Pulmonary fibrosis model and construction method and application thereof

Abstract

The invention relates to a pulmonary fibrosis model and a construction method and application thereof. The pulmonary fibrosis 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 a pulmonary fibrosis model and a construction method thereof, use of the pulmonary fibrosis model in drug detection, drug screening and / or detection of the effect of pathogen infection on the development process of pulmonary tissue fibrosis, a culture medium combination, and a drug detection method, a drug screening method and / or a method for detecting the effect of pathogen infection on the development process of pulmonary tissue fibrosis. Background Art

[0002] Pulmonary fibrosis is a type of end-stage interstitial lung disease. It is known that there are many factors that can cause pulmonary fibrosis, including toxicity, autoimmunity, drugs, infection, traumatic injury, etc. Pulmonary fibrosis is characterized by fibroblast proliferation, extracellular matrix aggregation, inflammatory damage, and destruction of tissue structure. Studies have shown that the median survival of patients with pulmonary fibrosis is only 2 to 5 years, and the 5-year survival rate is only 20%.

[0003] Previous studies on pulmonary fibrosis have mainly focused on in vitro 2D culture of fibroblasts and animal models. However, each of these aspects has its own drawbacks. Among them, when fibroblasts are cultured in culture dishes or slides (2D), the cell structure, adhesion, mechanical conduction and signal transduction of soluble cytokines cannot be well demonstrated, and the interaction between cells-cells / matrix in vivo cannot be simulated. For animal models, due to species differences and the fact that artificially induced fibrotic diseases can only simulate a certain stage in the course of fibrosis and cannot show the entire development process of the disease, most of the drugs that have been shown to alleviate fibrosis in animal models have failed in clinical Phase II / III trials.

[0004] Therefore, it is extremely urgent to find a new pulmonary fibrosis model that can better simulate the in vivo lung microenvironment for the study of pulmonary fibrosis diseases and drug screening. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a pulmonary fibrosis model. The pulmonary fibrosis model constructed by this construction method can better simulate the function and structural characteristics of the alveolar-capillary barrier and have an immune microenvironment, so as to be used for the research of pulmonary fibrosis diseases, drug screening, etc.

[0006] Specifically, in a first aspect, the present invention provides a method for constructing a pulmonary fibrosis model, comprising:

[0007] providing lung organoids and cell culture vessels;

[0008] 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;

[0009] 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;

[0010] Inoculating endothelial cells in the lower layer of the cell culture container, and then inoculating macrophages in the upper layer of the cell culture container and performing a second-stage air-liquid mode culture to obtain an in vitro lung model;

[0011] The lung in vitro model is treated with a pulmonary fibrosis inducing agent to obtain a pulmonary fibrosis model.

[0012] 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 attach to a membrane (e.g., a porous membrane) between the upper and lower layers according to needs. Accordingly, the obtained pulmonary fibrosis 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 pulmonary fibrosis model is also referred to as 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 202211680988.1, 202222130269.4 or 202222130382.2, and these application documents are incorporated herein by reference in their entirety.

[0013] 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.

[0014] 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.

[0015] In the construction method of the present invention, lung organoids are used instead of traditional cell lines to construct a pulmonary fibrosis 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 the order of each other, and the macrophages are cultured in the second stage of air-liquid mode. This is beneficial for making the pulmonary fibrosis model more mature and improving its simulation.

[0016] 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:

[0017] (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;

[0018] (2) Crushing: mince the lung tissue into minced meat, resuspend in buffer, let stand and discard the supernatant;

[0019] (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;

[0020] (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.

[0021] (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.

[0022] 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.

[0023] 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.

[0024] 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 ~10×10 5 cells / cm 2 .

[0025] 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 is, 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 -Ex Plus Medium).

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 ~5×10 5 cells / cm 2 .

[0032] In some embodiments, the endothelial cells are derived from umbilical vein endothelial cells, primary lung endothelial cells, or a lung endothelial cell line.

[0033] 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.

[0034] 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 2In 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In some embodiments, after the macrophages are inoculated in 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 in the lower layer of the cell culture container and culturing them is also included, so that the constructed pulmonary fibrosis model has immune function.

[0039] In some embodiments, the immune cells include PBMCs, T cells, B cells, NK cells.

[0040] In some embodiments, the immune cells are seeded at 0.5×10 5 ~1×10 6 cells / cm 2 .

[0041] 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.

[0042] 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.

[0043] In some embodiments, the lung in vitro model is treated with a pulmonary fibrosis inducing agent for 2 to 5 days, such as 2 days, 3 days, 4 days, 5 days, or any time within the range.

