Lung in-vitro model and construction method and application thereof

Abstract

The invention relates to a lung in-vitro model and a construction method and application thereof. The lung in-vitro model provided by the invention is good in stability and controllability and high in maturity, can better simulate functions and structural characteristics of pulmonary alveoli-capillary barriers, has the advantage of high simulation, and has a good application prospect in the aspects of disease simulation, drug detection, screening, evaluation, research and development and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an in vitro lung model and a construction method thereof, 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 Art

[0002] There are many known lung diseases. In order to study the occurrence and development mechanism of these diseases and screen corresponding drugs, it is necessary to construct lung models.

[0003] Lung models can mainly include animal models and in vitro models. Among them, animal models have disadvantages such as difficulty in sample acquisition, low success rate, and limited number of passages, while in vitro models can better overcome these disadvantages. However, the existing research on in vitro lung models mainly focuses on in vitro 2D culture. When cultured in culture dishes or slides (2D), it cannot well display cell structure, adhesion, mechanical transduction and soluble cytokine signaling, and cannot simulate the in vivo cell-cell / matrix interaction. Therefore, it is extremely urgent to find new lung models that can better simulate the in vivo lung microenvironment for the study of lung diseases and drug screening. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing an in vitro normal lung model. The in vitro lung model constructed by this construction method can better simulate the function and structural characteristics of the alveolar-capillary barrier, so as to be used for the research of lung diseases, drug screening, etc.

[0005] Specifically, in a first aspect, the present invention provides a method for constructing an in vitro lung model, comprising:

[0006] providing lung organoids and cell culture vessels;

[0007] Inoculating the lung organoids on the upper layer of the cell culture container, adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container, and culturing the lung organoids in a liquid-liquid mode;

[0008] The lung epithelial cell culture medium in the upper layer and the lower layer is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to perform the first stage of culturing the lung organoid in an air-liquid mode;

[0009] Endothelial cells are seeded in the lower layer of the cell culture container, and macrophages are seeded in the upper layer of the cell culture container and culture is carried out in the second stage of air-liquid mode to obtain an in vitro lung model.

[0010] In the present invention, the cell culture container is a culture container that can be divided into at least an upper layer and a lower layer (sometimes also called an upper chamber and a lower chamber) culture unit, so as to achieve cell migration or make different cells grow in different layers or even adhere to a membrane (e.g., a porous membrane) between the upper and lower layers according to needs. Accordingly, the obtained lung in vitro model can also be divided into an upper layer (i.e., an upper layer of the model) and a lower layer (i.e., a 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. Wherein, when a cell culture chip is used, the obtained lung in vitro model is also referred to as a lung chip (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 202211680988.1, 202222130269.4 or 202222130382.2, and these application documents are incorporated herein by reference in their entirety.

[0011] In the present invention, the lung organoids may be primary extracted lung organoids or differentiated lung organoids from induced pluripotent stem cells (IPS). The primary extraction may be extracted from lung tissue of a mammal, such as a human, domesticated animal, farm animal, zoo, sports field or pet animal, such as dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, cow, etc.

[0012] In the present invention, "liquid-liquid mode 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 mode 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.

[0013] In the construction method of the present invention, lung organoids are used instead of traditional cell lines to construct an in vitro lung model. The lung organoids are first cultured in a liquid-liquid mode and then in an air-liquid mode (i.e., the first stage of air-liquid mode culture), and then endothelial cells and macrophages are inoculated in sequence. The macrophages are cultured in the second stage of air-liquid mode. This helps to make the in vitro lung model more mature and improve its simulation.

[0014] In some embodiments, the lung organoid is a primary extracted lung organoid. The extraction can be performed using methods known in the art, preferably using the following steps:

[0015] (1) Washing: Wash the normal lung tissue with a buffer solution to remove the surface mucosa; the buffer solution preferably contains penicillin-streptomycin and gentamicin;

[0016] (2) Crushing: mince the lung tissue into minced meat, resuspend in buffer, let stand and discard the supernatant;

[0017] (3) Digestion: add lung primary tissue digestion solution, blow and mix, and then use a metal bath shaker at 37°C (other similar equipment can be used instead), shake and digest for 30 to 60 minutes, digest into small cell clusters, and finally add buffer to terminate digestion; the lung primary tissue digestion solution can use commercial lung primary tissue digestion solution, preferably, the lung primary tissue digestion solution contains collagenase and culture medium, wherein the collagenase includes: one or more of collagenase I, collagenase II, and collagenase IV, and the final concentration of any one of collagenase I, collagenase II, and collagenase IV in the lung primary digestion solution is 1 to 2 mg / mL, and the culture medium is DMEM or Advanced DMEM / F12;

[0018] (4) Resuspension: The digested cell suspension is filtered through a 100 μm cell mesh, and the cells are resuspended in lung organoid culture medium and counted; wherein the lung organoid culture medium can be a commercial lung organoid culture medium, such as the respiratory organoid expansion medium (Cat. No. A01-001) produced by Biogen, or the lung organoid culture kit (Cat. No. KLU0201) produced by Avid.

[0019] (5) Plating: Centrifuge, resuspend the lung organoids in extracellular matrix (Matrigel, BME, etc.), and plate. Add lung organoid culture medium and place in a 37°C incubator for culture.

[0020] In some embodiments, the cell culture container may be pretreated before inoculating various cells to facilitate cell attachment. The pretreatment method may be those known in the art, for example, pretreatment with a solution including an extracellular matrix, wherein the extracellular matrix may be Matrigel, collagen, fibronectin, etc., which are not limited here.

[0021] In some embodiments, before inoculation, the lung organoids are digested with an organoid passage digestion solution (trypsin, Tryple or Accutase cell digestion solution, etc.) to form single cells, and then lung organoid culture medium is added to terminate the digestion, and then inoculation is performed.

[0022] In some embodiments, in the step of inoculating the lung organoids on the upper layer of the cell culture container, the lung organoids are inoculated with 1×10 5 ~10×105 cells / cm 2 .

