A digestion solution was used for pre-digestion. Pre-digestion with collagenase A solution loosened the mouse lung tissue, ensuring more complete and thorough subsequent trypsin-EDTA digestion, resulting in a higher yield of lung fibroblasts.

[0015] Furthermore, the method of dissociating mouse lung tissue using digestive enzymes is as follows: first, the mouse lung tissue is pre-digested using collagenase A digestion solution, and then digested and dissociated using trypsin-EDTA digestion solution.

[0016] Furthermore, the mouse lung tissue was pre-digested for 20-40 minutes using a collagenase A digestion solution containing 2 mg / mL collagenase A.

[0017] The method for culturing airway-like structures using mouse pulmonary glomeruli and gas-liquid interface differentiation medium is as follows:

[0018] In a preferred embodiment, mouse pulmonary glomeruli are dissociated using cell digestive enzymes, and the dissociated pulmonary glomeruli are resuspended in lung epithelial stem cell expansion culture medium. The obtained cell suspension is added to the upper chamber of a Transwell chamber, and lung epithelial stem cell expansion culture medium is added to the lower chamber. After the cells have covered the bottom surface of the upper chamber, the remaining culture medium in the upper and lower chambers is aspirated, and gas-liquid interface differentiation culture medium is added to the lower chamber for continued culture until the mouse lung epithelial stem cells differentiate into airway-like structures. The pulmonary glomeruli are primary epithelial stem cell glomeruli from mouse lung tissue.

[0019] As a preferred embodiment, the Transwell chambers are co-cultured in a carbon dioxide cell culture incubator, with the culture medium being changed every two days.

[0020] As a preferred embodiment, the mouse lung glomeruli are prepared as follows:

[0021] a. Take a block of mouse lung tissue and digest it in a preheated collagenase digestion solution;

[0022] b. Add an equal volume of DMEM / F12 medium containing FBS to terminate digestion, mix well by pipetting, and filter through a cell sieve; centrifuge the filtrate and discard the supernatant;

[0023] c. Resuspend the cell pellet in red blood cell lysis buffer, incubate at room temperature, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant;

[0024] d. Repeat step c until the cell pellet turns white;

[0025] e. Resuspend the cell pellet in DNase solution, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant;

[0026] f. Resuspend the cell pellet in DMEM / F12 medium, mix well by pipetting, filter through a cell sieve, and discard the supernatant after centrifugation;

[0027] g. Resuspend the cells in pulmonary glomerular culture medium, adjust the cell density, and then culture in suspension.

[0028] Furthermore, the pulmonary globulin culture medium is a DMEM / F12 medium containing 1×B-27 additive, 1×penicillin / streptomycin solution, 4 μg / mL heparin, 20 ng / mL EGF, 10 ng / mL FGF2, and 10 μM Y-27632; the B-27 additive does not contain vitamin A.

[0029] This invention builds upon previously established methods for culturing primary epithelial stem cells from mouse lung tissue (CN111534477A) and mouse airway-like cells (CN114891725A). Using Transwell cell culture chambers as permeable membranes, a co-culture method for mouse airway-like cells and fibroblasts was successfully established through a 2D-ALI culture model, and its phenotypic and functional characteristics were validated. The establishment of this method provides strong technical support and an application platform for research on the pathological mechanisms of respiratory diseases such as pulmonary fibrosis, bronchial asthma, and chronic obstructive pulmonary disease, as well as related drug screening.

[0030] The present invention has the following beneficial effects:

[0031] (1) The system of the present invention is not co-cultured throughout the entire process. Instead, after the differentiation of the upper chamber airway-like cells is completed, the lower chamber is replaced with a lower chamber containing mouse lung fibroblasts. This ensures that the differentiation process of the upper chamber airway-like cells is not affected, and allows for dynamic observation of the phenotypic and functional changes of the airway-like cells and lung fibroblasts before and after co-culture. In addition, once the present invention is in the co-culture stage, the morphological characteristics, tissue structure, phenotype and function of the upper or lower chamber culture can be observed at any time. The co-culture time is flexible, controllable and unrestricted, which can meet the needs of research at different time stages. This system has a wider range of applications and application scenarios.