[0044] The pulmonary fibrosis inducing agent used in the present invention may be a substance known in the art that can induce fibrosis in lung tissue, and is not particularly limited herein. In some embodiments, the pulmonary fibrosis inducing agent is selected from at least one of the group consisting of TGF-β1, bleomycin, paraquat, and silicon dioxide.

[0045] In some embodiments, the pulmonary fibrosis inducing agent is added to a pulmonary epithelial cell culture medium to obtain a pulmonary fibrosis inducing culture medium, which is then used to treat a lung in vitro model to obtain a pulmonary fibrosis model. The pulmonary epithelial cell culture medium may be a commercially available pulmonary epithelial cell culture medium, such as PneumaCult produced by Stemcell. TM -ExPlus Medium.

[0046] According to the specific selection of the pulmonary fibrosis inducer, its addition amount in the pulmonary fibrosis induction medium can be adjusted accordingly. In some specific embodiments, in the pulmonary fibrosis induction medium, when the pulmonary fibrosis inducer contains TGF-β1, the addition amount of TGF-β1 is 5 to 200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the addition amount of bleomycin is 1 to 20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the addition amount of paraquat is 20 to 200 μM; when the pulmonary fibrosis inducer contains silica, the addition amount of silica is 20 to 200 μg / mL.

[0047] The pulmonary fibrosis induction medium provided by the present invention can be mixed with the endothelial cell medium and / or the immune cell medium to treat the lung in vitro model to obtain a pulmonary fibrosis model. Specifically, in some embodiments, when the lung in vitro model is not inoculated with immune cells, the pulmonary fibrosis induction medium and the endothelial cell medium are mixed in a volume ratio of (0.5-5):1 to obtain a third mixed medium for treating the lung in vitro model to obtain a pulmonary fibrosis model. In other embodiments, when the lung in vitro model is inoculated with immune cells, the pulmonary fibrosis induction medium is mixed with the immune cell medium and the endothelial cell medium in a volume ratio of (0.5-5):(0.5-5):1 to obtain a fourth mixed medium for treating the lung in vitro model to obtain a pulmonary fibrosis model.

[0048] In a second aspect, the present invention provides a pulmonary fibrosis model, which is constructed by the method for constructing the pulmonary fibrosis model provided in the first aspect.

[0049] In a third aspect, the present invention provides a culture medium combination, comprising a 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;

[0050] Among them, 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 an immune cell culture medium and the first mixed culture medium in a volume ratio of (0.5-3.5):1; the third mixed culture medium is obtained by mixing a lung fibrosis induction culture medium and an endothelial cell culture medium in a volume ratio of (1-2):1; the fourth mixed culture medium is obtained by mixing a lung fibrosis induction culture medium, an immune cell culture medium and an endothelial cell culture medium in a volume ratio of 1:(1-2):1; the lung fibrosis induction culture medium is obtained by adding a lung fibrosis inducer to a lung epithelial cell culture medium, and the lung fibrosis inducer is selected from at least one of the group consisting of TGF-β1, bleomycin, paraquat and silicon dioxide; 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.

[0051] In some specific embodiments, in the lung epithelial cell culture medium, when the pulmonary fibrosis inducer contains TGF-β1, the added amount of TGF-β1 is 5-200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the added amount of bleomycin is 1-20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the added amount of paraquat is 20-200 μM; when the pulmonary fibrosis inducer contains silica, the added amount of silica is 20-200 μg / mL.

[0052] In some embodiments, the culture medium combination is used in the construction method provided by the present invention, or for the construction of the pulmonary fibrosis 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, processing the lung in vitro model, and optionally third stage air-liquid mode culture.

[0053] 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.

[0054] In some preferred embodiments, the first mixed culture medium is used in the second stage gas-liquid mode.

[0055] In some preferred embodiments, the second mixed culture medium is used for the third stage gas-liquid mode culture.

[0056] In some preferred embodiments, when the lung in vitro model is not inoculated with immune cells, the third mixed culture medium is used to treat the lung in vitro model.

[0057] In some preferred embodiments, when the lung in vitro model is inoculated with immune cells, the fourth mixed culture medium is used to treat the lung in vitro model.

[0058] In some preferred embodiments, the culture medium combination comprises or consists of a lung epithelial cell culture medium, a first mixed culture medium, and a third mixed culture medium.

[0059] In some preferred embodiments, the culture medium combination comprises or consists of a lung epithelial cell culture medium, a first mixed culture medium, a second mixed culture medium, and a fourth mixed culture medium.