[0023] In some embodiments, in 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 commercial lung epithelial cell culture medium. In the present invention, the lung epithelial cell culture medium includes a lung organoid culture medium. In some specific embodiments, the lung epithelial cell culture medium may be, for example, a respiratory organoid expansion medium (item number A01-001) produced by Bogen, a lung organoid culture kit (item number KLU0201) produced by Avid, or an epithelial cell culture medium (PneumaCult TM -ExPlus Medium).

[0024] 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 to 300 μL of lung epithelial cell culture medium is added to the upper layer, and 800 μL to 1 mL of lung epithelial cell culture medium is added to the lower layer.

[0025] In some embodiments, the liquid-liquid mode culture time of the lung organoid is 3 to 7 days, such as 3 days, 4 days, 5 days, 6 days, 7 days or any time within this range. In some specific embodiments, the temperature of the liquid-liquid mode culture is 37° C. In some specific embodiments, during the liquid-liquid mode culture, the liquid is replaced every 3 days.

[0026] In some embodiments, when the lung organoids overgrow the upper layer, the lung epithelial cell culture medium in the upper layer and the lower layer is discarded.

[0027] 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.

[0028] In some embodiments, the first stage of gas-liquid mode culture of the lung organoid is 2 to 21 days, such as 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days or any time within this range. In some specific embodiments, the temperature of the first stage of gas-liquid mode culture is 37°C.

[0029] In some embodiments, endothelial cells are seeded in the lower layer of the cell culture container, and the endothelial cells are seeded at 0.5×10 5 ~5×105 cells / cm 2 .

[0030] In some embodiments, the endothelial cells are derived from umbilical vein endothelial cells, primary lung endothelial cells, or a lung endothelial cell line.

[0031] In some embodiments, in the step of inoculating endothelial cells in 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, wherein 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; for example, the lung epithelial cell culture medium and the endothelial cell culture medium are mixed in a ratio 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 commercial culture medium, such as the endothelial cell culture medium (item number #1001) produced by Sciencell. Preferably, the endothelial cells are digested, resuspended with the first mixed culture medium, the cell density is adjusted, and then inoculated. In some specific embodiments, 600-700 μL of the first mixed culture medium is added to the lower layer.

[0032] In some embodiments, in the step of inoculating macrophages on the upper layer of the cell culture container and performing the second-stage gas-liquid mode culture, the macrophages are inoculated at 0.1×10 5 ~5×10 5 cells / cm 2 In some specific embodiments, the macrophages are resuspended in a macrophage culture medium and the cell density is adjusted. The macrophage culture medium can be selected according to the specific source of the macrophages, such as the macrophage culture medium (Cat. No. 1921) produced by ScienCell.

[0033] In some embodiments, the macrophages are derived from PBMC-expanded macrophages, primary alveolar macrophages, passaged alveolar macrophages, or macrophages induced from THP-1 cell line.

[0034] In some embodiments, in the step of inoculating macrophages in the upper layer of the cell culture container and performing the second-stage gas-liquid mode culture, the macrophages are resuspended in macrophage culture medium, inoculated in the upper layer of the cell culture container and adhered to the wall, then the culture medium of the upper layer and the lower layer is discarded and the upper layer is kept dry, and a fresh first mixed culture medium is added to the lower layer to perform the second-stage gas-liquid mode culture, wherein 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. In some specific embodiments, 600-700 μL of fresh first mixed culture medium is added to the lower layer.

[0035] In some embodiments, the second stage gas-liquid mode culture time of the lung organoid is 1 to 5 days, such as 1 day, 2 days, 3 days, 4 days, 5 days or any time within the range.

[0036] In some embodiments, after the macrophages are inoculated into the upper layer of the cell culture container and cultured in the second stage of the gas-liquid mode, the step of inoculating immune cells into the lower layer of the cell culture container and culturing them is also included, so that the constructed in vitro lung model has immune function.

[0037] In some embodiments, the immune cells include PBMCs, T cells, B cells, NK cells.

[0038] In some embodiments, the immune cells are seeded at 0.5×10 5 ~1×10 6 cells / cm 2 .

[0039] In some embodiments, in the step of inoculating immune cells in the lower layer of the cell culture container and culturing, the immune cells are resuspended with 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 in a gas-liquid mode, and the second mixed culture medium is obtained by mixing the immune cell culture medium and the first mixed culture medium in a volume ratio of (0.5-3.5):1; for example, the immune cell culture medium and the first mixed culture medium are mixed in a ratio of 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1 or 3.5:1. Wherein, the immune cell culture medium can be a commercially available immune cell culture medium, for example, it can be a PBMC culture medium, using a commercial PBMC culture medium, such as SuperCulture L100 lymphocyte serum-free culture medium (Da Kewei, catalog number #6911011) supplemented with 5-10% SuperGrow cell culture additives (Da Kewei, catalog number #6122011) and 1-5‰ IL-2.

[0040] In some embodiments, in the step of inoculating the immune cells into the lower layer of the cell culture container and culturing them, the culturing time is 1 to 3 days, such as 1 day, 2 days, 3 days or any time within the range.

[0041] In a second aspect, the present invention provides an in vitro lung model, which is constructed by the construction method of the in vitro lung model provided in the first aspect.

[0042] In a third aspect, the present invention provides a culture medium combination, comprising a lung epithelial cell culture medium and a first mixed culture medium, and optionally a second mixed culture medium;

[0043] The first mixed culture medium is obtained by mixing lung epithelial cell culture medium and endothelial cell culture medium in 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 in a volume ratio of (0.5-3.5):1.

[0044] In some embodiments, the culture medium combination is used in the construction method provided by the present invention, or for the construction of the lung in vitro model provided by the present invention, including for the culture of lung organoids, liquid-liquid mode culture, first stage air-liquid mode culture, second stage air-liquid mode, and optionally third stage air-liquid mode culture.

[0045] In some preferred embodiments, the lung epithelial cell culture medium is used for the culture of the lung organoids, the liquid-liquid model culture and / or the first stage air-liquid model culture.

[0046] In some preferred embodiments, the first mixed culture medium is used in the second stage gas-liquid mode.

[0047] In some preferred embodiments, the second mixed culture medium is used for the third stage gas-liquid mode culture.

