Lung ex vivo model, construction method therefor, and use thereof

Abstract

The present invention relates to a lung ex vivo model, a construction method therefor, and a use thereof. The lung ex vivo model provided by the present invention uses lung organoids instead of traditional cell lines to construct the lung ex vivo model. The construction method provided by the present invention involves sequentially seeding lung organoids, endothelial cells, macrophages, and, optionally, immune cells. A normal lung ex vivo model is obtained by first culturing in a liquid-liquid mode, followed by culturing in an air-liquid mode. On this basis, a lung fibrosis model can optionally be obtained by treating the lung ex vivo model by using a lung fibrosis inducer.

Description

Lung in vitro models, their construction methods and applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an in vitro lung model and its construction method, the use of the in vitro lung model in drug detection, drug screening and / or detection of the effects of pathogen infection on lung tissue, a culture medium combination, and a drug detection method, a drug screening method and / or a method for detecting the effects of pathogen infection on lung tissue. Background Technology

[0002] Various lung diseases are known to exist. To study the mechanisms of their occurrence and development, and to screen for corresponding drugs, it is necessary to construct lung models.

[0003] Lung models mainly include animal models and in vitro models. Animal models suffer from drawbacks such as difficulty in obtaining samples, low success rates, and limited passage times, while in vitro models can overcome these limitations more effectively. However, current research on in vitro lung models primarily focuses on 2D in vitro culture. When cultured in petri dishes or slides (2D), cell structure, adhesion, mechanotransduction, and soluble cytokine signal transduction cannot be well visualized, and the interactions between cells and the matrix in vivo cannot be simulated.

[0004] Pulmonary fibrosis is a type of end-stage interstitial lung disease. Many factors are known to contribute to pulmonary fibrosis, including toxicity, autoimmune disorders, drug-induced fibrosis, infectious diseases, and traumatic injury. Pulmonary fibrosis is characterized by fibroblast proliferation, extracellular matrix accumulation, inflammatory damage, and destruction of tissue structure. Studies show that the median survival of patients with pulmonary fibrosis is only 2–5 years, with a 5-year survival rate of only 20%.

[0005] Previous research on pulmonary fibrosis has mainly focused on 2D in vitro culture of fibroblasts and animal models. However, these approaches each have their drawbacks. When fibroblasts are cultured in petri dishes or on slides (2D), they cannot adequately demonstrate cell structure, adhesion, mechanotransduction, and signal transduction of soluble cytokines, nor can they simulate cell-cell / matrix interactions in vivo. As for animal models, due to species differences and the fact that artificially induced fibrosis can only simulate a certain stage of the disease progression, failing to represent the entire development process, most drugs that have shown promise in alleviating fibrosis in animal models have failed in phase II / III clinical trials.

[0006] Therefore, it is extremely urgent to find new lung models (including in vitro normal lung models and pulmonary fibrosis models) that can better simulate the lung microenvironment in vivo for research on lung diseases (including pulmonary fibrosis) and drug screening. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing an in vitro lung model, which can better simulate the function and structural characteristics of the alveolar-capillary barrier and has an immune microenvironment, thereby enabling its use in research on lung diseases, drug screening, and other applications.

[0008] In this invention, unless otherwise stated, the term "in vitro lung model" includes both normal lung models and pulmonary fibrosis models.

[0009] Specifically, in a first aspect, the present invention provides a method for constructing an in vitro lung model, comprising:

[0010] Provides lung organoids and cell culture containers;

[0011] The lung organoids were seeded into the upper layer of the cell culture container, and lung epithelial cell culture medium was added to the upper and lower layers of the cell culture container to culture the lung organoids in a liquid-liquid mode.

[0012] The lung epithelial cell culture medium in the upper and lower layers is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to carry out the first stage of gas-liquid mode culture of the lung organoids.

[0013] Endothelial cells were seeded in the lower layer of the cell culture container, and macrophages were seeded in the upper layer of the cell culture container and cultured in a second stage of gas-liquid mode to obtain an in vitro lung model.

[0014] The lung model obtained through the above steps is a normal lung model. This invention also provides a method for constructing a pulmonary fibrosis model based on this normal lung model. Therefore, in some embodiments, the construction method further includes treating the lung model (i.e., the normal lung model) obtained through the above steps with a pulmonary fibrosis inducing agent to obtain a pulmonary fibrosis model.

[0015] In this invention, the cell culture container is a culture container that can be divided into at least upper and lower (sometimes also referred to as upper and lower chambers) culture units, thereby enabling cell migration or allowing different cells to grow in different layers or even attach to a membrane (e.g., a porous membrane) located between the upper and lower layers as needed. Correspondingly, the resulting in vitro lung model can also be divided into an upper layer (i.e., the upper layer of the model) and a lower layer (i.e., the lower layer of the model). In some embodiments, the cell culture container is a Transwell plate or a cell culture chip, such as a membrane chip or a barrier chip. When a cell culture chip is used, the resulting in vitro lung model is also called a lung-on-a-chip (LOC) model. In some specific embodiments, the membrane chip is, for example, those disclosed in application numbers 202321349258.3, 202330709231.X, or 202222062961.8, and the barrier chip is, for example, those disclosed in application numbers 202211680988.1, 202222130269.4, or 202222130382.2, and these application documents are incorporated herein by reference in their entirety.

[0016] In this invention, the lung organoids can be primary lung organoids or lung organoids differentiated from induced pluripotent stem cells (IPS). The primary extraction can be performed from the lung tissue of mammals, such as humans, domestic animals, farm animals, and zoo, sports, or pet animals, including dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0017] In this invention, "liquid-liquid culture" refers to a culture mode in which liquid (e.g., culture medium) is added to both the upper and lower layers of the cell culture container. "Gas-liquid culture" generally refers to a culture mode in which liquid (e.g., culture medium) is added only to the lower layer of the cell culture container, while the upper layer remains dry.

[0018] In the construction method of this invention, lung organoids are used to replace traditional cell lines to construct an in vitro lung model. The lung organoids are cultured in a liquid-liquid mode first, and then in a gas-liquid mode (i.e., the first stage gas-liquid mode culture). Then, endothelial cells and macrophages are seeded in sequence, and macrophages are cultured in a second stage gas-liquid mode. This helps to make the in vitro lung model more mature and improve its simulation.

[0019] In some embodiments, the lung organoids are primary lung organoids extracted. Extraction can be performed using methods known in the art, and preferably using the following steps:

[0020] (1) Washing: Normal lung tissue is washed with buffer solution to remove surface mucosa; preferably, penicillin and streptomycin are added to the buffer solution.

[0021] (2) Disintegration: Chop the lung tissue into a paste-like consistency, resuspend it in buffer solution, and discard the supernatant after standing.

[0022] (3) Digestion: Add lung primary tissue digestion solution, mix by pipetting, and then use a metal bath shaker at 37°C (other similar equipment can be used) to shake and digest for 30-60 minutes until small cell clusters are formed. Finally, add buffer solution to terminate the digestion. The lung primary tissue digestion solution can be a commercially available lung primary tissue digestion solution. Preferably, the lung primary tissue digestion solution contains collagenase and culture medium. The collagenase includes one or more of collagenase I, collagenase II, and collagenase IV. The final concentration of any one of collagenase I, collagenase II, and collagenase IV in the lung primary tissue digestion solution is 1-2 mg / mL. The culture medium is DMEM or Advanced DMEM / F12.

[0023] (4) Resuspension: The digested cell suspension was filtered through a 100 μm cell sieve, and the cells were resuspended in lung organoid culture medium and counted. The lung organoid culture medium can be a commercial lung organoid culture medium, such as respiratory organoid amplification medium (catalog number A01-001) produced by Bogeng Company, or lung organoid culture kit (catalog number KLU0201) produced by Ivyde Company.

[0024] (5) Plating: Centrifuge, resuspend lung organoids in extracellular matrix (Matrigel, BME, etc.), and plate them. Add lung organoid culture medium and incubate at 37°C.

[0025] In some embodiments, the cell culture vessel may be pretreated before inoculating various types of cells to facilitate cell adhesion. Pretreatment methods may be those known in the art, such as pretreatment with a solution comprising an extracellular matrix, wherein the extracellular matrix may be Matrigel, collagen, fibronectin, etc., and is not limited thereto.

[0026] In some implementations, prior to inoculation, the lung organoids are digested using organoid passage digestion solutions (trypsin, Tryple, or Accutase cell digestion solutions, etc.) to form single cells. After digestion is terminated by adding lung organoid culture medium, inoculation is then performed.

[0027] In some embodiments, in the step of seeding the lung organoids onto the upper layer of the cell culture vessel, the lung organoids are seeded at a rate of 1 × 10⁻⁶ m² / g², calculated by the seeding area (e.g., the area of ​​the membrane in a cell culture chip). 5 ~10×105 cells / cm 2 .

[0028] In some embodiments, during the step of adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container, the lung epithelial cell culture medium may be a commercially available lung epithelial cell culture medium. In this invention, the lung epithelial cell culture medium includes lung organoid culture medium. In some specific embodiments, the lung epithelial cell culture medium may be, for example, respiratory organoid amplification medium (catalog number A01-001) manufactured by Bogeng Company, lung organoid culture kit (catalog number KLU0201) manufactured by Ivytec Company, or epithelial cell culture medium (PneumaCult) manufactured by Stemcell Company. TM -Ex Plus Medium).