[0060] In a fourth aspect, the present invention provides a use of the pulmonary fibrosis model constructed by the construction method provided in the first aspect, or the pulmonary fibrosis model described in the second aspect in the following (a) or (b);

[0061] (a) drug testing and / or drug screening;

[0062] (b) Detect the effect of pathogen infection on the development of pulmonary fibrosis.

[0063] In some embodiments, the drug includes a small molecule drug, an antibody drug, an ADC drug, or an immune drug.

[0064] In some embodiments, the pathogens include bacteria, viruses, fungi, parasites, and the like.

[0065] In some embodiments, the drug testing and / or drug screening comprises drug testing and / or drug screening for the treatment of pulmonary fibrosis.

[0066] The pulmonary fibrosis model provided by the present invention can be used for the efficacy detection and drug mechanism exploration of drugs for treating lung diseases (e.g., drugs for treating pulmonary fibrosis diseases), and the response of the pulmonary fibrosis model to drugs is evaluated by changes in cell marker expression, changes in cell (e.g., 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, especially for the research and development of drugs for treating pulmonary fibrosis. For example, when a lung in vitro model is treated with a pulmonary fibrosis inducer to induce a pulmonary fibrosis model, a drug to be tested is added at the same time to detect whether the drug will cause acceleration or inhibition of the development process of pulmonary fibrosis, thereby detecting and / or screening drugs for treating pulmonary fibrosis; after the pathogen infects the pulmonary fibrosis model (upper or lower layer), the effect of the pathogen on the development process of pulmonary tissue fibrosis can be confirmed by indicator detection; after the pulmonary fibrosis model is induced, the drug can be administered and the indicator detection can be performed to detect whether the drug will accelerate or inhibit the development process of pulmonary fibrosis, thereby detecting and / or screening drugs for treating pulmonary fibrosis. The following are some examples (i) to (xii) of the use of the pulmonary fibrosis model of the present invention in (a) or (b). It should be understood that the application of the pulmonary fibrosis model of the present invention is not limited to these:

[0067] (i) Changes in the expression of pulmonary fibrosis markers: The drug to be tested is applied to the pulmonary fibrosis model or the pathogen is allowed to infect the pulmonary fibrosis model, and the changes in the expression of pulmonary fibrosis markers in the pulmonary fibrosis model are detected; wherein the pulmonary fibrosis markers are, for example, α-SMA, type I collagen (Collagen I), and fibronectin. In some specific embodiments, the method comprises: fixing the pulmonary fibrosis model with 4% paraformaldehyde at room temperature for 30 minutes, removing the paraformaldehyde, washing the sample with PBS, permeabilizing the sample with 0.1% Triton-X-100 and blocking the sample with 5% BSA, respectively dropping primary antibodies α-SMA, type I collagen, and fibronectin, incubating at 4°C overnight, dropping 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.

[0068] (ii) Flow cytometry detection of immune cell typing changes: administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the typing changes and / or dynamic immune response process of cells in the pulmonary fibrosis 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.

[0069] (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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance of the pulmonary fibrosis model; wherein the transmembrane resistance of a normal lung model is 200 to 1500 Ω·cm 2, the transmembrane resistance value of the pulmonary fibrosis model will be lower than that of the normal lung model. Therefore, the effect of the test drug or pathogen infection on the development process of pulmonary tissue fibrosis can be judged according to the change of the transmembrane resistance value. For example, when the test drug is added or infected by the pathogen, the transmembrane resistance value drops significantly to a statistically significant difference, then the test drug or pathogen infection will destroy the cell barrier; when the test drug is added, the transmembrane resistance value of the pulmonary fibrosis model is significantly increased to a statistically significant difference compared with before the addition, then the test drug can repair the cell barrier; when the test drug is added or infected by the pathogen, the transmembrane resistance value of the pulmonary fibrosis model is not statistically different from the change before the addition of the test drug or infection by the pathogen, then the test drug or pathogen infection has no significant effect on the integrity of the cell barrier. In some specific implementation schemes, the following steps are included: taking out the transmembrane resistance meter, plugging in the power supply, assembling the monitoring device, placing the chopstick detection head in PBS for 15 minutes for equilibrium; after the instrument value stabilizes at 0Ω, filling the inside and outside of the model with PBS; inserting the chopstick detection head into the model, and recording 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 pulmonary fibrosis model.

[0070] (iv) Detecting changes in barrier permeability and characterizing the cell barrier function of the model: applying the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in the barrier permeability of the pulmonary fibrosis 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.