[0048] In a fourth aspect, the present invention provides a use of the lung in vitro model constructed by the construction method provided in the first aspect, or the lung in vitro model described in the second aspect in the following (a) or (b);

[0049] (a) drug testing and / or drug screening;

[0050] (b) Detect the effects of pathogen infection on lung tissue.

[0051] In some embodiments, the drug includes a small molecule drug, an antibody drug, an ADC drug, or an immune drug.

[0052] In some embodiments, the pathogens include bacteria, viruses, fungi, parasites, etc.

[0053] The lung in vitro model provided by the present invention can be used for efficacy testing and drug mechanism exploration of drugs for treating lung diseases. The response of the lung in vitro model to drugs can be evaluated by changes in cell marker expression, changes in cell (such as alveolar cell) ratios, cell activity, inflammatory factor secretion, changes in immune cell typing, cell barrier function, etc., and then used for drug research and development. For example, drugs can be added to the upper layer or the lower layer of the model to detect whether the drugs will cause pulmonary fibrosis; after the pathogen infects the lung in vitro model (upper layer or lower layer), the effect of the pathogen on the lung tissue can be confirmed by indicator detection. The following are some examples (i) to (xii) of the use of the lung in vitro model described in the present invention in (a) or (b). It should be understood that the applicable methods of the lung in vitro model described in the present invention are not limited to these:

[0054] (i) Changes in the expression of pulmonary fibrosis markers: The drug to be tested is applied to the lung in vitro model or the pathogen is allowed to infect the lung in vitro model, and the expression changes of pulmonary fibrosis markers in the lung in vitro model are detected; wherein the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin. In some specific embodiments, it includes: fixing the lung in vitro model with 4% paraformaldehyde at room temperature for 30 minutes, removing the paraformaldehyde, and washing the sample with PBS, permeabilizing the sample with 0.1% Triton-X-100 and blocking the sample with 5% BSA, respectively adding primary antibodies α-SMA, type I collagen, and fibronectin, incubating at 4°C overnight, adding secondary antibodies, incubating at room temperature for 1 hour, performing nuclear staining with DAPI for 15 minutes, washing with PBS, and imaging using a confocal microscope.

[0055] (ii) Flow cytometry detection of immune cell typing changes: administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the typing changes and / or dynamic immune response process of the cells in the in vitro lung model by flow cytometry; wherein the detection markers of the 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 typing changes of the cells include the typing changes of macrophages and / or the typing changes of immune cells, and the typing changes of the macrophages are, for example, the typing changes of M1 macrophages and the typing changes of M2 macrophages; the typing changes of the immune cells are, for example, the typing changes of T cells, the typing changes of B cells, the typing changes of NK cells, and the typing changes of innate lymphocytes. In some specific embodiments, the flow cytometry includes: changes in the typing of M1 macrophages and M2 macrophages in the upper cells of the model, and the flow cytometry indicators include but are not limited to CD45, CD80, HLA-DR, CD163, CD68, CD206, etc.; or changes in the typing of immune cells such as T cells, B cells, NK cells, innate lymphocytes, etc. in the lower cells of the model, and the flow cytometry indicators include but are not limited to CD45, CD3, CD4, CXCR3, CCR4 (Th1, Th2 cells), CD19 (B cells), Lin, CD127, CD94, CD117, CRTH2 (ILC cells), etc. The operation steps are as follows: collect cells, digest into single cells with Tryple, Trypsin or Accutas, and resuspend cells in flow buffer. Use CD45, CD80, HLA-DR, CD163, CD3, CD4 and other antibodies for surface staining, 4°C, 30min. Add cell fixative and fix at room temperature in the dark for 10min. After centrifugation and washing with PBS, resuspend the cells with a membrane permeabilization reagent and incubate at room temperature for 10-15 minutes. Incubate the antibodies that need to be permeabilized, such as CD68 and CD206, at room temperature in the dark for 30 minutes. After incubation, wash twice with a membrane permeabilization reagent, add 200-500 μL of flow cytometry buffer to resuspend the cells, and detect using a flow cytometer.

[0056] (iii) detecting the transmembrane resistance of the model to characterize the cell barrier function of the model: administering the drug to be tested to the lung in vitro model or infecting the lung in vitro model with the pathogen, and detecting the transmembrane resistance of the lung in vitro model; wherein, before adding the drug to be tested or before being infected with the pathogen, the normal transmembrane resistance of the lung in vitro model is 200 to 1500 Ω·cm 2When the drug to be tested is added or the pathogen is infected, the transmembrane resistance value is significantly reduced to a statistically significant difference compared with the normal transmembrane resistance value, then the drug to be tested or the pathogen infection has damaged the cell barrier function of the lung tissue. In some specific embodiments, the following steps are included: take out the transmembrane resistance meter, plug in the power supply, assemble the monitoring device, place the chopstick detection head in PBS for 15 minutes; after the instrument value stabilizes at 0Ω, fill the model with PBS inside and outside; insert the chopstick detection head into the model, and record the value after the display value stabilizes; data processing: TEER = TΩ × A cm 2 , where T is the resistance value and A is the area of ​​the lung in vitro model.

[0057] (iv) Detecting changes in barrier permeability and characterizing the cell barrier function of the model: applying the drug to be tested to the lung in vitro model or infecting the pathogen to the lung in vitro model, and detecting changes in the barrier permeability of the lung in vitro model. In some specific embodiments, it includes: adding fluorescent permeable molecules, such as FITC-Dextran (molecular weight 10kDa, 30KDa, 70KDa), etc. to the upper layer of the model, incubating at different time points, using an ELISA instrument to detect the mass of the fluorescent molecules permeated in the lower layer of the model, and calculating the apparent permeability coefficient to reflect the changes in the barrier permeability of the model. The operation steps are as follows: discard all the culture medium in the well, add 50-100μM FITC-Dextran solution to the upper layer, add 400μL of fresh culture medium to the lower layer, and incubate in a 37°C incubator; collect 200μL of the lower culture medium at 2h, 4h, 8h, and 24h of incubation respectively. 200 μL of culture medium was taken out each time, and 200 μL of culture medium was added to each well to continue culturing; the fluorescence value in the culture medium was detected using an ELISA instrument; and the following calculation was performed: Papp = (dQ / dt) / (A*C0); wherein Papp is the apparent permeability coefficient, A is the membrane area of ​​the membrane chip, C0 is the initial concentration of the fluorescent dye, and dQ / dt is the amount of fluorescent dye that passes through per unit time.