[0029] In some embodiments, in the step of adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container, 200-300 μL of lung epithelial cell culture medium is added to the upper layer, and 800 μL-1 mL of lung epithelial cell culture medium is added to the lower layer.

[0030] In some embodiments, the liquid-liquid culture time for the lung organoids is 3 to 7 days, for example, 3, 4, 5, 6, 7 days or any time within this range. In some specific embodiments, the liquid-liquid culture temperature is 37°C. In some specific embodiments, the medium is changed every 3 days during liquid-liquid culture.

[0031] In some implementations, when the lung organoids have grown to fill the upper layer, the culture medium for the lung epithelial cells in the upper and lower layers is discarded.

[0032] In some embodiments, in the step of adding fresh lung epithelial cell culture medium to the lower layer, 600-700 μL of lung epithelial cell culture medium is added to the lower layer.

[0033] In some embodiments, the first stage of gas-liquid culture of the lung organoids is carried out for 2 to 21 days, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or any time within this range. In some specific embodiments, the temperature for the first stage of gas-liquid culture is 37°C.

[0034] In some embodiments, endothelial cells are seeded in a lower step of the cell culture vessel, with the seeding area calculated as 0.5 × 10⁻⁶ m² / m². 5 ~5×105 cells / cm 2 .

[0035] In some implementations, the endothelial cells are derived from umbilical vein endothelial cells, primary lung endothelial cells, or lung endothelial cell lines.

[0036] In some embodiments, during the step of seeding endothelial cells into the lower layer of the cell culture vessel, after the endothelial cells have adhered to the vessel, a first mixed culture medium is added to the lower layer. The first mixed culture medium is a mixture of lung epithelial cell culture medium and endothelial cell culture medium at a ratio of (0.5–3.5):1; for example, the lung epithelial cell culture medium and endothelial cell culture medium are mixed at ratios of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, or 3.5:1. The endothelial cell culture medium can be a commercially available medium, such as endothelial cell culture medium (catalog number #1001) manufactured by Sciencell. Preferably, the endothelial cells are digested, resuspended in the first mixed culture medium, and their cell density is adjusted before seeding. In some specific embodiments, 600–700 μL of the first mixed culture medium is added to the lower layer.

[0037] In some embodiments, in the step of seeding macrophages onto the upper layer of the cell culture vessel and performing a second-stage gas-liquid culture, the macrophage seeding area is calculated as 0.1 × 10⁻⁶ m² / g². 5 ~5×10 5 cells / cm 2 In some specific implementations, the macrophages are resuspended and their density adjusted using macrophage culture medium. The macrophage culture medium can be selected according to the specific source of the macrophages, such as macrophage culture medium (catalog number #1921) manufactured by ScienCell.

[0038] In some implementations, the macrophages are derived from macrophages expanded from PBMCs, primary alveolar macrophages, passaged alveolar macrophages, or macrophages induced by the THP-1 cell line.

[0039] In some embodiments, in the step of seeding macrophages into the upper layer of the cell culture vessel and performing a second-stage gas-liquid culture, the macrophages are resuspended in macrophage culture medium, seeded into the upper layer of the cell culture vessel, and after adhering to the vessel wall, the culture media of the upper and lower layers are discarded and the upper layer is kept dry. Fresh first mixed culture medium is added to the lower layer for the second-stage gas-liquid culture. The first mixed culture medium is a mixture of lung epithelial cell culture medium and endothelial cell culture medium at a ratio of (0.5–3.5):1. In some specific embodiments, 600–700 μL of fresh first mixed culture medium is added to the lower layer.

[0040] In some implementations, the second-stage gas-liquid culture of the lung organoids is carried out for 1 to 5 days, such as 1 day, 2 days, 3 days, 4 days, 5 days, or any time within that range.

[0041] In some implementations, after the macrophages are seeded in the upper layer of the cell culture container and cultured in a second-stage gas-liquid mode, the method further includes the step of seeding immune cells in the lower layer of the cell culture container and culturing them, thereby enabling the constructed in vitro lung model to have immune function.

[0042] In some implementations, the immune cells include PBMCs, T cells, B cells, and NK cells.

[0043] In some implementations, the inoculation area (e.g., the area of ​​the membrane in a cell culture chip) is calculated as 0.5 × 10⁻⁶. 5 ~1×10 6 cells / cm 2 .

[0044] In some embodiments, in the step of seeding immune cells into the lower layer of the cell culture container and culturing them, the immune cells are resuspended in a second mixed culture medium to obtain a suspension. The upper layer is kept dry, the culture medium in the lower layer is discarded, and then the suspension is added. A third-stage gas-liquid culture is then performed in a gas-liquid mode. The second mixed culture medium is obtained by mixing the immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5 to 3.5):1; for example, the immune cell culture medium and the first mixed culture medium are mixed at a ratio of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, or 3.5:1. The immune cell culture medium can be a commercially available immune cell culture medium, such as PBMC medium. A commercially available PBMC medium, such as SuperCulture L100 serum-free lymphocyte medium (Dakco, catalog number #6122011) supplemented with 5-10% SuperGrow cell culture additive and 1-5‰ IL-2, is used.

[0045] In some embodiments, the step of seeding immune cells into the lower layer of the cell culture container and culturing them for 1 to 3 days, such as 1 day, 2 days, 3 days or any time within that range.

[0046] In some implementations, the in vitro lung model (i.e., normal lung model) is treated with a pulmonary fibrosis inducer for 2 to 5 days, for example, 2 days, 3 days, 4 days, 5 days or any time within that range.

[0047] The pulmonary fibrosis inducer used in this invention can be any substance known in the art that can induce fibrosis in lung tissue, and is not particularly limited herein. In some embodiments, the pulmonary fibrosis inducer is selected from at least one of the group consisting of TGF-β1, bleomycin, paraquat, and silica.

[0048] In some embodiments, the pulmonary fibrosis inducing agent is added to a lung epithelial cell culture medium to obtain a pulmonary fibrosis inducing medium, which is then used to treat an in vitro lung model (i.e., a normal lung model) to obtain a pulmonary fibrosis model. The lung epithelial cell culture medium can be a commercially available lung epithelial cell culture medium, such as PneumaCult produced by Stemcell. TM -Ex Plus Medium.

[0049] Depending on the specific choice of the pulmonary fibrosis inducer, its addition amount in the pulmonary fibrosis induction medium can be adjusted accordingly. In some specific embodiments, when the pulmonary fibrosis inducer contains TGF-β1, the addition amount of TGF-β1 is 5–200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the addition amount of bleomycin is 1–20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the addition amount of paraquat is 20–200 μM; and when the pulmonary fibrosis inducer contains silica, the addition amount of silica is 20–200 μg / mL.

[0050] The pulmonary fibrosis induction medium provided by this invention can be mixed with the endothelial cell culture medium and / or the immune cell culture medium to treat the in vitro lung model (i.e., the normal lung model) to obtain a pulmonary fibrosis model. Specifically, in some embodiments, when the in vitro lung model (i.e., the normal lung model) is not inoculated with immune cells, the pulmonary fibrosis induction medium and the endothelial cell culture medium are mixed at a volume ratio of (0.5–5):1 to obtain a third mixed culture medium, which is used to treat the in vitro lung model (i.e., the normal lung model) to obtain a pulmonary fibrosis model. In other embodiments, when the in vitro lung model (i.e., the normal lung model) is inoculated with immune cells, the pulmonary fibrosis induction medium is mixed with the immune cell culture medium and the endothelial cell culture medium at a volume ratio of (0.5–5):(0.5–5):1 to obtain a fourth mixed culture medium, which is used to treat the in vitro lung model (i.e., the normal lung model) to obtain a pulmonary fibrosis model.

[0051] Secondly, the present invention provides an in vitro lung model, which is constructed by the method for constructing the in vitro lung model provided in the first aspect.

[0052] Thirdly, the present invention provides a culture medium combination comprising lung epithelial cell culture medium, a first mixed culture medium and a third mixed culture medium, and optionally a second mixed culture medium and a fourth mixed culture medium;

[0053] The first mixed culture medium is obtained by mixing lung epithelial cell culture medium and endothelial cell culture medium at a volume ratio of (0.5-3.5):1; the second mixed culture medium is obtained by mixing immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5-3.5):1; the third mixed culture medium is obtained by mixing pulmonary fibrosis induction culture medium and endothelial cell culture medium at a volume ratio of (1-2):1; the fourth mixed culture medium is obtained by mixing pulmonary fibrosis induction culture medium, immune cell culture medium and endothelial cell culture medium at a volume ratio of 1:(1-2):1; the pulmonary fibrosis induction culture medium is obtained by adding a pulmonary fibrosis inducing agent to the lung epithelial cell culture medium, wherein the pulmonary fibrosis inducing agent is selected from at least one of the group consisting of TGF-β1, bleomycin, paraquat and silica; the lung epithelial cell culture medium, the endothelial cell culture medium, the immune cell culture medium and the lung epithelial cell culture medium are as described above, and will not be repeated here.