[0071] (v) Detecting the activity of model cells: administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in cell activity in the pulmonary fibrosis 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.

[0072] (vi) Detecting cell apoptosis in the model: administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting cell apoptosis in the pulmonary fibrosis 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.

[0073] (vii) Detecting cytokine content: administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pulmonary fibrosis model; wherein the inflammatory factors are, for example, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-13, CXCL13, and 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.

[0074] (viii) Detecting cell types and expressions associated with lung tissue: administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the cell phenotype or cell content associated with lung tissue in the pulmonary fibrosis 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).

[0075] (ix) Detecting the level of oxidative stress: administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the level of cellular oxidative stress in the pulmonary fibrosis 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 the drug on oxidative stress. For example, using Invitrogen TM The 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.

[0076] (x) Detecting the expression of genes related to the development of pulmonary fibrosis: administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis-related genes in the pulmonary fibrosis 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. In some specific embodiments, RNA-seq or RT-PCR is used to detect the changes in the transcription levels of pulmonary fibrosis-related genes.

[0077] (xi) Detecting the expression of receptors associated with pathogen infection: The pathogen is allowed to infect the pulmonary fibrosis model, and the expression changes of the receptors associated with the pathogen infection in the pulmonary fibrosis model are detected. In some specific embodiments, the pulmonary fibrosis model is infected with avian influenza virus, and the expression of influenza virus receptor α-2,6 linked to sialic acid is detected using a kit.

[0078] (xii) Detecting the anti-pathogen effect of the drug: the pathogen is infected with the pulmonary fibrosis model, and then the drug to be tested is applied to the pulmonary fibrosis model to detect the inhibitory effect of the drug to be tested on the pathogen. In some specific embodiments, the anti-viral effect of the drug is detected by detecting the viral titer at different time points after the pathogen is infected with the pulmonary fibrosis 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.

[0079] 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:

[0080] (i) applying the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis markers in the pulmonary fibrosis 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 pulmonary fibrosis 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 by staining and imaging;

[0081] (ii) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the phenotypic changes and / or dynamic immune response process of cells in the pulmonary fibrosis 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 pulmonary fibrosis 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 by a flow cytometer;

[0082] (iii) applying the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance of the pulmonary fibrosis model; the effect of the drug to be tested or the pathogen infection on the development of pulmonary tissue fibrosis can be determined based on the change in the transmembrane resistance. When the drug to be tested is added or infected by the pathogen, the transmembrane resistance value significantly decreases to a statistically significant difference, then the drug to be tested or the pathogen infection can destroy the cell barrier; when the drug to be tested is added, the transmembrane resistance value of the pulmonary fibrosis model is significantly increased to a statistically significant difference compared with before the addition, then the drug to be tested can repair the cell barrier; when the drug to be tested is added or infected by the pathogen, the transmembrane resistance value of the pulmonary fibrosis model is not statistically different from the change before the addition of the drug to be tested or the pathogen infection, then the drug to be tested or the 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;

[0083] (iv) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in the barrier permeability of the pulmonary fibrosis model. In some embodiments, the detection comprises: adding a fluorescent molecule (e.g., FITC-Dextran) to the upper layer of the pulmonary fibrosis model and incubating, detecting the amount of the fluorescent molecule permeated in the lower layer of the pulmonary fibrosis model, and calculating the apparent permeability coefficient;

[0084] (v) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in cell activity in the pulmonary fibrosis 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 pulmonary fibrosis model and incubating, and detecting the chemiluminescence value;

[0085] (vi) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting cell apoptosis in the pulmonary fibrosis 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;

[0086] (vii) Applying the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pulmonary fibrosis 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 standard solution of inflammatory cytokines with gradient concentrations, 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;

[0087] (viii) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the cell phenotype or cell content associated with the lung tissue in the pulmonary fibrosis model. In some embodiments, the cell phenotype is, for example, α-tubulin, MUC5AC, AQP5, SFTPC, CC10, or KRT5;

[0088] (ix) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the level of cellular oxidative stress in the pulmonary fibrosis model;

[0089] (x) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis-related genes in the pulmonary fibrosis 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;

[0090] (xi) allowing the pathogen to infect a pulmonary fibrosis model, and detecting changes in the expression of receptors associated with the pathogen infection in the pulmonary fibrosis model;

[0091] (xii) allowing the pathogen to infect the pulmonary fibrosis model, then administering the drug to be tested to the pulmonary fibrosis model, and detecting the inhibitory effect of the drug to be tested on the pathogen;

[0092] Wherein, the pulmonary fibrosis model is the pulmonary fibrosis model constructed by the construction method of the first aspect, or the pulmonary fibrosis model described in the second aspect.