[0058] (v) Detecting the activity of model cells: administering the drug to be tested to the in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting changes in the cell activity in the in vitro lung model; wherein the cell activity is, for example, cell proliferation activity. In some specific embodiments, the activity of model cells is detected using methods such as MTT, CCK8, and CTG. Taking CTG detection as an example: add an equal amount of CTG activity detection reagent stock solution to the model as that of the culture medium, mix by pipetting for 5 minutes, incubate at room temperature for 30 minutes, and use an enzyme marker to detect its chemiluminescence value.

[0059] (vi) Detecting cell apoptosis in the model: administering the drug to be tested to the in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting cell apoptosis in the in vitro lung model; wherein the detection method is, for example, immunofluorescence or flow cytometry; and the markers detected by flow cytometry are, for example, Caspase 3, AnnexinV, Live / Dead, and TUNEL.

[0060] (vii) Detecting cytokine content: administering the drug to be tested to the lung in vitro model or infecting the pathogen to the lung in vitro model, and detecting changes in the content of inflammatory cytokines in the lung in vitro model; wherein the inflammatory factors are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, CCL18. In some specific embodiments, detection is performed using methods such as ELISA, CBA multifactor detection, and Luminex multifactor detection. Take ELISA detection of inflammatory factor TNF-α content as an example: prepare 0, 11.7, 23.4, 46.9, 93.8, 187.5, 375 and 750pg / mL standard solutions, add 100μL standard solution and sample solution to each well, incubate at room temperature for 1h, wash each well with 300μL cleaning solution 4 times, 2min each time, add 100μL antibody of inflammatory cytokine to be tested to each well, incubate at room temperature for 30min, wash each well with 300μL cleaning solution 4 times, 2min each time, add 100μL color development solution to each well, incubate at room temperature for 20min, add 50μL stop solution to each well, and detect the OD value at 450nm within 10min.

[0061] (viii) Detecting cell types and expressions related to lung tissue: administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the cell phenotype or cell content related to lung tissue in the in vitro lung model; wherein the cell phenotype is, for example, α-tubulin (ciliary cell marker), MUC5AC (goblet cell marker), AQP5 (AT1 cell marker), SFTPC (AT2 cell marker), CC10 (club cell marker), and KRT5 (basal cell marker).

[0062] (ix) Detecting the level of oxidative stress: administering the drug to be tested to the in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting the level of cellular oxidative stress in the in vitro lung model. In some specific embodiments, the method includes using a ROS oxidative stress detection kit to detect the level of ROS / RNS and evaluate the regulation of oxidative stress by the drug. For example, using Invitrogen TMThe CellROX kit is used to test the model. CellROX is a fluorescent probe that can measure generalized oxidative stress in cells using fluorescence microscopy, high-content imaging systems, microplate fluorometers, or flow cytometry platforms. The dye is non-fluorescent in the reduced state and emits bright green, orange, or deep red fluorescence when oxidized.

[0063] (x) Detecting the expression of genes related to the development of pulmonary fibrosis: administering the drug to be tested to the lung in vitro model or infecting the pathogen to the lung in vitro model, and detecting the expression changes of genes related to pulmonary fibrosis in the lung in vitro model; 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. In some specific embodiments, RNA-seq or RT-PCR is included to detect the changes in the transcription levels of genes related to pulmonary fibrosis.

[0064] (xi) Detecting the expression of receptors associated with pathogen infection: the pathogen is allowed to infect the in vitro lung model, and the expression changes of the receptors associated with the pathogen infection in the in vitro lung model are detected. In some specific embodiments, the in vitro lung model is infected with avian influenza virus, and the expression of influenza virus receptor α-2,6 linked sialic acid is detected using a kit.

[0065] (xii) Detecting the anti-pathogen effect of the drug: the pathogen is infected with the in vitro lung model, and then the drug to be tested is applied to the in vitro lung model to detect the inhibition of the pathogen by the drug to be tested. In some specific embodiments, the anti-viral effect of the drug to be tested is detected at different time points after the pathogen is infected with the in vitro lung model. Alternatively, the number of viral RNA copies and the 50% tissue culture infection dose are determined by RT-PCR after the model is infected with the virus.

[0066] In a fifth aspect, 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:

[0067] (i) Applying the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting the expression changes of pulmonary fibrosis markers in the lung in vitro model; wherein the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin. In some embodiments, the detection method is an immunofluorescence detection method; preferably, the detection method comprises: after fixing, washing, and sealing the lung in vitro model, adding a primary antibody of a pulmonary fibrosis marker and incubating, then adding a secondary antibody and incubating, and obtaining the expression change results of the pulmonary fibrosis marker through staining and imaging;

[0068] (ii) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the phenotypic changes and / or dynamic immune response process of cells in the in vitro lung model by flow cytometry. In some embodiments, the detection markers of the 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 typing changes of the cells include typing changes of macrophages and / or typing changes of immune cells, and the typing changes of the macrophages are, for example, typing changes of M1 macrophages and typing changes of M2 macrophages; the typing changes of the immune cells are, for example, typing changes of T cells, typing changes of B cells, typing changes of NK cells, and typing changes of innate lymphocytes; preferably, the detection method comprises: collecting cells in the lung in vitro model and resuspending them with a flow buffer, staining the surface with an antibody for the detection marker, fixing, centrifuging, and washing, adding a flow buffer to resuspend the cells, and detecting the fluorescence signal with a flow cytometer;

[0069] (iii) applying the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting the transmembrane resistance value of the lung in vitro model; wherein the normal transmembrane resistance value of the lung in vitro model before adding the drug to be tested or before being infected by the pathogen is 200 to 1500 Ω·cm 2 When the drug to be tested is added or the lung tissue is infected by a pathogen, the transmembrane resistance value significantly decreases to a statistically significant difference compared with the normal transmembrane resistance value, then the drug to be tested or the pathogen infection damages the cell barrier function of the lung tissue. In some embodiments, the detection method includes: using a transmembrane resistance meter to detect the resistance value of the lung in vitro 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 lung in vitro model;