[0054] In some specific embodiments, in the lung epithelial cell culture medium, when the pulmonary fibrosis inducer contains TGF-β1, the amount of TGF-β1 added is 5–200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the amount of bleomycin added is 1–20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the amount of paraquat added is 20–200 μM; and when the pulmonary fibrosis inducer contains silica, the amount of silica added is 20–200 μg / mL.

[0055] In some embodiments, the culture medium combination is used in the construction method provided by the present invention, or in the construction of the in vitro lung model provided by the present invention, including for the culture of lung organoids, liquid-liquid mode culture, first-stage gas-liquid mode culture, second-stage gas-liquid mode, treatment of the in vitro lung model (i.e., normal lung model), and optionally a third-stage gas-liquid mode culture.

[0056] In some preferred embodiments, the lung epithelial cell culture medium is used for the culture of the lung organoids, the liquid-liquid mode culture, and / or the first-stage gas-liquid mode culture.

[0057] In some preferred embodiments, the first mixed culture medium is used in the second stage gas-liquid mode.

[0058] In some preferred embodiments, the second mixed culture medium is used for the third stage of gas-liquid mode culture.

[0059] In some preferred embodiments, when the in vitro lung model (i.e., a normal lung model) is not inoculated with immune cells, the in vitro lung model is treated with the third mixed culture medium.

[0060] In some preferred embodiments, when the in vitro lung model (i.e., a normal lung model) is inoculated with immune cells, the in vitro lung model is treated with the fourth mixed culture medium.

[0061] In some preferred embodiments, the culture medium combination comprises or consists of lung epithelial cell culture medium, a first mixed culture medium, and a third mixed culture medium.

[0062] In some preferred embodiments, the culture medium combination comprises or consists of lung epithelial cell culture medium, a first mixed culture medium, a second mixed culture medium, and a fourth mixed culture medium.

[0063] Fourthly, the present invention provides an in vitro lung model constructed by the construction method provided in the first aspect, or the use of the in vitro lung model described in the second aspect in the following (a), (b) or (c);

[0064] (a) Drug testing and / or drug screening;

[0065] (b) Detecting the effects of pathogen infection on lung tissue;

[0066] (c) Detect the impact of pathogen infection on the progression of pulmonary fibrosis.

[0067] In some implementations, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs.

[0068] In some implementations, the pathogens include bacteria, viruses, fungi, parasites, etc.

[0069] In some implementations, the drug testing and / or drug screening includes drug testing and / or drug screening for the treatment of pulmonary fibrosis.

[0070] The in vitro lung model provided by this invention can be used for the efficacy testing and mechanism exploration of drugs for treating lung diseases (e.g., drugs for treating pulmonary fibrosis). By evaluating changes in cell marker expression, changes in cell (e.g., alveolar cell) ratio, cell activity, secretion of inflammatory factors, changes in immune cell typing, and cell barrier function, the response of the in vitro lung model to drugs can be assessed, and thus used for drug research and development, especially for the research and development of drugs for treating pulmonary fibrosis. For example, drugs can be added to the upper or lower layer of the model to detect whether the drugs induce pulmonary fibrosis; the effect of pathogens on lung tissue can be confirmed by index detection after pathogens infect the in vitro lung model (upper or lower layer); the test drug can be added simultaneously when treating the in vitro lung model (i.e., normal lung model) with pulmonary fibrosis inducers to induce pulmonary fibrosis, and the drug can be detected to see whether it accelerates or inhibits the progression of pulmonary fibrosis, thereby detecting and / or screening drugs for the treatment of pulmonary fibrosis; the effect of pathogens on the progression of pulmonary fibrosis can be confirmed by index detection after pathogens infect the pulmonary fibrosis model (upper or lower layer); and drugs can be administered and index detection performed after the pulmonary fibrosis model is induced to detect whether the drug accelerates or inhibits the progression of pulmonary fibrosis, thereby detecting and / or screening drugs for the treatment of pulmonary fibrosis. The following are some examples (i) to (xiii) of the use of the in vitro lung model of the present invention in (a), (b), or (c). It should be understood that the application of the in vitro lung model of the present invention is not limited to these:

[0071] (i) Changes in the expression of pulmonary fibrosis markers: The test drug is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and the changes in the expression of pulmonary fibrosis markers in the in vitro lung model are detected; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin. In some specific embodiments, the method includes: fixing the in vitro lung model with 4% paraformaldehyde at room temperature for 30 min, removing the paraformaldehyde, washing the sample with PBS, permeabilizing with 0.1% Triton-X-100 and blocking the sample with 5% BSA, adding primary antibodies against α-SMA, type I collagen, and fibronectin respectively, incubating overnight at 4°C, adding secondary antibodies, incubating at room temperature for 1 h, performing nuclear staining with DAPI for 15 min, washing with PBS, and imaging using confocal microscopy.

[0072] (ii) Flow cytometry detection of changes in immune cell typing: The drug to be tested is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model. Flow cytometry is used to detect changes in cell typing and / or dynamic immune response processes in the in vitro lung model. The biomarkers detected by flow cytometry include, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2. The changes in cell typing include changes in macrophage typing and / or changes in immune cell typing. The changes in macrophage typing include, for example, changes in M1 macrophage typing and M2 macrophage typing. The changes in immune cell typing include, for example, changes in T cell typing, changes in B cell typing, changes in NK cell typing, and changes in intrinsic lymphocyte typing. In some specific implementation schemes, the following methods are included: flow cytometry analysis of changes in M1 and M2 macrophage typing in the upper layer of cells of the model, with flow cytometry indicators including, but not limited to, CD45, CD80, HLA-DR, CD163, CD68, and CD206; or flow cytometry analysis of changes in immune cell typing (T cells, B cells, NK cells, and intrinsic lymphocytes) in the lower layer of cells of the model, with flow cytometry indicators including, but not limited to, CD45, CD3, CD4, CXCR3, CCR4 (Th1 and Th2 cells), CD19 (B cells), Lin, CD127, CD94, CD117, and CRTH2 (ILC cells). The procedure is as follows: collect cells, digest them into single cells using Tryple, Trypsin, or Accutas, and resuspend the cells in flow cytometry buffer. Stain the surface with antibodies against CD45, CD80, HLA-DR, CD163, CD3, and CD4 at 4°C for 30 minutes. Add cell fixation solution and fix at room temperature in the dark for 10 minutes. After centrifugation and washing with PBS, resuspend the cells using a cell permeation reagent and incubate at room temperature for 10-15 minutes. Incubate the cells with the required cell permeation antibodies, such as CD68 and CD206, at room temperature in the dark for 30 minutes. After incubation, wash twice with a cell permeation reagent, resuspend the cells in 200-500 μL of flow cytometry buffer, and perform analysis using a flow cytometer.

[0073] (iii) Detecting the transmembrane resistance of the model to characterize its cell barrier function: The test drug is applied to the in vitro lung model or the pathogen is introduced into the in vitro lung model, and the transmembrane resistance of the in vitro lung model is detected; wherein, before the addition of the test drug or before infection by the pathogen, the normal transmembrane resistance of the in vitro lung model is 200–1500 Ω·cm. 2If, after the addition of the test drug or infection by a pathogen, the transmembrane resistance value decreases significantly to a statistically significant level compared to the normal transmembrane resistance value, then the test drug or pathogen infection impairs the cell barrier function of the lung tissue. In some specific implementation schemes, the following steps are included: remove the transmembrane resistance meter, plug in the power supply, assemble the monitoring device, and place the chopstick detection tip in PBS for equilibration for 15 minutes; after the instrument value stabilizes at 0Ω, fill the model inside and outside with PBS; insert the chopstick detection tip into the model, and record the value after the display value stabilizes; data processing: TEER = TΩ × Acm 2 Where T is the resistance value and A is the area of ​​the lung in vitro model.

[0074] (iv) Detecting the transmembrane resistance value of the model to characterize the cell barrier function of the model: The test drug is applied to the pulmonary fibrosis model or the pathogen is introduced into the pulmonary fibrosis model, and the transmembrane resistance value of the pulmonary fibrosis model is detected; wherein, the transmembrane resistance value of the normal lung model is 200–1500 Ω·cm. 2 The transmembrane resistance value of a pulmonary fibrosis model is lower than that of a normal lung model. Therefore, changes in transmembrane resistance can be used to determine the impact of a test drug or pathogen infection on the progression of pulmonary fibrosis. For example, if the transmembrane resistance value decreases significantly to a statistically significant degree after the addition of a test drug or infection with a pathogen, then the test drug or pathogen infection damages the cell barrier; if the transmembrane resistance value of the pulmonary fibrosis model increases significantly to a statistically significant degree after the addition of a test drug compared to before the addition, then the test drug can repair the cell barrier; if the change in the transmembrane resistance value of the pulmonary fibrosis model after the addition of a test drug or infection with a pathogen is not statistically significant, then the test drug or pathogen infection has no significant impact on the integrity of the cell barrier. In some specific implementation schemes, the following steps are included: Remove the transmembrane resistance meter, plug in the power supply, assemble the monitoring device, and place the chopstick probe in PBS for equilibration for 15 minutes; after the instrument reading stabilizes at 0Ω, fill the model inside and out with PBS; insert the chopstick probe into the model, and record the reading after the display stabilizes; data processing: TEER = TΩ × A cm 2 Where T is the resistance value and A is the area of ​​the pulmonary fibrosis model.