[0093] In some embodiments, the drug includes a small molecule drug, an antibody drug, an ADC drug, or an immune drug.

[0094] First, the pulmonary fibrosis model provided by the present invention is constructed by replacing traditional cell lines with lung organoids, which has a shorter experimental cycle compared with animal models, is conducive to reducing experimental costs, simplifies the operation difficulty, and enhances the stability and controllability of the model; secondly, the construction method provided by the present invention is inoculated in sequence according to the order of lung organoids, endothelial cells, macrophages and optionally immune cells, and obtains a lung in vitro model by first liquid-liquid mode culture and then gas-liquid mode culture (including first stage gas-liquid mode culture, second stage gas-liquid mode culture and optionally third stage gas-liquid mode culture), and then obtains a lung fibrosis model by treating the lung in vitro model with a pulmonary fibrosis inducer, which is not only conducive to making the pulmonary fibrosis model have a higher degree of maturity, but also can better simulate the function and structural characteristics of the alveolar-capillary barrier, thereby improving its simulation. In addition, the pulmonary fibrosis model provided by the present invention comprises lung organoids, endothelial cells, macrophages and optionally immune cells, which can well simulate the microenvironment associated with these cells and cell-to-cell interactions. It also has the advantages of being able to be passaged multiple times and maintaining the phenotype in vitro for a long time, providing a good platform for disease simulation, drug testing, screening, evaluation and research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 The results of the detection of the expression of pulmonary fibrosis markers in each group of models in Example 2 of the present invention are shown.

[0096] Figure 2 The transmembrane resistance values ​​of each group of models in Example 2 of the present invention are shown.

[0097] Figure 3 The expression of inflammatory factors IL-6 and TNF-α in each group of models in Example 3 of the present invention is shown.

[0098] Figure 4A and Figure 4B The figure shows the proportion of helper T cells Th1 and Th2 in the control group in Example 4 of the present invention; Figure 4C and Figure 4D The figure shows the proportion of helper T cells Th1 and Th2 in the induction group to which TGF-β1 was added in Example 4 of the present invention. DETAILED DESCRIPTION

[0099] 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.

[0100] Example 1

[0101] This example provides a specific construction process of the pulmonary fibrosis model:

[0102] Primary extraction and liquid-liquid culture of lung organoids:

[0103] (1) Cleaning: Use primary tissue cleaning solution to clean normal human lung tissue and remove the surface mucosa. The primary tissue cleaning solution is a buffer solution to which penicillin-streptomycin and gentamicin are added.

[0104] (2) Disintegration: Chop the tissue into minced meat, resuspend in buffer, let stand and discard the supernatant.

[0105] (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.

[0106] (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.

[0107] (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.

[0108] (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 add lung organoid culture medium to terminate the digestion.

[0109] (7) Inoculation: Centrifuge and remove the supernatant, resuspend in lung epithelial cell culture medium, and adjust the density to 1×10 6 / 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.

[0110] (8) Change the medium every 3 days and culture for 5 days.

[0111] Air-liquid culture:

[0112] (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.

[0113] (2) Cultivation in the gas-liquid mode for 2 days (first stage gas-liquid mode cultivation).

[0114] Seeding of endothelial cells:

[0115] (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).

[0116] (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.

[0117] Inoculation of macrophages:

[0118] (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 0.33cm 2 , inoculated with 1×10 4 Macrophages, cell density was 0.3×10 5 Pieces / cm 2 .

[0119] (2) The macrophages were cultured overnight, and then the macrophage culture medium on the membrane was discarded.

[0120] (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)).

[0121] (4) Continue the gas-liquid mode culture for 2 days (second stage gas-liquid mode culture).

[0122] Vaccination of immune cells (if immune function is not required, this step can be omitted):

[0123] (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.

[0124] #6911011).

[0125] (2) Continue the air-liquid mode culture for 1 day (the third stage of air-liquid mode culture) to obtain an in vitro lung model.

[0126] Inducing pulmonary fibrosis:

[0127] TGF-β1 was used as a pulmonary fibrosis inducer and mixed with a lung epithelial cell culture medium to obtain a pulmonary fibrosis induction medium (the content of the pulmonary fibrosis inducer was 10 ng / mL). The pulmonary fibrosis induction medium was then mixed with an immune cell culture medium and an 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 pulmonary fibrosis model.