[0070] (iv) administering the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting changes in the barrier permeability of the lung in vitro model. In some embodiments, the detection comprises: adding fluorescent molecules (such as FITC-Dextran) to the upper layer of the lung in vitro model and incubating, detecting the amount of the fluorescent molecules permeated into the lower layer of the lung in vitro model, and calculating the apparent permeability coefficient;

[0071] (v) administering the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting changes in cell activity in the lung in vitro model. In some embodiments, the cell activity is, for example, cell proliferation activity; preferably, the detection method comprises: adding MTT, CCK8 or CTG activity detection reagent to the lung in vitro model and incubating, and detecting the chemiluminescence value;

[0072] (vi) administering the drug to be tested to the in vitro lung model or infecting the pathogen to the in vitro lung model, and detecting apoptosis in the in vitro lung model. In some embodiments, the detection method is, for example, immunofluorescence or flow cytometry; the markers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, TUNEL;

[0073] (vii) Apply the drug to be tested to the lung in vitro model or allow the pathogen to infect the lung in vitro model, and detect the changes in the content of inflammatory cytokines in the lung in vitro model. In some embodiments, the inflammatory factors 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: preparing a gradient concentration of inflammatory cytokine standard solution, adding the standard solution and sample culture medium to a culture container, incubating and washing, adding an antibody to the inflammatory cytokine to be tested, and then incubating and washing, adding a color developing solution and incubating, and then adding a stop solution and detecting the OD value;

[0074] (viii) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the cell phenotype or cell content associated with the lung tissue in the in vitro lung model. In some embodiments, the cell phenotype is, for example, α-tubulin, MUC5AC, AQP5, SFTPC, CC10, or KRT5;

[0075] (ix) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the level of cellular oxidative stress in the in vitro lung model;

[0076] (x) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the expression changes of pulmonary fibrosis-related genes in the in vitro lung model. 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;

[0077] (xi) allowing the pathogen to infect an in vitro lung model, and detecting changes in the expression of receptors associated with the pathogen infection in the in vitro lung model;

[0078] (xii) allowing the pathogen to infect an in vitro lung model, then administering the drug to be tested to the in vitro lung model, and detecting the inhibitory effect of the drug to be tested on the pathogen;

[0079] Wherein, the in vitro lung model is the in vitro lung model constructed by the construction method of the first aspect, or the in vitro lung model described in the second aspect.

[0080] In some embodiments, the drug includes a small molecule drug, an antibody drug, an ADC drug, or an immune drug.

[0081] First, the lung in vitro model provided by the present invention replaces the traditional cell line with lung organoids to construct the lung in vitro model, which has a shorter experimental cycle than the animal model, is conducive to reducing the experimental cost, simplifies the difficulty of operation, and enhances the stability and controllability of the model; secondly, the construction method provided by the present invention is inoculated in the order of lung organoids, endothelial cells, macrophages and optionally immune cells, and is first cultured in a liquid-liquid mode and then in a gas-liquid mode (including the first stage gas-liquid mode culture, the second stage gas-liquid mode culture and the optional third stage gas-liquid mode culture), which is not only conducive to making the lung in vitro model have a higher degree of maturity, but also can better simulate the function and structural characteristics of the alveolar-capillary barrier, and improve its simulation. In addition, the lung in vitro model provided by the present invention includes lung organoids, endothelial cells, macrophages and optionally immune cells, which can well simulate these cell-related microenvironments and cell-to-cell interactions, and provide a good platform for disease simulation, drug detection, screening, evaluation and research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The transmembrane resistance values ​​of the lung in vitro model of the present invention are shown.

[0083] Figure 2 Bright field images and fluorescent staining images of the in vitro lung model of the present invention are shown.

[0084] Figure 3 The results of the detection of the expression of pulmonary fibrosis markers in Example 3 of the present invention are shown.

[0085] Figure 4 The results of ELISA in Example 3 of the present invention for detecting the secretion level of the inflammatory factor TNF-α are shown.

[0086] Figure 5 The results of detecting cell activity of the lung in vitro model in Example 4 of the present invention are shown. DETAILED DESCRIPTION

[0087] The materials, methods and examples described herein are illustrative only and are not intended to be limiting. Other features, objects and advantages of the present invention will be apparent from the specification, drawings and claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art.

[0088] Example 1

[0089] This example provides a specific construction process of the lung in vitro model:

[0090] Primary extraction and liquid-liquid culture of lung organoids:

[0091] (1) Cleaning: Use primary tissue cleaning solution to clean normal human lung tissue and remove the surface mucosa, wherein the primary tissue cleaning solution is a buffer solution to which penicillin-streptomycin and gentamicin are added.

[0092] (2) Disintegration: Chop the tissue into minced meat, resuspend in buffer, let stand and discard the supernatant.

[0093] (3) Digestion: Add lung primary tissue digestion solution, mix by blowing, and then use a metal bath shaker at 37°C for 30-60 minutes to digest into small cell clusters, and finally add buffer to terminate digestion. The lung primary tissue digestion solution can be a commercially available lung primary tissue digestion solution. In this embodiment, the lung primary tissue digestion solution contains collagenase and culture medium, wherein the collagenase is collagenase II, the final concentration is 1-2 mg / mL, and the culture medium is DMEM or Advanced DMEM / F12.

[0094] (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 cell density was adjusted to 5 × 10 5 / mL; Among them, lung organoid culture medium: produced by Avid, the name is lung organoid culture kit, product number: KLU0201.

[0095] (5) Lung organoid plating culture: Centrifuge and remove the supernatant, use extracellular matrix Matrigel to resuspend the lung organoids, 30 μL gel droplets per well, and plate the lung organoids on a 24-well plate. After the gel is formed, add lung organoid culture medium and culture in a 37°C incubator for 7 days.

[0096] (6) Digestion of lung organoids: After the lung organoids have grown for 7 days, use Tryple, a lung organoid digestion solution, to digest the lung organoids into single cells, and then add lung organoid culture medium to terminate the digestion.