[0075] (v) Detecting changes in barrier permeability to characterize the cell barrier function of the model: The test drug is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and changes in the barrier permeability of the in vitro lung model are detected. In some specific implementations, this includes: adding fluorescent permeating molecules, such as FITC-Dextran (molecular weight 10kDa, 30kDa, 70kDa), to the upper layer of the model, incubating at different time points, and using a microplate reader to detect the mass of the fluorescent molecules permeating into the lower layer of the model. The apparent permeability coefficient is calculated to reflect changes in the barrier permeability of the model. The operation steps are as follows: Discard all culture medium in the wells, add 50-100 μM FITC-Dextran solution to the upper layer, add 400 μL of fresh culture medium to the lower layer, and incubate at 37°C; collect 200 μL of the lower layer culture medium at 2 h, 4 h, 8 h, and 24 h of incubation, respectively. Take out 200 μL of culture medium each time, and add another 200 μL of culture medium to each well to continue culturing; use an ELISA reader to detect the fluorescence value in the culture medium; calculate: Papp=(dQ / dt) / (A*C0); where Papp is the apparent permeability coefficient, A is the membrane area of ​​the membrane chip, C0 is the initial concentration of fluorescent dye, and dQ / dt is the amount of fluorescent dye that permeates per unit time.

[0076] (vi) Detection of model cell viability: The test drug is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and changes in cell viability in the in vitro lung model are detected; wherein, the cell viability is, for example, cell proliferation activity. In some specific embodiments, methods such as MTT, CCK8, and CTG are used to detect model cell viability. Taking CTG detection as an example: An equal volume of CTG activity detection reagent stock solution is added to the model, mixed by pipetting for 5 min, incubated at room temperature with shaking for 30 min, and its chemiluminescence value is detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0077] (vii) Detection of apoptosis in model cells: The drug to be tested is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and the apoptosis status in the in vitro lung model is detected; wherein, the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL.

[0078] (viii) Detection of cytokine levels: The test drug is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and changes in the levels of inflammatory cytokines in the in vitro lung model are detected; wherein the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18. In some specific implementations, detection is performed using methods such as ELISA, CBA multifactor assay, and Luminex multifactor assay. Taking the detection of inflammatory factor TNF-α content by ELISA as an example: Prepare standard solutions of 0, 11.7, 23.4, 46.9, 93.8, 187.5, 375 and 750 pg / mL. Add 100 μL of standard solution and sample solution to each well, and incubate at room temperature with shaking for 1 h. Wash each well 4 times with 300 μL of washing buffer for 2 min each time. Add 100 μL of antibody of the inflammatory cytokine to be tested to each well, and incubate at room temperature with shaking for 30 min. Wash each well 4 times with 300 μL of washing buffer for 2 min each time. Add 100 μL of chromogenic solution to each well, and incubate at room temperature with shaking for 20 min. Add 50 μL of stop solution to each well, and detect the OD value at 450 nm within 10 min.

[0079] (ix) Detection of lung tissue-related cell types and expression: The test drug is applied to the lung in vitro model or the pathogen is used to infect the lung in vitro model, and the lung tissue-related cell phenotypes or cell contents in the lung in vitro model are detected; wherein, the cell phenotypes are, for example, α-tubulin (ciliated cell marker), MUC5AC (goblet cell marker), AQP5 (AT1 cell marker), SFTPC (AT2 cell marker), CC10 (club cell marker), and KRT5 (basal cell marker).

[0080] (x) Detection of oxidative stress levels: The test drug is administered to the in vitro lung model or the pathogen is introduced into the in vitro lung model, and the cellular oxidative stress levels in the in vitro lung model are detected. In some specific embodiments, this includes using a ROS oxidative stress assay kit to detect ROS / RNS levels to assess the drug's modulatory effect on oxidative stress. For example, using Invitrogen... TM The CellROX kit is used to analyze the model. CellROX is a fluorescent probe that can measure generalized oxidative stress within cells using fluorescence microscopy, high-content imaging systems, microplate fluorometers, or flow cytometry platforms. The dye is non-fluorescent in its reduced state and emits bright green, orange, or deep red fluorescence upon oxidation.

[0081] (xi) Detection of the expression of genes related to the development of pulmonary fibrosis: The test drug is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and changes in the expression of pulmonary fibrosis-related genes in the in vitro lung model are detected; wherein, the pulmonary fibrosis-related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene. In some specific embodiments, RNA-seq or RT-PCR is used to detect changes in the transcriptional level of pulmonary fibrosis-related genes.

[0082] (xii) Detection of pathogen infection-related receptor expression: The lung model is infected with the pathogen, and changes in the expression of pathogen infection-related receptors in the lung model are detected. In some specific embodiments, this includes infecting the lung model with avian influenza virus and using a kit to detect the expression of influenza virus receptor α-2,6-linked sialic acid.

[0083] (xiii) Detecting the antipathogenic effect of the drug: The lung model is infected with the pathogen, and then the test drug is applied to the lung model to detect the inhibitory effect of the test drug on the pathogen. In some specific embodiments, this includes detecting the viral titer at different time points after infecting the lung model with the pathogen to detect the antiviral effect of the test drug. Alternatively, RT-PCR is used to detect the viral RNA copy number after the model is infected with the virus and to determine the infection dose in 50% tissue culture.

[0084] Fifthly, the present invention provides a drug detection method, a drug screening method, and / or a method for detecting the effect of pathogen infection on lung tissue, comprising:

[0085] (i) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the expression changes of pulmonary fibrosis markers in the in vitro lung model are detected; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin. In some embodiments, the detection method is immunofluorescence detection; preferably, the detection method includes: fixing, washing, and blocking the in vitro lung model, adding primary antibodies against pulmonary fibrosis markers and incubating, then adding secondary antibodies and incubating, and obtaining the expression changes of pulmonary fibrosis markers by staining and imaging;

[0086] (ii) The drug to be tested is applied to an in vitro lung model or the pathogen is infecting an in vitro lung model, and the cell typing changes and / or dynamic immune response process in the in vitro lung model are detected by flow cytometry. In some embodiments, the flow cytometry detection markers are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2; the cell typing changes include macrophage typing changes and / or immune cell typing changes, the macrophage typing changes being, for example, M1 type macrophage typing changes and M2 type macrophage typing changes; the immune cell typing changes being, for example, T cell typing changes, B cell typing changes, NK cell typing changes, and intrinsic lymphocyte typing changes; preferably, the detection method includes: collecting cells from an in vitro lung model and resuspending them in flow cytometry buffer, staining the surface with antibodies against the detection markers, fixing, centrifuging, washing, resuspending the cells in flow cytometry buffer, and detecting fluorescence signals using a flow cytometer;

[0087] (iii) The test drug is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the transmembrane resistance of the in vitro lung model is measured; wherein, before the test drug is added or before infection by the pathogen, the normal transmembrane resistance of the in vitro lung model is 200–1500 Ω·cm. 2 If, after the addition of the test drug or infection by a pathogen, the transmembrane resistance value decreases significantly to a statistically significant degree compared to the normal transmembrane resistance value, then the test drug or pathogen infection impairs the cell barrier function of the lung tissue. In some embodiments, the detection method includes: measuring the resistance value of an in vitro lung model using a transmembrane resistance meter, and using the formula TEER = TΩ × A cm. 2 Calculate the transmembrane resistance, where T is the resistance and A is the area of ​​the in vitro lung model;

[0088] (iv) The test drug is applied to a pulmonary fibrosis model or the pathogen is used to infect the pulmonary fibrosis model, and the transmembrane resistance value of the pulmonary fibrosis model is detected. The change in transmembrane resistance value can be used to determine the effect of the test drug or pathogen infection on the progression of pulmonary fibrosis. If the transmembrane resistance value decreases significantly to a statistically significant level after the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection can disrupt the cell barrier. If the transmembrane resistance value of the pulmonary fibrosis model increases significantly to a statistically significant level after the addition of the test drug compared to before the addition, then the test drug can repair the cell barrier. If the change in the transmembrane resistance value of the pulmonary fibrosis model after the addition of the test drug or infection with the pathogen is not statistically significant compared to before the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection has no significant effect on the integrity of the cell barrier. In some embodiments, the detection method includes: using a transmembrane resistance meter to detect the resistance value of the pulmonary fibrosis model, using the formula TEER = TΩ × Acm. 2 Calculate the transmembrane resistance value, where T is the resistance value and A is the area of ​​the pulmonary fibrosis model;

[0089] (v) Applying the test drug to an in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting changes in the barrier permeability of the in vitro lung model. In some embodiments, the detection includes: adding a fluorescent molecule (e.g., FITC-Dextran) to the upper layer of the in vitro lung model and incubating it, detecting the amount of the fluorescent molecule permeating into the lower layer of the in vitro lung model, and calculating the apparent permeability coefficient;

[0090] (vi) Applying the drug to an in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting changes in cell activity in the in vitro lung model. In some embodiments, the cell activity is, for example, cell proliferation activity; preferably, the detection method includes: adding an MTT, CCK8, or CTG activity detection reagent to the in vitro lung model and incubating, and detecting the chemiluminescence value;

[0091] (vii) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the apoptosis status in the in vitro lung model is detected. In some embodiments, the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL;