[0128] Example 2

[0129] In this example, drug efficacy tests were performed on an in vitro lung model and a pulmonary fibrosis model, and the steps and results are as follows:

[0130] (1) A lung in vitro model was constructed according to the method of Example 1. A control group, an induction group, and a drug treatment group were set. The control group was a lung in vitro model, and the second mixed culture medium was added to the lower layer of the chip; the induction group was a lung fibrosis induction culture medium containing 10 ng / mL TGF-β1 was added to the lower layer of the chip of the lung in vitro model; the drug treatment group was a lung fibrosis induction culture medium containing 10 ng / mL TGF-β1 and 10 μM Nintedanib was added to the lower layer of the chip of the lung in vitro model.

[0131] (2) Drug treatment for 3 days.

[0132] (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, the induced group, and the drug treatment group was compared to see if there was any difference.

[0133] (4) Use a transmembrane resistance meter to detect the transmembrane resistance value of each group of models.

[0134] The results are as follows Figure 1 and 2 shown.

[0135] pass Figure 1 It can be seen that the addition of TGF-β1 in the induction group can significantly induce pulmonary fibrosis, and the expression of α-SMA and type I collagen is significantly increased compared with the control group. TGF-β1 and nintedanib were added to the drug treatment group, among which nintedanib can significantly inhibit pulmonary fibrosis, and the expression of α-SMA and type I collagen is significantly reduced compared with the induction group.

[0136] pass Figure 2 It can be seen from the transmembrane resistance value that the cell barrier of the induced group was damaged, and the transmembrane resistance value was significantly decreased compared with the control group. In the drug-treated group, since the addition of nintedanib can alleviate the damage to the cell barrier, its transmembrane resistance value was significantly increased compared with the induced group.

[0137] Example 3

[0138] In this example, drug efficacy tests were performed on an in vitro lung model and a pulmonary fibrosis model, and the steps and results are as follows:

[0139] (1) A lung in vitro model was constructed according to the method of Example 1. A control group, an induction group, and a drug treatment group were set. The control group was a lung in vitro model, and the second mixed culture medium was added to the lower layer of the chip; the induction group was a lung fibrosis induction culture medium containing 10 ng / mL TGF-β1 was added to the lower layer of the chip of the lung in vitro model; the drug treatment group was a lung fibrosis induction culture medium containing 10 ng / mL TGF-β1 and 10 μM nintedanib was added to the lower layer of the chip of the lung in vitro model.

[0140] (2) Drug treatment for 3 days.

[0141] (3) After drug treatment, the levels of inflammatory factors IL-6 and TNF-α in the pulmonary fibrosis model were detected using an ELISA kit to compare whether there was any difference in the expression of inflammatory cytokines in the control group, the induced group, and the drug treatment group.

[0142] The results are as follows Figure 3 shown.

[0143] pass Figure 3 It can be seen that compared with the control group, the expression of IL-6 and TNF-α in the induction group with the addition of TGF-β1 was significantly increased. TGF-β1 and nintedanib were added to the drug treatment group, among which nintedanib could significantly inhibit pulmonary fibrosis, and the expression of IL-6 and TNF-α was significantly reduced compared with the induction group.

[0144] Example 4

[0145] In this example, immune cell detection was performed on an in vitro lung model and a pulmonary fibrosis model, and the steps and results are as follows:

[0146] (1) A lung in vitro model was constructed according to the method of Example 1. A control group and an induction group were set up. The control group was a lung in vitro model, and the second mixed culture medium was added to the lower layer of the chip; the induction group was a lung in vitro model, and the lower layer of the chip was added with a lung fibrosis induction culture medium containing 10 ng / mL TGF-β1.

[0147] (2) Treatment with inducer for 3 days.

[0148] (3) After induction, flow cytometry was used to detect the expression of Th1 and Th2 immune cells in the lung in vitro model and pulmonary fibrosis model. The typing changes of Th1 and Th2 immune cells in the control group and the induced group were compared.

[0149] The results are as follows 4A to 4D shown.

[0150] pass 4A to 4DIt can be seen that in the control group, B cells accounted for 12.4% of the total cells, CD3 T cells accounted for 28.4% of the total cells, Th1 cells accounted for 2.65% of the total cells, and Th2 cells accounted for 2.8% of the total cells. In the TGF-β1 group, B cells accounted for 10.4% of the total cells, CD3 T cells accounted for 25.3% of the total cells, Th1 cells accounted for 2.1% of the total cells, and Th2 cells accounted for 2.6% of the total cells. It can be seen that after TGF-β1 induction, there was no significant difference in the content of immune cells B cells and T cells.