[0032] (2) In the 2D-ALI co-culture system of the present invention, there is no culture medium in the upper chamber during the co-culture stage, and only the culture medium is added in the lower chamber. The airway-like epithelium in the upper chamber covers the permeable membrane of the upper chamber of Transwell and forms a tight junction. The culture medium in the lower chamber cannot penetrate into the upper chamber, but the cytokines secreted by the airway-like epithelium in the upper chamber can penetrate into the culture medium in the lower chamber and act on the fibroblasts. The cytokines secreted by the fibroblasts can also act on the airway-like epithelium in the upper chamber through the permeable membrane of Transwell.

[0033] (3) The gas-liquid interface differentiation culture medium in this invention can be used for the culture of airway epithelial cells and lung fibroblasts. Using the same culture medium has good batch stability and reproducibility, and has a wide range of applications. No additional adjustments to the experimental conditions are required. The operation is simple and easy.

[0034] (4) In the preparation of lung fibroblasts in this invention, collagenase A digestion solution is used for pre-digestion. After pre-digestion with collagenase A digestion solution, the mouse lung tissue is loose, which can ensure that the subsequent trypsin-EDTA digestion is more complete and sufficient, and the yield of lung fibroblasts is higher.

[0035] (5) In the early stage of the 2D-ALI co-culture model, the differentiation of airway epithelial cells in the present invention requires the use of a Transwell chamber with a pore size of 0.4 μm. The subsequent co-culture system is an extension of this system and can match the original 2D-ALI differentiation model to the greatest extent. In addition, the stacking growth of airway epithelial cells in the upper chamber, the rhythmic beating of cilia, and the distribution of surface mucus in the 2D-ALI co-culture system can be directly observed by optical microscope during the co-culture stage without special treatment (such as cell digestion, fixation, or clearing), which reduces the difficulty of operation and can be flexibly applied according to the needs of subsequent research.

[0036] (6) The co-culture method of the present invention can not only dynamically study the hierarchical structure, morphological characteristics and regenerative function of the upper chamber airway of Transwell, but also dynamically detect the phenotype, function and activation level of related signaling pathways of lower chamber fibroblasts, providing a new technical platform for a comprehensive and in-depth study of the cellular and molecular mechanisms of epithelial-mesenchymal cell interaction in related lung diseases. Attached Figure Description

[0037] Figure 1 This is a flowchart of the co-culture process of mouse airway and lung fibroblasts.

[0038] Figure 2 The results are the morphology of mouse third-generation lung fibroblasts under a light microscope (100×) and the identification results of Vimentin immunofluorescence staining.

[0039] Figure 3The results are H&E and PAS staining of paraffin sections of upper ventricular airway z-axis after 0 hours and 72 hours of co-culture of mouse airway and lung fibroblasts.

[0040] Figure 4 The results show the morphology (100×) and EdU staining of lower ventricular lung fibroblasts under a light microscope after 0 hours and 72 hours of co-culture with mouse airway and lung fibroblasts. Detailed Implementation

[0041] The main materials used in the embodiments and their sources are as follows:

[0042] Transwell cell culture chamber: Corning Incorporated, USA, catalog number 3460.

[0043] TrypLE cellular digestive enzyme: Thermo Fisher Scientific (China) Co., Ltd., item number 12604021.

[0044] Lung epithelial stem cell expansion culture medium: [added with 0.096 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, and 20 ng / mL EGF PneumaCult] TM [Ex Plus Culture Medium]: Hydrocortisone solution: STEMCELL Ltd., Canada, catalog number 07925; Penicillin and streptomycin solution: ScienCell Ltd., USA, catalog number 0503; EGF: PeproTech Ltd., USA, catalog number 315-09; PneumaCult TM -Ex Plus culture medium: STEMCELL Ltd, Canada, catalog number 05040.

[0045] Gas-liquid interface differentiation medium: [added with 0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, 4 μg / mL heparin, 20 ng / mL EGF PneumaCult] TM -ALI culture medium]: Hydrocortisone solution: STEMCELL Ltd., Canada, catalog number 07925; Penicillin and streptomycin solution: ScienCell Ltd., USA, catalog number 0503; Heparin: APExBIO Ltd., USA, catalog number B3602; EGF: PeproTech Ltd., USA, catalog number 315-09; PneumaCult TM -ALI culture medium: STEMCELL Ltd, Canada, catalog number 05001.