[0092] (viii) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and changes in the levels of inflammatory cytokines in the in vitro lung model are detected. In some embodiments, the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; the detection method includes ELISA, CBA multifactor assay, and Luminex multifactor assay; preferably, the detection method includes: preparing standard solutions of inflammatory cytokines with gradient concentrations, adding the standard solutions and sample culture medium to a culture vessel, incubating and washing, adding antibodies to the inflammatory cytokines to be tested, incubating and washing again, adding chromogenic solution and incubating, then adding stop solution and detecting the OD value;

[0093] (ix) The test drug is administered to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the lung tissue-related cell phenotypes or cell contents in the in vitro lung model are detected. In some embodiments, the cell phenotypes are, for example, α-tubulin, MUC5AC, AQP5, SFTPC, CC10, KRT5;

[0094] (x) Apply the drug to be tested to an in vitro lung model or infect the in vitro lung model with the pathogen, and detect the level of cellular oxidative stress in the in vitro lung model;

[0095] (xi) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the expression changes of pulmonary fibrosis-related genes in the in vitro lung model are detected. In some embodiments, the pulmonary fibrosis-related genes are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene;

[0096] (xii) Infect the lung in vitro model with the pathogen and detect the expression changes of the pathogen infection-related receptors in the lung in vitro model;

[0097] (xiii) Infect the lung in vitro model with the pathogen, then apply the test drug to the lung in vitro model, and detect the inhibitory effect of the test drug on the pathogen;

[0098] The lung in vitro model is either the lung in vitro model constructed by the construction method of the first aspect or the lung in vitro model described in the second aspect.

[0099] In some implementations, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs.

[0100] First, the lung in vitro model provided by this invention uses lung organoids instead of traditional cell lines to construct the lung in vitro model, which has a shorter experimental cycle compared with animal models, thus reducing experimental costs. It also simplifies operation and enhances model stability and controllability. Second, the construction method provided by this invention involves sequentially seeding lung organoids, endothelial cells, macrophages, and optionally immune cells. A normal in vitro lung model is obtained through liquid-liquid culture followed by gas-liquid culture (including a first stage of gas-liquid culture, a second stage of gas-liquid culture, and optionally a third stage of gas-liquid culture). Optionally, a pulmonary fibrosis model can be obtained by treating the in vitro lung model with a pulmonary fibrosis inducing agent. This not only helps to achieve a higher level of maturity in the in vitro lung model but also better simulates the function and structural characteristics of the alveolar-capillary barrier, improving its simulation accuracy. Furthermore, the in vitro lung model provided by this invention includes lung organoids, endothelial cells, macrophages, and optionally immune cells, which can effectively simulate the cell-related microenvironment and intercellular interactions. It also has the advantages of being able to be passaged multiple times and maintaining the phenotype for a long time in vitro, providing a good platform for disease simulation, drug detection, screening, evaluation, and research and development. Attached Figure Description

[0101] Figure 1 shows the transmembrane resistance values ​​of the lung in vitro model of the present invention.

[0102] Figure 2 shows the bright field image and fluorescence staining image of the lung in vitro model of the present invention.

[0103] Figure 3 shows the detection results of pulmonary fibrosis marker expression in Example 4 of the present invention.

[0104] Figure 4 shows the results of ELISA detection of the secretion level of the inflammatory factor TNF-α in Example 4 of the present invention.

[0105] Figure 5 shows the detection results of cell viability in the in vitro lung model in Example 5 of the present invention.

[0106] Figure 6 shows the detection results of pulmonary fibrosis marker expression in each group of models in Example 6 of the present invention.

[0107] Figure 7 shows the transmembrane resistance values ​​of each group of models in Embodiment 6 of the present invention.

[0108] Figure 8 shows the expression of inflammatory factors IL-6 and TNF-α in each group of models in Example 7 of the present invention.

[0109] Figures 9A and 9B show the proportions of Th1 and Th2 helper T cells in the control group in Example 8 of the present invention; Figures 9C and 9D show the proportions of Th1 and Th2 helper T cells in the induction group with added TGF-β1 in Example 8 of the present invention. Detailed Implementation

[0110] The materials, methods, and embodiments described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from this specification, the drawings, and the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0111] Example 1

[0112] This embodiment provides a detailed process for constructing an in vitro lung model (normal lung model):

[0113] Primary extraction and liquid-liquid culture of lung organoids:

[0114] (1) Cleaning: Normal human lung tissue was cleaned with primary tissue cleaning solution to remove surface mucosa. The primary tissue cleaning solution was a buffer solution containing penicillin and streptomycin.

[0115] (2) Disintegration: Chop the tissue into a paste, resuspend it in buffer solution, let it stand, and then discard the supernatant.

[0116] (3) Digestion: Add primary lung tissue digestion solution, mix well by pipetting, and then digest using a metal bath shaker at 37°C for 30-60 minutes until small cell clusters are formed. Finally, add buffer solution to terminate the digestion. Commercially available primary lung tissue digestion solution can be used. In this example, the primary lung tissue digestion solution contains collagenase and culture medium, wherein the collagenase is collagenase II with a final concentration of 1-2 mg / mL, and the culture medium is DMEM or Advanced DMEM / F12.

[0117] (4) Resuspension: The digested cell suspension was filtered through a 100 μm cell sieve, the cells were resuspended in lung organoid culture medium, counted, and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 5 The lung organoid culture medium is named Lung Organoid Culture Kit, manufactured by Ivytech, catalog number: KLU0201.

[0118] (5) Lung organoid plating culture: After centrifugation and removal of supernatant, lung organoids were resuspended in Matrigel extracellular matrix, with 30 μL of gel dropper per well, and plated into 24-well plates. After gel formation, lung organoid culture medium was added, and the plates were incubated at 37°C for 7 days.

[0119] (6) Digesting lung organoids: After the lung organoids have grown for 7 days, use lung organoid passage digestion solution Tryple to digest the lung organoids into single cells, and add lung organoid culture medium to stop the digestion.

[0120] (7) Inoculation: Centrifuge to remove supernatant, resuspend in lung epithelial cell culture medium, and adjust density to 1×10⁶. 6 Cells / mL. The chip membrane area is 0.33 cm². 2 2×10 5 10 cells, cell density 6×10 5 pcs / cm 2 The lung organoid suspension was seeded onto the upper layer of the membrane. 1 mL of lung epithelial cell culture medium (BorgH Group's Respiratory Organoid Expansion Medium (Catalog No. A01-001), Ivylink's Lung Organoid Culture Kit (Catalog No. KLU0201), or Stemcell's PneumaCult) was added to the lower layer. TM -Ex Plus Medium)) was cultured in a liquid-liquid incubator at 37°C.

[0121] (8) Change the medium every 3 days and culture for 5 days.

[0122] Gas-liquid culture mode:

[0123] (1) Once the lung organoids have grown to the upper membrane layer, discard the culture medium in both the upper and lower membrane layers, and add 600 μL of lung epithelial cell culture medium to the lower membrane layer.

[0124] (2) Cultured in gas-liquid mode for 2 days (first stage of gas-liquid mode culture).

[0125] Inoculation with endothelial cells:

[0126] (1) Digest the endothelial cells and resuspend them in a primary mixed culture medium (lung epithelial cell culture medium: endothelial cell culture medium volume ratio 2:1). Count the cells and adjust the endothelial cell density to 6 × 10⁶ cells / year. 5 Cells / mL. The chip membrane area is 0.33 cm². 2 3×10 4 There are 10 endothelial cells, with a cell density of 0.9 × 10⁻⁶. 5 pcs / cm 2 The endothelial cell culture medium used was Sciencell (catalog number #1001).

[0127] (2) Place the chip in a 37°C incubator for 2 hours. After the cells adhere to the wall, place the chip upright and add 0.6 mL of the first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium are mixed at a volume ratio of 2:1) to the lower chamber.

[0128] Inoculation with macrophages:

[0129] (1) Collect macrophages and resuspend them in macrophage culture medium (ScienCell, #1921). Count the cells and adjust the cell density to 1×10⁶.5 Cells / mL. The chip membrane area is 0.33 cm². 2 Administer 1×10 4 There are 10 macrophages, with a cell density of 0.3 × 10⁶. 5 pcs / cm 2 .

[0130] (2) Macrophages were cultured overnight, and then the macrophage culture medium on the membrane was discarded.

[0131] (3) Change the culture medium under the membrane (first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium are mixed at a volume ratio of 2:1)).

[0132] (4) Continue gas-liquid mode culture for 2 days (second stage gas-liquid mode culture).

[0133] Inoculate immune cells (this step can be omitted if immune function is not required):

[0134] (1) Collect PBMCs and resuspend them in a second mixed medium (immune cell culture medium and the first mixed medium mixed at a volume ratio of 2:1). Discard the submucosa medium. The bottom area of ​​the lower chamber of the chip should be 2 cm². 2 2×10 5 One PBMC, with a cell density of 1×10⁶ cells. 5 pcs / cm 2 For example, commercially available PBMC media can be used, such as SuperCulture L100 serum-free lymphocyte medium (Dakorway, catalog number #6911011) supplemented with 5-10% SuperGrow cell culture additive (Dakorway, catalog number #6122011) and 1-5‰ IL-2.