Claims

1. A method for constructing a pulmonary fibrosis 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; Inoculating endothelial cells in the lower layer of the cell culture container, and then inoculating macrophages in the upper layer of the cell culture container and performing a second-stage air-liquid mode culture to obtain an in vitro lung model; The lung in vitro model is treated with a pulmonary fibrosis inducing agent to obtain a pulmonary fibrosis model.

2. The construction method according to claim 1, wherein: The liquid-liquid mode culture time of the lung organoid is 3 to 7 days; The time for culturing the lung organoids in the air-liquid mode in the first stage is 2 to 21 days; The duration of the second stage gas-liquid mode culture is 1 to 5 days; and / or The pulmonary fibrosis inducer is used to treat the lung in vitro model 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 inducer is added to a lung epithelial cell culture medium to obtain a pulmonary fibrosis induction medium, and the pulmonary fibrosis induction medium is mixed with an endothelial cell culture medium and / or an immune cell culture medium for treating the lung in vitro model; more preferably, in the pulmonary fibrosis induction medium, when the pulmonary fibrosis inducer contains TGF-β1, the added amount of TGF-β1 is 5 to 200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the added amount of bleomycin is 1 to 20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the added amount of paraquat is 20 to 200 μM; when the pulmonary fibrosis inducer contains silica, the added amount of silica is 20 to 200 μg / mL.

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

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. A pulmonary fibrosis 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, a first mixed culture medium and a third mixed culture medium, and optionally a second mixed culture medium and a fourth 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; The third mixed culture medium is obtained by mixing the pulmonary fibrosis induction culture medium and the endothelial cell culture medium in a volume ratio of (0.5-5):1; The fourth mixed culture medium is obtained by mixing the pulmonary fibrosis induction culture medium, the immune cell culture medium and the endothelial cell culture medium in a volume ratio of (0.5-5):(0.5-5):1; The pulmonary fibrosis induction medium is obtained by adding a pulmonary fibrosis inducer to a pulmonary epithelial cell culture medium, wherein the pulmonary fibrosis inducer is at least one selected from the group consisting of TGF-β1, bleomycin, paraquat, and silicon dioxide; Preferably, in the lung epithelial cell culture medium, when the pulmonary fibrosis inducer contains TGF-β1, the added amount of TGF-β1 is 5 to 200 ng / mL; when the pulmonary fibrosis inducer contains bleomycin, the added amount of bleomycin is 1 to 20 μg / mL; when the pulmonary fibrosis inducer contains paraquat, the added amount of paraquat is 20 to 200 μM; when the pulmonary fibrosis inducer contains silicon dioxide, the added amount of silicon dioxide is 20 to 200 μg / mL; 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 fibrosis model according to claim 7, including for the culture of lung organoids, liquid-liquid mode culture, first stage air-liquid mode culture, second stage air-liquid mode, treatment of the lung in vitro model, 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 air-liquid mode culture; when the lung in vitro model is not inoculated with immune cells, the third mixed culture medium is used to treat the lung in vitro model; when the lung in vitro model is inoculated with immune cells, the fourth mixed culture medium is used to treat the lung in vitro model; More preferably, the culture medium combination comprises or consists of a 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 a lung epithelial cell culture medium, a first mixed culture medium, a second mixed culture medium and a fourth mixed culture medium.

9. Use of the pulmonary fibrosis model constructed by the construction method according to any one of claims 1 to 6, or the pulmonary fibrosis 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, immune drugs; preferably; the drug detection and / or drug screening includes drug detection and / or drug screening for the treatment of pulmonary fibrosis; (b) Detect the effect of pathogen infection on the development of pulmonary fibrosis.