[0097] (7) Inoculation: Centrifuge and remove the supernatant, resuspend in lung epithelial cell culture medium, and adjust the density to 1×106 / mL. The chip membrane area is 0.33cm 2 , inoculated with 2×10 5 cells, and the cell density was 6×10 5 Pieces / cm 2 . The lung organoids were inoculated on the upper layer of the membrane. 1 mL of lung epithelial cell culture medium (Respiratory Organoid Expansion Medium (Cat. No. A01-001) produced by Bogen, lung organoid culture kit (Cat. No. KLU0201) produced by Avid, or epithelial cell culture medium (PneumaCult TM -Ex Plus Medium)) and cultured in a 37°C incubator in liquid-liquid mode.

[0098] (8) Change the medium every 3 days and culture for 5 days.

[0099] Air-liquid culture:

[0100] (1) When the lung organoids have grown all over the upper layer of the membrane, discard the culture medium on the upper and lower layers of the membrane and add 600 μL of lung epithelial cell culture medium to the lower layer of the membrane.

[0101] (2) Cultivation in the gas-liquid mode for 2 days (first stage gas-liquid mode cultivation).

[0102] Seeding of endothelial cells:

[0103] (1) Digest endothelial cells and resuspend the cells in the first mixed culture medium (lung epithelial cell culture medium: endothelial cell culture medium volume ratio 2:1). Count and adjust the endothelial cell density to 6×10 5 / mL. The chip membrane area is 0.33cm 2 , inoculated with 3×10 4 Endothelial cells, with a cell density of 0.9×10 5 Pieces / cm 2 The endothelial cell culture medium is the endothelial cell culture medium produced by Sciencell (Cat. No. #1001).

[0104] (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 mixed in a volume ratio of 2:1) to the lower chamber.

[0105] Inoculation of macrophages:

[0106] (1) Collect macrophages and resuspend them in macrophage culture medium (ScienCell, #1921). Count and adjust the cell density to 1×10 5 / mL. The chip membrane area is 0.33cm2 , inoculated with 1×10 4 Macrophages, cell density was 0.3×10 5 Pieces / cm 2 .

[0107] (2) The macrophages were cultured overnight, and then the macrophage culture medium on the membrane was discarded.

[0108] (3) The culture medium under the membrane is replaced (first mixed cell culture medium (lung epithelial cell culture medium and endothelial cell culture medium mixed in a volume ratio of 2:1)).

[0109] (4) Continue the gas-liquid mode culture for 2 days (second stage gas-liquid mode culture).

[0110] Vaccination of immune cells (if immune function is not required, this step can be omitted):

[0111] (1) Collect PBMCs and resuspend them in the second mixed culture medium (immune cell culture medium and first mixed culture medium mixed in a volume ratio of 2:1). Discard the culture medium under the membrane. The bottom area of ​​the chip lower chamber is 2 cm 2 , inoculated with 2×10 5 PBMCs, with a cell density of 1×10 5 Pieces / cm 2 Among them, for example, a commercial PBMC culture medium is used, such as SuperCulture L100 lymphocyte serum-free culture medium (Dakoway, catalog number 6122011) supplemented with 5-10% SuperGrow cell culture supplement (Dakoway, catalog number 6122011) and 1-5‰ IL-2.

[0112] #6911011).

[0113] (2) Continue the gas-liquid mode culture for 1 day (the third stage of gas-liquid mode culture).

[0114] Model detection:

[0115] (1) Use a transmembrane resistance meter to measure the transmembrane resistance of the model. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that the resistance value of the lung in vitro model constructed in this embodiment is 550-600Ω·cm 2 .

[0116] (2) Immunofluorescence detection of the expression of E-cadherin, endothelial cadherin (VE-Cadherin), AQP5 in type I alveolar cells, and SFTPB in type II alveolar cells in lung organoids. The results are as follows Figure 2 As shown. Figure 2It can be seen that the cells of the in vitro lung model constructed in this example are tightly connected and express markers related to lung tissue.

[0117] Example 2

[0118] In this example, ADC drug side effects were detected on the lung in vitro model constructed in Example 1, and the steps and results were as follows:

[0119] (1) Drug testing was performed on an in vitro lung model. A control group, an ADC drug group, and a payload group were set up. The first mixed culture medium was added to the lower layer of the chip in the control group, a culture medium containing 50 μg / mL Trastuzumab deruxtecan was added to the lower layer of the chip in the ADC drug group, and a culture medium containing 50 μg / mL ADC drug-linker conjugate Deruxtecan was added to the lower layer of the chip in the payload group.

[0120] (2) Drug treatment for 5 days.

[0121] (3) After drug treatment, immunofluorescence staining was used to detect the expression of α-SMA and type I collagen in the model, and photos were taken using a fluorescence microscope or confocal microscope. The average fluorescence intensity and the total fluorescence intensity were calculated. The expression of pulmonary fibrosis markers in the control group, ADC drug group, and Payload group was compared to see if there was any difference.

[0122] (4) ELISA was used to detect the secretion levels of inflammatory factors TNF-α and IL-6.

[0123] Example 3

[0124] In this example, the side effects of bleomycin were detected on the lung in vitro model constructed in Example 1, and the steps and results were as follows:

[0125] (1) Drug testing was performed 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 lower layer of the chip in the control group, the first mixed culture medium containing 1 μg / mL bleomycin was added to the lower layer of the chip in the low-concentration bleomycin group, and the first mixed culture medium containing 10 μg / mL bleomycin was added to the lower layer of the chip in the high-concentration bleomycin group.

[0126] (2) Drug treatment for 3 days.

[0127] (3) After drug treatment, immunofluorescence staining was used to detect the expression of α-SMA and type I collagen in the model, and photos were taken using a fluorescence microscope or confocal microscope. The average fluorescence intensity and the total fluorescence intensity 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.

[0128] (4) ELISA was used to detect the secretion level of inflammatory factor TNF-α.

[0129] The results are as follows Figure 3 and Figure 4 shown.

[0130] pass Figure 3 It can be seen that there was no expression of α-SMA and type I collagen in the control group, the low-concentration bleomycin group could cause the expression of α-SMA and type I collagen, and the lungs showed fibrosis; the high-concentration bleomycin group had high expression of α-SMA and type I collagen, and severe lung fibrosis.