[0135] (2) Continue gas-liquid culture for 1 day (third stage gas-liquid culture) to obtain an in vitro lung model (normal lung model).

[0136] Model Detection:

[0137] (1) The transmembrane resistance of the model was measured using a transmembrane ohmmeter, and the results are shown in Figure 1. As can be seen from Figure 1, the resistance of the lung model constructed in this embodiment is 550–600 Ω·cm. 2 .

[0138] (2) Immunofluorescence was used to detect the expression of E-cadherin in lung organoids, VE-cadherin in endothelial cells, AQP5 in type I alveolar cells, and SFTPB in type II alveolar cells. The results are shown in Figure 2. As can be seen from Figure 2, the cells in the in vitro lung model constructed in this embodiment are tightly connected and express lung tissue-related markers.

[0139] Example 2

[0140] This embodiment constructs a lung fibrosis model based on the in vitro lung model (normal lung model) constructed in Example 1. The steps are as follows:

[0141] Inducing pulmonary fibrosis:

[0142] TGF-β1 was used as a lung fibrosis inducer and mixed with lung epithelial cell culture medium to obtain lung fibrosis induction medium (the content of lung fibrosis inducer was 10 ng / mL). Then, the lung fibrosis induction medium was mixed with immune cell culture medium and endothelial cell culture medium at a volume ratio of 1:1:1 to obtain a fourth mixed culture medium. The lower chamber of the lung in vitro model was treated with the fourth mixed culture medium for 3 days to obtain a lung fibrosis model.

[0143] Example 3

[0144] This embodiment uses the in vitro lung model (normal lung model) constructed in Example 1 to detect the side effects of ADC drugs. The steps and results are as follows:

[0145] (1) Drug testing was conducted on an in vitro lung model. A control group, an ADC drug group, and a payload group were set up. The control group was placed in the lower layer of the chip with the first mixed culture medium, the ADC drug group was placed in the lower layer of the chip with culture medium containing 50 μg / mL trastuzumab deruxtecan, and the payload group was placed in the lower layer of the chip with culture medium containing 50 μg / mL ADC drug-adaptor conjugate Deruxtecan.

[0146] (2) Drug treatment for 5 days.

[0147] (3) After drug treatment, immunofluorescence staining was used to detect the expression of α-SMA and type I collagen in the model. Images were taken using a fluorescence microscope or confocal microscope, and the average fluorescence intensity and the sum of fluorescence intensities were calculated. The expression of pulmonary fibrosis markers was compared between the control group, the ADC drug group, and the Payload group.

[0148] (4) ELISA was used to detect the secretion levels of inflammatory factors TNF-α and IL-6.

[0149] Example 4

[0150] This embodiment tests the side effects of bleomycin on the in vitro lung model (normal lung model) constructed in Example 1. The steps and results are as follows:

[0151] (1) Drug testing was conducted on an in vitro lung model. A control group, a low-concentration bleomycin group (1 μg / mL), and a high-concentration bleomycin group (10 μg / mL) were set up. The first mixed culture medium was added to the bottom layer of the control chip, the first mixed culture medium containing 1 μg / mL bleomycin was added to the bottom layer of the low-concentration bleomycin group, and the first mixed culture medium containing 10 μg / mL bleomycin was added to the bottom layer of the high-concentration bleomycin group.

[0152] (2) Drug treatment for 3 days.

[0153] (3) After drug treatment, immunofluorescence staining was used to detect the expression of α-SMA and type I collagen in the model. Images were taken using a fluorescence microscope or confocal microscope, and the average fluorescence intensity and the sum of fluorescence intensities were calculated. The expression of pulmonary fibrosis markers was compared between the control group, the low-concentration bleomycin group, and the high-concentration bleomycin group.

[0154] (4) ELISA was used to detect the secretion level of the inflammatory factor TNF-α.

[0155] The results are shown in Figures 3 and 4.

[0156] As shown in Figure 3, the control group showed no expression of α-SMA and type I collagen, while the low-concentration bleomycin group could induce the expression of α-SMA and type I collagen, and the lungs showed fibrosis; the high-concentration bleomycin group showed high expression of α-SMA and type I collagen, and severe lung fibrosis.

[0157] As shown in Figure 4, the control group did not secrete TNF-α, while both the low-concentration and high-concentration bleomycin groups induced TNF-α secretion, and the higher the concentration of bleomycin, the higher the level of TNF-α secretion.

[0158] Example 5

[0159] This embodiment tests the side effects of nintedanib on the in vitro lung model (normal lung model) constructed in Example 1. The steps and results are as follows:

[0160] (1) Drug testing was conducted on an in vitro lung model. A control group, a low-concentration nintedanib group (1 μM), and a high-concentration nintedanib group (10 μM) were set up. The first mixed culture medium was added to the bottom layer of the control chip, the first mixed culture medium containing 1 μM nintedanib was added to the bottom layer of the low-concentration nintedanib group, and the first mixed culture medium containing 10 μM nintedanib was added to the bottom layer of the high-concentration nintedanib group.

[0161] (2) Drug treatment for 3 days.

[0162] (3) After drug treatment, the cell viability of the model cells was detected using the CTG method to investigate the effect of nintedanib on cell viability. The cell viability was compared between the control group, the low-concentration nintedanib group, and the high-concentration nintedanib group to determine if there were any differences.

[0163] The results are shown in Figure 5. As can be seen from Figure 5, there was no significant difference in activity between the control group, the low-concentration nintedanib group, and the high-concentration nintedanib group, indicating that nintedanib does not affect cell activity.

[0164] Example 6

[0165] In this embodiment, the efficacy of the drug was tested in an in vitro lung model and a pulmonary fibrosis model. The steps and results are as follows:

[0166] (1) A lung in vitro model (normal lung model) was constructed according to the method in Example 1 and a pulmonary fibrosis model was constructed according to the method in Example 2. A control group, an induction group, and a drug treatment group were set up. The control group was a lung in vitro model (normal lung model) with a second mixed culture medium added to the lower layer of the chip; the induction group was a pulmonary fibrosis induction culture medium containing 10 ng / m LTGF-β1 added to the lower layer of the lung in vitro model (normal lung model); the drug treatment group was a pulmonary fibrosis induction culture medium containing 10 ng / m LTGF-β1 and 10 μM nintedanib added to the lower layer of the lung in vitro model (normal lung model).

[0167] (2) Drug treatment for 3 days.

[0168] (3) After drug treatment, immunofluorescence staining was used to detect the expression of α-SMA and type I collagen in the model. Images were taken using a fluorescence microscope or confocal microscope, and the average fluorescence intensity and the sum of fluorescence intensities were calculated. The expression of pulmonary fibrosis markers was compared between the control group, the induction group, and the drug-treated group.

[0169] (4) Use a transmembrane resistance meter to detect the transmembrane resistance value of each model group.

[0170] The results are shown in Figures 6 and 7.

[0171] As shown in Figure 6, the induction group, with the addition of TGF-β1, significantly induced pulmonary fibrosis, and the expression of α-SMA and type I collagen was significantly increased compared to the control group. The drug treatment group, with the addition of TGF-β1 and nintedanib, showed that nintedanib significantly inhibited pulmonary fibrosis, and the expression of α-SMA and type I collagen was significantly reduced compared to the induction group.

[0172] As shown in Figure 7, the transmembrane resistance values ​​in the induction group were impaired, and the transmembrane resistance values ​​were significantly lower compared to the control group. In the drug treatment group, the addition of nintedanib alleviated the cell barrier damage, resulting in a significantly higher transmembrane resistance value compared to the induction group.

[0173] Example 7

[0174] In this embodiment, the efficacy of the drug was tested in an in vitro lung model and a pulmonary fibrosis model. The steps and results are as follows:

[0175] (1) An in vitro lung model (normal lung model) was constructed according to the method in Example 1. A control group, an induction group, and a drug treatment group were set up. The control group was an in vitro lung model (normal lung model) with a second mixed culture medium added to the lower layer of the chip; the induction group was a lung fibrosis induction culture medium containing 10 ng / mL TGF-β1 added to the lower layer of the in vitro lung model (normal lung model); the drug treatment group was a lung fibrosis induction culture medium containing 10 ng / mL TGF-β1 and 10 μM nintedanib added to the lower layer of the in vitro lung model (normal lung model).

[0176] (2) Drug treatment for 3 days.

[0177] (3) After the drug treatment, the levels of inflammatory factors IL-6 and TNF-α in the model were detected by ELISA kit, and the expression of inflammatory cytokines in the control group, the induction group and the drug treatment group were compared to see if there was any difference.

[0178] The results are shown in Figure 8.

[0179] As shown in Figure 8, compared with the control group, the expression of IL-6 and TNF-α was significantly increased in the TGF-β1-induced group. The drug-treated groups received TGF-β1 and nintedanib, with nintedanib significantly inhibiting pulmonary fibrosis, and the expression of IL-6 and TNF-α was significantly reduced compared with the induced group.

[0180] Example 8

[0181] This embodiment describes immune cell detection in an in vitro lung model and a pulmonary fibrosis model. The steps and results are as follows:

[0182] (1) An in vitro lung model (normal lung model) was constructed according to the method in Example 1. A control group and an induction group were set up. The control group was an in vitro lung model (normal lung model) with a second mixed culture medium added to the lower layer of the chip; the induction group was a lung fibrosis induction culture medium containing 10 ng / mLTGF-β1 added to the lower layer of the in vitro lung model (normal lung model).