[0046] Collagenase A digestion solution (2 mg / mL collagenase A in DMEM / F12 medium): Collagenase A: Shanghai Sigma-Aldrich Trading Co., Ltd., catalog number C0130; DMEM / F12 medium: Thermo Fisher Scientific (China) Co., Ltd., catalog number 11039021.

[0047] Trypsin-EDTA digestion solution (0.25%): Shanghai Beyotime Biotechnology Co., Ltd., product number C0203.

[0048] DMEM / F12 Complete Medium: [DMEM / F12 medium supplemented with 10% FBS (fetal bovine serum)]: FBS: ScienCell, Inc., USA, catalog number 0010; DMEM / F12 medium: Thermo Fisher Scientific (China) Co., Ltd., catalog number 11039021.

[0049] 40μm cell sieve: Hefei Biosharp Co., Ltd., product number BS-40-XBS.

[0050] 12-well cell culture plate: Corning Incorporated, USA, catalog number 3513.

[0051] T25 cell culture flask: Corning Incorporated, USA, catalog number 430639.

[0052] Sterile PBS (phosphate buffered solution): Beijing Solarbio Science & Technology Co., Ltd., catalog number P1020.

[0053] Example 1

[0054] First, mouse-like airways were cultured using the method shown in CN114891725A (i.e., steps 1 to 20 below).

[0055] (1) Thoroughly disinfect the skin of mice with 75% alcohol, aseptically separate the lung tissue and rinse it in pre-cooled sterile PBS (phosphate buffer solution) to remove connective tissue and the main bronchus in the lungs. At the same time, separate the lung lobes and wash them 2-3 times to remove blood.

[0056] (2) Use sterile forceps to transfer the cleaned lung lobes to a new cell culture dish, remove any residual PBS, and use ophthalmic surgical scissors to cut the lung tissue into pieces of approximately 1 mm. 3 The tissue blocks were transferred to a preheated collagenase digestion solution and digested at 37°C in a shaker (100 rpm) for 45-60 minutes.

[0057] (3) Add an equal amount of DMEM / F12 medium containing 10% FBS to stop digestion, mix well by pipetting, and filter through a 100μm cell sieve; centrifuge the filtrate at 700-900g for 10 minutes at 4℃ and discard the supernatant;

[0058] (4) Add 2-3 mL of red blood cell lysis buffer to resuspend the cell pellet, incubate at room temperature for 1-2 minutes, add 6 mL of DMEM / F12 medium and mix well, centrifuge at 700-900g at 4℃ for 10 minutes, and discard the supernatant;

[0059] (5) Repeat step (4) 1-2 times until the cell pellet turns white;

[0060] (6) Add 4 mL of DNase solution (20 U / mL) to resuspend the cell pellet, shake by hand for 3-5 minutes at room temperature, add 6 mL of LDM / F12 medium and mix well, centrifuge at 700-900g for 10 minutes at 4℃, and discard the supernatant;

[0061] (7) Add 6 mL of DMEM / F12 medium to resuspend the cell pellet, mix thoroughly by pipetting, filter through a 40 μm cell sieve, centrifuge at 700-900 g for 10 minutes at 4℃, and discard the supernatant;

[0062] (8) Resuspend the pulmonary glomeruli in culture medium and adjust the cell density to 2×10⁻⁶. 6 - 3×10 6 / mL;

[0063] (9) Culture in poly-HEMA-coated T25 cell culture flasks, 3-5 mL of pulmonary glomeruli culture medium / flask;

[0064] (10) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), and change the culture medium every three days;

[0065] (11) After culturing for 12-15 days, collect lung glomeruli, centrifuge at 4℃ for 200-300g for 5-10 minutes, and discard the supernatant;

[0066] (12) Add 5 mL of DMEM / F12 medium to resuspend the lung glomeruli, centrifuge at 200-300g for 5-10 minutes at 4℃, and discard the supernatant;

[0067] (13) Repeat step (12) 1-2 times;

[0068] (14) Add 5mL of LTrypLE cell digestive enzymes to dissociate the lung glomeruli and incubate at 37°C for 5-10 minutes;

[0069] (15) Filter through a 40μm cell sieve to remove incompletely digested cell clumps, centrifuge at 200-300g for 5-10 minutes at 4℃, and discard the supernatant;