[0131] pass Figure 4 It can be seen that there is no secretion of TNF-α in the control group, while both the low-concentration bleomycin group and the high-concentration bleomycin group can cause the secretion of TNF-α, and the higher the bleomycin concentration, the higher the secretion level of the inflammatory factor TNF-α.

[0132] Example 4

[0133] In this example, the side effects of nintedanib were detected on the lung in vitro model constructed in Example 1, and the steps and results were as follows:

[0134] (1) Drug testing was performed 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 lower layer of the chip in the control group, the first mixed culture medium containing 1 μM nintedanib was added to the lower layer of the chip in the low-concentration nintedanib group, and the first mixed culture medium containing 10 μM nintedanib was added to the lower layer of the chip in the high-concentration nintedanib group.

[0135] (2) Drug treatment for 3 days.

[0136] (3) After drug treatment, the CTG method was used to detect the activity of the model cells to explore the effect of nintedanib on cell activity. The control group, the low-concentration nintedanib group, and the high-concentration nintedanib group were compared to see whether there was any difference in cell activity.

[0137] The results are as follows Figure 5 As shown. Figure 5 It can be seen that there is no significant difference in activity among the control group, the low-concentration nintedanib group and the high-concentration nintedanib group, indicating that the nintedanib drug does not affect cell activity.

Claims

1. A method for constructing an in vitro lung model, comprising: providing lung organoids and cell culture vessels; Inoculating the lung organoids on the upper layer of the cell culture container, adding lung epithelial cell culture medium to the upper and lower layers of the cell culture container, and culturing the lung organoids in a liquid-liquid mode; The lung epithelial cell culture medium in the upper layer and the lower layer is discarded, the upper layer is kept dry, and fresh lung epithelial cell culture medium is added to the lower layer to perform the first stage of culturing the lung organoid in an air-liquid mode; Endothelial cells are seeded in the lower layer of the cell culture container, and macrophages are seeded in the upper layer of the cell culture container and culture is carried out in the second stage of air-liquid mode to obtain an in vitro lung model.

2. The construction method according to claim 1, wherein: The time for culturing the lung organoids in the liquid-liquid mode is 3 to 7 days; the time for culturing the lung organoids in the first-stage air-liquid mode is 2 to 21 days; and the time for culturing the lung organoids in the second-stage air-liquid mode is 1 to 5 days.

3. The construction method according to claim 1 or 2, wherein: Based on the inoculation area, the lung organoids were inoculated with 1×10 5 ~10×10 5 cells / cm 2 ; Calculated by the inoculation area, the endothelial cells were inoculated with 0.5×10 5 ~5×10 5 cells / cm 2 ; Calculated by the inoculation area, the macrophages were inoculated with 0.1×10 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 PBMC-expanded macrophages, primary alveolar macrophages, passaged alveolar macrophages, or macrophages induced by THP-1 cell line.

4. The construction method according to any one of claims 1 to 3, wherein: In the step of inoculating endothelial cells in 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, wherein the first mixed culture medium is obtained by mixing the lung epithelial cell culture medium and the endothelial cell culture medium in a volume ratio of (0.5-3.5):1; In the step of inoculating macrophages in the upper layer of the cell culture container and performing a second-stage gas-liquid mode culture, after the macrophages adhere to the wall, the culture medium in the upper layer and the lower layer is discarded and the upper layer is kept dry, and a fresh first mixed culture medium is added to the lower layer to perform a second-stage gas-liquid mode culture, wherein 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.

5. The construction method according to any one of claims 1 to 4, wherein: After the macrophages are inoculated in the upper layer of the cell culture container and cultured in the second stage of gas-liquid mode, the method further includes inoculating immune cells in the lower layer of the cell culture container and culturing them; Preferably, the immune cells include PBMC, T cells, B cells, and NK cells; Preferably, the immune cells are inoculated at 0.5×10 5 ~1×10 6 cells / cm 2 ; Preferably, in the step of inoculating the immune cells in the lower layer of the cell culture container and culturing, the immune cells are resuspended with 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 of gas-liquid mode culture, wherein the second mixed culture medium is obtained by mixing the immune cell culture medium and the first mixed culture medium in a volume ratio of (0.5-3.5):1; More preferably, the gas-liquid mode culture time of the third stage is 1 to 3 days.

6. The construction method according to any one of claims 1 to 5, wherein: The cell culture container is a Transwell or a cell culture chip, such as a membrane chip or a barrier chip.

7. An in vitro lung model constructed by the construction method according to any one of claims 1 to 6.

8. A culture medium combination comprising a lung epithelial cell culture medium and a first mixed culture medium, and optionally a second mixed culture medium; in, The first mixed culture medium is obtained by mixing a lung epithelial cell culture medium and an endothelial cell culture medium in a volume ratio of (0.5-3.5):1; The second mixed culture medium is obtained by mixing the immune cell culture medium and the first mixed culture medium in a volume ratio of (0.5-3.5):1; Preferably, the culture medium combination is used for the construction method according to any one of claims 1 to 6, or for the construction of the lung in vitro model according to claim 7, including culture for lung organoids, liquid-liquid mode culture, first stage air-liquid mode culture, second stage air-liquid mode, and optionally third stage air-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.

9. Use of the lung in vitro model constructed by the construction method according to any one of claims 1 to 6, or the lung in vitro model according to claim 7 in the following (a) or (b); (a) drug detection and / or drug screening; preferably, the drug includes small molecule drugs, antibody drugs, ADC drugs, and immune drugs; (b) Detect the effects of pathogen infection on lung tissue.