[0183] (2) Treat with inducer for 3 days.

[0184] (3) After induction, the expression levels of Th1 and Th2 immune cells in the in vitro lung model (normal lung model) and pulmonary fibrosis model were detected by flow cytometry. The changes in Th1 and Th2 phenotypes of immune cells in the control group and the induction group were compared.

[0185] The results are shown in Figures 9A to 9D.

[0186] As shown in Figures 9A to 9D, in the control group, B cells accounted for 12.4% of the total cells, CD3 T cells accounted for 28.4%, Th1 cells accounted for 2.65%, and Th2 cells accounted for 2.8%. In the TGF-β1 group, B cells accounted for 10.4% of the total cells, CD3 T cells accounted for 25.3%, Th1 cells accounted for 2.1%, and Th2 cells accounted for 2.6%. Therefore, after TGF-β1 induction, there was no significant difference in the content of B cells and T cells among immune cells.

Claims

1. A method for constructing an in vitro lung model, comprising: Provides lung organoids and cell culture containers; The lung organoids were seeded into the upper layer of the cell culture container, and lung epithelial cell culture medium was added to the upper and lower layers of the cell culture container to culture the lung organoids in a liquid-liquid mode. The lung epithelial cell culture medium in the upper and lower layers is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to carry out the first stage of gas-liquid mode culture of the lung organoids. Endothelial cells were seeded in the lower layer of the cell culture container, and macrophages were seeded in the upper layer of the cell culture container and cultured in a second stage of gas-liquid mode to obtain an in vitro lung model. Optionally, the in vitro lung model is a pulmonary fibrosis model, and the construction method includes the step of treating the in vitro lung model with a pulmonary fibrosis inducing agent to obtain a pulmonary fibrosis model.

2. The construction method according to claim 1, wherein, The lung organoids are cultured in liquid-liquid mode for 3 to 7 days; the lung organoids are cultured in gas-liquid mode for 2 to 21 days in the first stage; and the lung organoids are cultured in gas-liquid mode for 1 to 5 days in the second stage.

3. The construction method according to claim 1 or 2, wherein, The lung in vitro model was treated with the pulmonary fibrosis inducing agent for 2 to 5 days. Preferably, the pulmonary fibrosis inducer is selected from at least one of the group consisting of TGF-β1, bleomycin, paraquat, and silica; Preferably, the pulmonary fibrosis inducing agent is added to the lung epithelial cell culture medium to obtain a pulmonary fibrosis inducing culture medium, and the pulmonary fibrosis inducing culture medium is mixed with endothelial cell culture medium and / or immune cell culture medium for treating the in vitro lung model; More preferably, in the pulmonary fibrosis induction culture medium, when the pulmonary fibrosis inducer contains TGF-β1, the amount of TGF-β1 added is 5-200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the amount of bleomycin added is 1-20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the amount of paraquat added is 20-200 μM; and when the pulmonary fibrosis inducer contains silica, the amount of silica added is 20-200 μg / mL.

4. The construction method according to any one of claims 1-3, wherein, Based on the inoculation area, the lung organoid inoculation is 1×10 5 ~10×10 5 cells / cm 2 ; Based on the inoculation area, the endothelial cells were inoculated at a rate of 0.5 × 10⁻⁶. 5 ~5×10 5 cells / cm 2 ; Based on the inoculation area, the macrophage inoculation was 0.1 × 10⁻⁶. 5 ~5×10 5 cells / cm 2 ; Preferably, the endothelial cells are derived from umbilical vein endothelial cells, primary lung endothelial cells, or lung endothelial cell lines; Preferably, the macrophages are derived from macrophages expanded from PBMCs, primary alveolar macrophages, passaged alveolar macrophages, or macrophages induced by the THP-1 cell line.

5. The construction method according to any one of claims 1 to 4, wherein, In the step of seeding endothelial cells into the lower layer of the cell culture container, after the endothelial cells adhere to the wall, a first mixed culture medium is added to the lower layer. The first mixed culture medium is obtained by mixing the lung epithelial cell culture medium and the endothelial cell culture medium at a volume ratio of (0.5-3.5):

1. In the step of seeding macrophages into the upper layer of the cell culture container and performing the second stage of gas-liquid culture, after the macrophages adhere to the wall, the culture media of the upper and lower layers are discarded and the upper layer is kept dry. Fresh first mixed culture medium is added to the lower layer for the second stage of gas-liquid culture. The first mixed culture medium is obtained by mixing the lung epithelial cell culture medium and the endothelial cell culture medium in a ratio of (0.5-3.5):

1.

6. The construction method according to any one of claims 1 to 5, wherein, After seeding the macrophages into the upper layer of the cell culture container and performing a second-stage gas-liquid culture, the method further includes the step of seeding immune cells into the lower layer of the cell culture container and culturing them. Preferably, the immune cells include PBMCs, T cells, B cells, and NK cells; Preferably, the number of immune cells inoculated is 0.5 × 10⁻⁶, calculated based on the inoculation area. 5 ~1×10 6 cells / cm 2 ; Preferably, in the step of seeding immune cells into the lower layer of the cell culture container and culturing them, the immune cells are resuspended in a second mixed culture medium to obtain a suspension, the upper layer is kept dry, the culture medium in the lower layer is discarded, and then the suspension is added to perform a third-stage gas-liquid mode culture. The second mixed culture medium is obtained by mixing immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5-3.5):

1. More preferably, the third stage gas-liquid mode culture time is 1 to 3 days; Preferably, when the in vitro lung model is not inoculated with immune cells, the pulmonary fibrosis induction medium and the endothelial cell medium are mixed at a volume ratio of (1-2):1 to obtain a third mixed medium for treating the in vitro lung model; or, when the in vitro lung model is inoculated with immune cells, the pulmonary fibrosis induction medium is mixed with the immune cell medium and the endothelial cell medium at a volume ratio of 1:(1-2):1 to obtain a fourth mixed medium for treating the in vitro lung model.

7. The construction method according to any one of claims 1 to 6, wherein, The cell culture container is a Transwell or a cell culture chip, such as a membrane chip or a barrier chip.

8. An in vitro lung model, which is constructed by the construction method according to any one of claims 1 to 7.

9. A culture medium assembly comprising a lung epithelial cell culture medium and a first mixed culture medium, and optionally a second mixed culture medium, a third mixed culture medium and a fourth mixed culture medium; in, The first mixed culture medium is obtained by mixing lung epithelial cell culture medium and endothelial cell culture medium at a volume ratio of (0.5-3.5):1; The second mixed culture medium is obtained by mixing immune cell culture medium and the first mixed culture medium at a volume ratio of (0.5-3.5):1; The third mixed culture medium is obtained by mixing pulmonary fibrosis induction culture medium and endothelial cell culture medium at a volume ratio of (0.5-5):1; The fourth mixed culture medium is obtained by mixing pulmonary fibrosis induction culture medium with immune cell culture medium and endothelial cell culture medium in a volume ratio of (0.5-5):(0.5-5):

1. The pulmonary fibrosis induction culture medium is obtained by adding a pulmonary fibrosis inducer to a lung epithelial cell culture medium, wherein the pulmonary fibrosis inducer is selected from at least one of the group consisting of TGF-β1, bleomycin, paraquat, and silica. Preferably, in the lung epithelial cell culture medium, when the pulmonary fibrosis inducer contains TGF-β1, the amount of TGF-β1 added is 5–200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the amount of bleomycin added is 1–20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the amount of paraquat added is 20–200 μM; when the pulmonary fibrosis inducer contains silica, the amount of silica added is 20–200 μg / mL. Preferably, the culture medium combination is used in the construction method according to any one of claims 1 to 7, or in the construction of the lung in vitro model according to claim 8, including culturing lung organoids, liquid-liquid mode culture, first stage gas-liquid mode culture, second stage gas-liquid mode, treating the lung in vitro model, and optionally a third stage gas-liquid mode culture. More preferably, the lung epithelial cell culture medium is used for the culture of the lung organoids, the liquid-liquid mode culture, and / or the first stage gas-liquid mode culture; The first mixed culture medium is used in the second stage gas-liquid mode; The second mixed culture medium is used for the third stage gas-liquid mode culture; when the lung in vitro model is not inoculated with immune cells, the lung in vitro model is treated with the third mixed culture medium; when the lung in vitro model is inoculated with immune cells, the lung in vitro model is treated with the fourth mixed culture medium. More preferably, the culture medium combination comprises or consists of lung epithelial cell culture medium, a first mixed culture medium, and a third mixed culture medium; or, the culture medium combination comprises or consists of lung epithelial cell culture medium, a first mixed culture medium, a second mixed culture medium, and a fourth mixed culture medium.

10. The use of the in vitro lung model constructed by the construction method according to any one of claims 1 to 7, or the use of the in vitro lung model according to claim 8 in the following (a), (b) or (c); (a) Drug detection and / or drug screening; preferably, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs; preferably, the drug detection and / or drug screening includes drug detection and / or drug screening for the treatment of pulmonary fibrosis; (b) Detecting the effects of pathogen infection on lung tissue; (c) Detect the impact of pathogen infection on the progression of pulmonary fibrosis.