[0070] (16) Resuspend the lung epithelial stem cell expansion culture medium and adjust the cell density to 2×10⁻⁶. 5 - 3×10 5 / mL;

[0071] (17) Add 0.5 mL of cell suspension to the upper chamber of the Transwell chamber and 1 mL of lung epithelial stem cell expansion culture medium to the lower chamber;

[0072] (18) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), and change the culture medium in the upper and lower chambers every two days;

[0073] (19) After the cells have spread to cover the bottom of the upper chamber, the culture medium in the upper and lower chambers is aspirated, and only 1 mL of gas-liquid interface differentiation medium is added to the lower chamber.

[0074] (20) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), change the culture medium in the lower chamber every two days, and dynamically observe the differentiation of mouse lung epithelial stem cells, airway-like formation, and morphological changes;

[0075] (21) Thoroughly disinfect the skin of mice with 75% alcohol, aseptically separate the lung tissue and rinse it in pre-cooled sterile PBS (phosphate buffer solution) to remove connective tissue and the main bronchus in the lungs. At the same time, separate the lung lobes and wash them 2-3 times to remove blood.

[0076] (22) Use sterile forceps to transfer the cleaned lung lobes to a new cell culture dish, remove any residual PBS, and use ophthalmic surgical scissors to cut the lung tissue into pieces of approximately 1 mm. 3 The tissue blocks were transferred to a preheated collagenase A digestion solution and digested at 37°C on a shaker (100 rpm) for 30 minutes. Then, they were centrifuged at 700-900g for 10 minutes at 4°C, and the supernatant was discarded.

[0077] (23) Add 1 mL of sterile PBS to wash the precipitate, centrifuge at 700-900g for 10 minutes at 4℃, and discard the supernatant;

[0078] (24) Add 1 mL of trypsin-EDTA digestion solution (0.25%), digest at 37°C in a shaker (100 rpm) for 20 minutes, and terminate digestion with an equal volume of DMEM / F12 complete medium;

[0079] (25) Filter through a 40μm cell sieve to remove incompletely digested cell clumps, centrifuge at 900g for 5 minutes at 4℃, and discard the supernatant;

[0080] (26) Resuspend in DMEM / F12 complete medium and adjust cell density to 1×10⁻⁶. 5 - 2×10 5 / mL, seeded in T25 cell culture flasks;

[0081] (27) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), and change the culture medium every three days;

[0082] (28) Based on the cell proliferation and fusion status, use trypsin-EDTA digestion solution (0.25%) to digest and separate the cells, and passage them at a ratio of 1:3;

[0083] (29) Third generation lung fibroblasts were seeded in 12-well plates. After the cells adhered, the culture medium was aspirated and the Transwell chamber containing airway-like structures was inserted into the 12-well plates. Then, air-liquid interface differentiation medium was added to the lower chamber (the 12-well plate seeded with fibroblasts).

[0084] (30) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), and change the culture medium every two days. During this period, the phenotypic and functional changes of airway epithelial cells (upper chamber) and lung fibroblasts (lower chamber) in this co-culture system under different intervention conditions can be dynamically detected according to the research objectives.

[0085] Figure 2 Under a light microscope (100×), mouse lung fibroblasts (left) with large, multi-protruding, spindle-shaped or star-shaped flat cells and regular oval nuclei are visible, and the cell outline is indistinct. They are identified by immunofluorescence staining with the fibroblast marker Vimentin (right).

[0086] Figure 3 After 0 and 72 hours of co-culture of mouse airway and lung fibroblasts, histological staining of the Z-axis section of the upper ventricular airway of Transwell revealed mucus secreted by ciliated cells (H&E staining, left) and goblet cells (PAS staining, right).

[0087] Figure 4 After 0 and 72 hours of co-culture of mouse airway and lung fibroblasts, Transwell lower ventricular lung fibroblasts showed typical light microscopic morphology (left) and cell proliferation (EdU staining, right).

[0088] As can be seen from the attached diagram, the co-culture system does not affect the normal growth of airway-like cells and fibroblasts, and is indeed feasible.