10. The use according to claim 9, wherein Said (a) and (b) include at least one of the following (i) to (xii): (i) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the expression changes of pulmonary fibrosis markers in the in vitro lung model; wherein the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin; (ii) applying the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the typing changes and / or dynamic immune response process of cells in the in vitro lung model by flow cytometry; wherein the detection markers of the flow cytometry are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, CRTH2; the typing changes of the cells include the typing changes of macrophages and / or the typing changes of immune cells, and the typing changes of the macrophages are, for example, the typing changes of M1 macrophages and the typing changes of M2 macrophages; the typing changes of the immune cells are, for example, the typing changes of T cells, the typing changes of B cells, the typing changes of NK cells, and the typing changes of innate lymphocytes; (iii) applying the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting the transmembrane resistance value of the lung in vitro model; wherein the normal transmembrane resistance value of the lung in vitro model before adding the drug to be tested or before being infected by the pathogen is 200 to 1500 Ω·cm 2 When the drug to be tested is added or the lung tissue is infected by a pathogen, the transmembrane resistance value significantly decreases to a statistically significant difference compared with the normal transmembrane resistance value, then the drug to be tested or the pathogen infection has damaged the cell barrier function of the lung tissue; (iv) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting changes in the barrier permeability of the in vitro lung model; (v) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting changes in cell activity in the in vitro lung model; wherein the cell activity is, for example, cell proliferation activity; (vi) administering the drug to be tested to the in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting cell apoptosis in the in vitro lung model; wherein the detection method is, for example, immunofluorescence or flow cytometry; and the markers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL; (vii) administering the drug to be tested to the in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting changes in the content of inflammatory cytokines in the in vitro lung model; wherein the inflammatory factors are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and CCL18; (viii) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the cell phenotype or cell content related to the lung tissue in the in vitro lung model; 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; (ix) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the level of cellular oxidative stress in the in vitro lung model; (x) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the expression changes of genes related to the process of pulmonary fibrosis in the in vitro lung model; 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; (xi) allowing the pathogen to infect the in vitro lung model, and detecting changes in the expression of receptors associated with the pathogen infection in the in vitro lung model; (xii) allowing the pathogen to infect the in vitro lung model, then administering the drug to be tested to the in vitro lung model, and detecting the inhibitory effect of the drug to be tested on the pathogen.

11. A method for drug detection, drug screening and / or detection of the effects of pathogen infection on lung tissue, comprising: (i) applying the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting the expression changes of pulmonary fibrosis markers in the lung in vitro model; wherein the pulmonary fibrosis markers are, for example, α-SMA, type I collagen, and fibronectin; preferably, the detection method is an immunofluorescence detection method; more preferably, the detection method comprises: after fixing, washing, and sealing the lung in vitro model, adding a primary antibody of the pulmonary fibrosis marker and incubating, then adding a secondary antibody and incubating, and obtaining the expression change results of the pulmonary fibrosis marker by staining and imaging; (ii) administering the drug to be tested to the in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting the typing changes and / or dynamic immune response process of cells in the in vitro lung model by flow cytometry; wherein the detection markers of the flow cytometry are, for example, CD45, CD80, HLA-DR, CD163, CD68, CD206, CD45, CD3, CD4, CXCR3, CCR4, CD19, Lin, CD127, CD94, CD117, CRTH2; the typing changes of the cells include the typing of macrophages; The typing change of the macrophages is, for example, a typing change of M1 macrophages and a typing change of M2 macrophages; the typing change of the immune cells is, for example, a typing change of T cells, a typing change of B cells, a typing change of NK cells, and a typing change of innate lymphocytes; preferably, the detection method comprises: collecting cells in the lung in vitro model and resuspending them with a flow cytometric buffer, staining with an antibody for detecting a marker, fixing, centrifuging, washing, adding a flow cytometric buffer to resuspend the cells, and detecting the fluorescence signal with a flow cytometer; (iii) applying the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting the transmembrane resistance value of the lung in vitro model; wherein the normal transmembrane resistance value of the lung in vitro model before adding the drug to be tested or before being infected by the pathogen is 200 to 1500 Ω·cm 2 When the drug to be tested is added or the lung tissue is infected by a pathogen, the transmembrane resistance value is significantly reduced to a statistically significant difference compared with the normal transmembrane resistance value, then the drug to be tested or the pathogen infection has damaged the cell barrier function of the lung tissue; preferably, the detection method comprises: using a transmembrane resistance meter to detect the resistance value of the lung in vitro 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 lung in vitro model; (iv) administering the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting changes in the barrier permeability of the lung in vitro model; preferably, the detection comprises: adding fluorescent molecules (such as FITC-Dextran) to the upper layer of the lung in vitro model and incubating, detecting the amount of the fluorescent molecules permeated in the lower layer of the lung in vitro model, and calculating the apparent permeability coefficient; (v) administering the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting changes in cell activity in the lung in vitro model; wherein the cell activity is, for example, cell proliferation activity; preferably, the detection method comprises: adding MTT, CCK8 or CTG activity detection reagent to the lung in vitro model and incubating, and detecting the chemiluminescence value; (vi) administering the drug to be tested to the in vitro lung model or infecting the in vitro lung model with the pathogen, and detecting cell apoptosis in the in vitro lung model; wherein the detection method is, for example, immunofluorescence or flow cytometry; and the markers detected by flow cytometry are, for example, Caspase 3, Annexin V, Live / Dead, and TUNEL; (vii) applying the drug to be tested to the lung in vitro model or allowing the pathogen to infect the lung in vitro model, and detecting changes in the content of inflammatory cytokines in the lung in vitro model; wherein the inflammatory factors 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, and then incubating and washing, adding a color developing solution and incubating, and then adding a stop solution and detecting the OD value; (viii) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the cell phenotype or cell content related to the lung tissue in the in vitro lung model; 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; and the detection method includes: flow cytometry, immunofluorescence; (ix) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the level of cellular oxidative stress in the in vitro lung model; (x) administering the drug to be tested to the in vitro lung model or allowing the pathogen to infect the in vitro lung model, and detecting the expression changes of genes related to the process of pulmonary fibrosis in the in vitro lung model; 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; (xi) allowing the pathogen to infect an in vitro lung model, and detecting changes in the expression of receptors associated with the pathogen infection in the in vitro lung model; (xii) allowing the pathogen to infect an in vitro lung model, then administering the drug to be tested to the in vitro lung model, and detecting the inhibitory effect of the drug to be tested on the pathogen; Wherein, the in vitro lung model is the in vitro lung model constructed by the construction method according to any one of claims 1 to 6, or the in vitro lung model according to claim 7; Preferably, the drugs include small molecule drugs, antibody drugs, ADC drugs, and immune drugs.

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