11. The use according to claim 10, wherein, The terms (a) to (c) include at least one of the following (i) to (xiii): (i) The drug to be tested is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and the expression changes of pulmonary fibrosis markers in the in vitro lung model are detected; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin; (ii) The drug to be tested is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and the cell typing changes and / or dynamic immune response process in the in vitro lung model are detected by flow cytometry; wherein, the biomarkers detected by flow cytometry are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2; the cell typing changes include macrophage typing changes and / or immune cell typing changes, the macrophage typing changes are, for example, M1 type macrophage typing changes and M2 type macrophage typing changes; the immune cell typing changes are, for example, T cell typing changes, B cell typing changes, NK cell typing changes, and innate lymphocyte typing changes; (iii) The test drug is applied to the in vitro lung model or the pathogen is introduced into the in vitro lung model, and the transmembrane resistance value of the in vitro lung model is measured; wherein, before the test drug is added or before infection by the pathogen, the normal transmembrane resistance value of the in vitro lung model is 200–1500 Ω·cm. 2 If, after the addition of the test drug or infection by a pathogen, the transmembrane resistance value decreases significantly to a statistically significant level compared to the normal transmembrane resistance value, then the test drug or pathogen infection impairs the cell barrier function of the lung tissue. (iv) Apply the test drug to the pulmonary fibrosis model or infect the pulmonary fibrosis model with the pathogen, and detect the transmembrane resistance value of the pulmonary fibrosis model; wherein, if the transmembrane resistance value of the pulmonary fibrosis model decreases significantly to a statistically significant degree after the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection can damage the cell barrier; if the transmembrane resistance value of the pulmonary fibrosis model increases significantly to a statistically significant degree after the addition of the test drug compared to before the addition, then the test drug can repair the cell barrier; if the change in the transmembrane resistance value of the pulmonary fibrosis model after the addition of the test drug or infection with the pathogen is not statistically significant compared to before the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection has no significant effect on the integrity of the cell barrier; (v) Apply the drug to be tested to the in vitro lung model or infect the in vitro lung model with the pathogen, and detect changes in the barrier permeability of the in vitro lung model; (vi) The drug to be tested is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and changes in cell activity in the in vitro lung model are detected; wherein, the cell activity is, for example, cell proliferation activity; (vii) The drug to be tested is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and the apoptosis status in the in vitro lung model is detected; wherein the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL; (viii) The test drug is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and the changes in the levels of inflammatory cytokines in the in vitro lung model are detected; wherein the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; (ix) The test drug is applied to the in vitro lung model or the pathogen is used to infect the in vitro lung model, and the lung tissue-related cell phenotype or cell content in the in vitro lung model is detected; wherein the cell phenotype is, for example, a ciliated cell marker, a goblet cell marker, an AT1 cell marker, an AT2 cell marker, a club cell marker, or a basal cell marker. (x) The drug to be tested is applied to the lung in vitro model or the pathogen is infected in the lung in vitro model, and the level of cellular oxidative stress in the lung in vitro model is detected; (xi) The drug to be tested is applied to the in vitro lung model or the pathogen is infected in the in vitro lung model, and the expression changes of genes related to the process of pulmonary fibrosis in the in vitro lung model are detected; wherein, the genes related to pulmonary fibrosis are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene. (xii) Infect the lung model with the pathogen and detect changes in the expression of pathogen-related receptors in the lung model. (xiii) Infect the lung in vitro model with the pathogen, then apply the test drug to the lung in vitro model, and detect the inhibitory effect of the test drug on the pathogen.

12. A method for drug detection, drug screening, and / or a method for detecting the effect of pathogen infection on lung tissue, comprising: (i) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the expression changes of pulmonary fibrosis markers in the in vitro lung model are detected; wherein, the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin; preferably, the detection method is immunofluorescence detection; more preferably, the detection method includes: fixing, washing, and blocking the in vitro lung model, adding primary antibodies against pulmonary fibrosis markers and incubating, then adding secondary antibodies and incubating, and obtaining the expression changes of pulmonary fibrosis markers by staining and imaging; (ii) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model. Flow cytometry is used to detect cell typing changes and / or dynamic immune response processes in the in vitro lung model. The flow cytometry markers used for detection include, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, and CRTH2. The cell typing changes include macrophage typing. The changes in cell type and / or the changes in immune cell type, wherein the changes in macrophage type are, for example, changes in M1 macrophage type or M2 macrophage type; the changes in immune cell type are, for example, changes in T cell type, changes in B cell type, changes in NK cell type, and changes in intrinsic lymphocyte type; preferably, the detection method includes: collecting cells from an in vitro lung model and resuspending them in flow cytometry buffer, staining with antibodies against the detection marker, fixing, centrifuging, washing, resuspending the cells in flow cytometry buffer, and detecting fluorescence signals using a flow cytometer; (iii) The test drug is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the transmembrane resistance of the in vitro lung model is measured; wherein, before the test drug is added or before infection by the pathogen, the normal transmembrane resistance of the in vitro lung model is 200–1500 Ω·cm. 2 If, after the addition of the test drug or infection by a pathogen, the transmembrane resistance value decreases significantly to a statistically significant level compared to the normal transmembrane resistance value, then the test drug or pathogen infection impairs the cell barrier function of the lung tissue. Preferably, the detection method includes: measuring the resistance value of an in vitro lung model using a transmembrane resistance meter, and using the formula TEER = TΩ × A cm. 2 Calculate the transmembrane resistance, where T is the resistance and A is the area of ​​the in vitro lung model; (iv) Applying the test drug to a pulmonary fibrosis model or infecting a pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance value of the pulmonary fibrosis model; wherein, if the transmembrane resistance value of the pulmonary fibrosis model decreases significantly to a statistically significant degree after the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection can damage the cell barrier; if the transmembrane resistance value of the pulmonary fibrosis model increases significantly to a statistically significant degree after the addition of the test drug compared to before the addition, then the test drug can repair the cell barrier; if the change in the transmembrane resistance value of the pulmonary fibrosis model after the addition of the test drug or infection with the pathogen is not statistically significant compared to before the addition of the test drug or infection with the pathogen, then the test drug or pathogen infection has no significant effect on the integrity of the cell barrier; preferably, the detection method includes: using a transmembrane resistance meter to detect the resistance value of the pulmonary fibrosis model, using the formula TEER=TΩ×A cm. 2 Calculate the transmembrane resistance value, where T is the resistance value and A is the area of ​​the pulmonary fibrosis model; (v) Apply the drug to be tested to an in vitro lung model or infect the in vitro lung model with the pathogen, and detect changes in the barrier permeability of the in vitro lung model; preferably, the detection includes: adding a fluorescent molecule (e.g., FITC-Dextran) to the upper layer of the in vitro lung model and incubating it, detecting the amount of the fluorescent molecule permeating into the lower layer of the in vitro lung model, and calculating the apparent permeability coefficient. (vi) Apply the drug to be tested to an in vitro lung model or infect the in vitro lung model with the pathogen, and detect changes in cell activity in the in vitro lung model; wherein, the cell activity is, for example, cell proliferation activity; preferably, the detection method includes: adding MTT, CCK8 or CTG activity detection reagent to the in vitro lung model and incubating, and detecting chemiluminescence value; (vii) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the apoptosis of cells in the in vitro lung model is detected; wherein the detection method is, for example, immunofluorescence or flow cytometry; the biomarkers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL; (viii) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the changes in the content of inflammatory cytokines in the in vitro lung model are detected; wherein the inflammatory cytokines are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; the detection method includes ELISA, CBA multifactor detection, and Luminex multifactor detection; preferably, the detection method includes: adding sample culture medium to a culture container, incubating and washing, adding antibodies to the inflammatory cytokines to be tested, incubating and washing again, adding chromogenic solution and incubating, then adding stop solution and detecting OD value; (ix) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the lung tissue-related cell phenotypes or cell contents in the in vitro lung model are detected; wherein, the cell phenotypes are, for example, ciliated cell markers, goblet cell markers, AT1 cell markers, AT2 cell markers, club cell markers, and basal cell markers; the detection methods include: flow cytometry and immunofluorescence. (x) Apply the drug to be tested to an in vitro lung model or infect the in vitro lung model with the pathogen, and detect the level of cellular oxidative stress in the in vitro lung model; (xi) The drug to be tested is applied to an in vitro lung model or the pathogen is used to infect an in vitro lung model, and the expression changes of genes related to the process of pulmonary fibrosis in the in vitro lung model are detected; wherein, the genes related to pulmonary fibrosis are, for example, surfactant protein B gene, type II alveolar cell surface antigen gene, and matrix metalloproteinase MMP7 gene. (xii) Infect the lung in vitro model with the pathogen and detect the expression changes of the pathogen infection-related receptors in the lung in vitro model; (xiii) Infect the lung in vitro model with the pathogen, then apply the test drug to the lung in vitro model, and detect the inhibitory effect of the test drug on the pathogen; Wherein, the lung in vitro model is the lung in vitro model constructed by the construction method of any one of claims 1 to 7, or the lung in vitro model of claim 8; Preferably, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs; Preferably, the drug detection method and drug screening method include drug detection and / or drug screening for the treatment of pulmonary fibrosis.

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