[0089] The co-culture system of mouse airway-like cells and fibroblasts established by the method described in the examples, in which the two types of cells are separated by a permeable membrane with a pore size of 0.4 μm in the Transwell chamber, is a non-contact co-culture model that can better simulate the paracrine effect or synergistic effect between cells in the native environment of lung tissue in vivo. It has a stronger physiological relevance and can also be used to study the phenotype, function and activation level of related signaling pathways of airway-like cells (upper chamber) and lung fibroblasts (lower chamber) separately. It is particularly suitable for the study of respiratory diseases caused by the combined action of epithelial and interstitial cells.

Claims

1. A method for co-culturing mouse airway cells and fibroblasts, characterized in that, include: In the upper chamber of a Transwell chamber, airway-like cultures were performed using mouse pulmonary glomeruli and air-liquid interface differentiation medium. After the airway-like differentiation is completed, lung fibroblasts are seeded in a multi-well plate. The lower chamber of the Transwell chamber is replaced with the multi-well plate seeded with lung fibroblasts, and gas-liquid interface differentiation medium is added to the new lower chamber for co-culture of airway-like cells and lung fibroblasts. The lung spheres are primary epithelial stem cell spheres from mouse lung tissue; The gas-liquid interface differentiation culture medium consisted of: 0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, 4 μg / mL heparin, and 20 ng / mL EGF in PneumaCult. TM -ALI medium.

2. The method according to claim 1, characterized in that, The membrane pore size of the Transwell chamber is 0.4 μm.

3. The method according to claim 1, characterized in that, The lung fibroblasts mentioned are third-generation lung fibroblasts from mice.

4. The method according to claim 1 or 3, characterized in that, The lung fibroblasts were obtained in the following manner: Mouse lung tissue was dissociated using digestive enzymes. The resulting single-cell suspension was filtered and then induced to adhere and expand using DMEM / F12 complete culture medium to obtain lung fibroblasts.

5. The method according to claim 4, characterized in that, The method for dissociating mouse lung tissue using digestive enzymes is as follows: first, the mouse lung tissue is pre-digested using collagenase A digestion solution, and then digested and dissociated using trypsin-EDTA digestion solution.

6. The method according to claim 5, characterized in that, The mouse lung tissue was pre-digested for 20-40 minutes using a collagenase A digestion solution containing 2 mg / mL collagenase A.

7. The method according to claim 1, characterized in that, The method for culturing airway-like structures using mouse pulmonary glomeruli and gas-liquid interface differentiation medium is as follows: Mouse lung glomeruli were dissociated using cell digestive enzymes. The dissociated lung glomeruli were resuspended in lung epithelial stem cell expansion culture medium. The obtained cell suspension was added to the upper chamber of a Transwell chamber, and lung epithelial stem cell expansion culture medium was added to the lower chamber. After the cells covered the bottom surface of the upper chamber, the remaining culture medium in the upper and lower chambers was aspirated, and gas-liquid interface differentiation culture medium was added to the lower chamber for continued culture until the mouse lung epithelial stem cells differentiated into airway-like structures. The lung glomeruli were primary epithelial stem cell glomeruli from mouse lung tissue.

8. The method according to claim 1, characterized in that, The Transwell chambers were co-cultured in a CO2 cell culture incubator, with the culture medium changed every two days.

9. The method according to claim 1, characterized in that, The mouse lung glomeruli were prepared as follows: a. Take a block of mouse lung tissue and digest it in a preheated collagenase digestion solution; b. Add an equal volume of DMEM / F12 medium containing FBS to terminate digestion, mix well by pipetting, and filter through a cell sieve; centrifuge the filtrate and discard the supernatant; c. Resuspend the cell pellet in red blood cell lysis buffer, incubate at room temperature, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant; d. Repeat step c until the cell pellet turns white; e. Resuspend the cell pellet in DNase solution, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant; f. Resuspend the cell pellet in DMEM / F12 medium, mix well by pipetting, filter through a cell sieve, and discard the supernatant after centrifugation; g. Resuspend the cells in pulmonary glomerular culture medium, adjust the cell density, and then culture in suspension.

10. The method according to claim 9, characterized in that, The pulmonary glomeruli culture medium is a DMEM / F12 medium containing 1×B-27 additive, 1×penicillin / streptomycin solution, 4 μg / mL heparin, 20 ng / mL EGF, 10 ng / mL FGF2, and 10 μM Y-27632; the B-27 additive does not contain vitamin A.

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