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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis markers in the pulmonary fibrosis 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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the typing changes and / or dynamic immune response process of cells in the pulmonary fibrosis 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 pulmonary fibrosis model or allowing the pathogen to infect the pulmonary fibrosis model, and detecting the transmembrane resistance of the pulmonary fibrosis model; wherein, when the transmembrane resistance of the pulmonary fibrosis model significantly decreases to a statistically significant level after the drug to be tested is added or the pulmonary fibrosis model is infected by the pathogen, the drug to be tested or the pathogen infection will damage the cell barrier; when the transmembrane resistance of the pulmonary fibrosis model significantly increases to a statistically significant level compared with before the drug to be tested is added after the drug to be tested is added, the drug to be tested can repair the cell barrier; when the transmembrane resistance of the pulmonary fibrosis model does not have a statistically significant change compared with before the drug to be tested is added or the pathogen is infected after the drug to be tested is added or the pathogen is infected, the drug to be tested or the pathogen infection has no significant effect on the integrity of the cell barrier; (iv) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in the barrier permeability of the pulmonary fibrosis model; (v) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in cell activity in the pulmonary fibrosis model; wherein the cell activity is, for example, cell proliferation activity; (vi) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting cell apoptosis in the pulmonary fibrosis 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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pulmonary fibrosis 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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the cell phenotype or cell content related to the lung tissue in the pulmonary fibrosis 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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the level of cellular oxidative stress in the pulmonary fibrosis model; (x) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the expression changes of genes related to the development of pulmonary fibrosis in the pulmonary fibrosis model; wherein the 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 pulmonary fibrosis model, and detecting changes in the expression of receptors associated with the pathogen infection in the pulmonary fibrosis model; (xii) allowing the pathogen to infect the pulmonary fibrosis model, then administering the drug to be tested to the pulmonary fibrosis 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 effect of pathogen infection on the progression of pulmonary fibrosis, comprising: (i) administering the drug to be tested to a pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the expression changes of pulmonary fibrosis markers in the pulmonary fibrosis 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 blocking the pulmonary fibrosis 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 by staining and imaging; (ii) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the cell typing changes and / or dynamic immune response process in the pulmonary fibrosis 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 cell typing changes include macrophages Typing changes and / or typing changes of immune cells, the typing changes of macrophages are, for example, typing changes of M1 macrophages and M2 macrophages; the typing changes of immune cells are, for example, typing changes of T cells, B cells, NK cells, and innate lymphocytes; preferably, the detection method comprises: collecting cells in the pulmonary fibrosis 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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the transmembrane resistance of the pulmonary fibrosis model; wherein, when the transmembrane resistance of the pulmonary fibrosis model significantly decreases to a statistically significant level after the drug to be tested is added or the pulmonary fibrosis model is infected by the pathogen, the drug to be tested or the pathogen infection will damage the cell barrier; when the transmembrane resistance of the pulmonary fibrosis model significantly increases to a statistically significant level compared with before the drug to be tested is added after the drug to be tested is added or the pathogen is infected, the drug to be tested can repair the cell barrier; when the transmembrane resistance of the pulmonary fibrosis model does not change significantly compared with before the drug to be tested is added or the pathogen is infected after the drug to be tested is added or the pathogen is infected, the drug to be tested or the pathogen infection has no significant effect on the integrity of the cell barrier; preferably, the detection method comprises: using a transmembrane resistance meter to detect the resistance 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; (iv) administering the drug to be tested to the pulmonary fibrosis model or allowing the pathogen to infect the pulmonary fibrosis model, and detecting changes in the barrier permeability of the pulmonary fibrosis model; preferably, the detection comprises: adding fluorescent molecules (such as FITC-Dextran) to the upper layer of the pulmonary fibrosis model and incubating, detecting the amount of the fluorescent molecules permeated in the lower layer of the pulmonary fibrosis model, and calculating the apparent permeability coefficient; (v) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in cell activity in the pulmonary fibrosis 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 pulmonary fibrosis model and incubating, and detecting the chemiluminescence value; (vi) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting cell apoptosis in the pulmonary fibrosis 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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting changes in the content of inflammatory cytokines in the pulmonary fibrosis 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 a pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the cell phenotype or cell content associated with the lung tissue in the pulmonary fibrosis 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 pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the level of cellular oxidative stress in the pulmonary fibrosis model; (x) administering the drug to be tested to the pulmonary fibrosis model or infecting the pulmonary fibrosis model with the pathogen, and detecting the expression changes of genes related to the development of pulmonary fibrosis in the pulmonary fibrosis model; wherein the 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 a pulmonary fibrosis model, and detecting changes in the expression of receptors associated with the pathogen infection in the pulmonary fibrosis model; (xii) allowing the pathogen to infect the pulmonary fibrosis model, then administering the drug to be tested to the pulmonary fibrosis model, and detecting the inhibitory effect of the drug to be tested on the pathogen; Wherein, the pulmonary fibrosis model is a pulmonary fibrosis model constructed by the construction method according to any one of claims 1 to 6, or a pulmonary fibrosis model according to claim 7; Preferably, the drugs include small molecule drugs, antibody drugs, ADC drugs, and immune drugs; Preferably, the drug detection method and drug screening method include drug detection and / or drug screening for treating pulmonary fibrosis.

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