A method for constructing an in vitro model of immune checkpoint inhibitor-associated pneumonia based on human lung organoids

Abstract

The application provides a construction method of an immune checkpoint inhibitor related pneumonia in vitro model based on a human lung organoid, and belongs to the technical field of biotechnology.The method is to co-culture bronchoalveolar lavage fluid of a CIP patient with a lung organoid to obtain an immune checkpoint inhibitor related pneumonia in vitro model.The application successfully constructs a stable immune checkpoint inhibitor related pneumonia in vitro model by filtering and centrifugally separating BALF directly derived from a CIP patient, co-culturing supernatant with an organoid, and lays a foundation for studying a CIP pathological mechanism and related drug evaluation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for constructing an in vitro model of pneumonia based on human lung organoids and immune checkpoint inhibitors. Background Technology

[0002] Immune checkpoint inhibitor-associated pneumonitis (CIP) is a serious pulmonary immune-related adverse reaction that occurs during cancer treatment with immune checkpoint inhibitors (ICIs). With the widespread use of immune checkpoint inhibitors, the incidence of CIP is gradually increasing, and in severe cases, it can lead to treatment interruption, respiratory failure, and even death. The pathogenesis of CIP is complex and highly heterogeneous; relying solely on clinical case observation or clinical testing makes it difficult to systematically elucidate the mechanisms of CIP's development and to standardize the evaluation of candidate interventional drugs.

[0003] Currently, existing research on CIP mainly includes retrospective clinical studies, animal models, and in vitro cell models. However, all of these have certain limitations. Clinical studies struggle to conduct controlled mechanistic and intervention experiments. Animal models suffer from species differences, animal tolerance to ICIs, and low stability. Traditional two-dimensional lung epithelial cells are insufficient to simulate the three-dimensional spatial structure and functional characteristics of lung tissue. In vitro models that solely use inflammatory factors or immune cell stimulation often fail to fully reflect the complex interactions between lung epithelial tissue and the local inflammatory microenvironment of CIP-affected lungs.

[0004] Organoid technology provides a new tool for constructing human in vitro disease models. Lung organoids can preserve the three-dimensional structure, differentiation characteristics, and some tissue functions of lung epithelial cells to a certain extent, and are considered important in vitro models for studying the pathogenesis and drug response of lung diseases. However, simple lung organoid culture systems usually lack the lung local inflammatory environment specific to CIP patients, making it difficult to directly simulate the pathological damage process of lung epithelial tissue in CIP. Existing technology (LUO T, CHENW, HUANG D, et al. Preclinical models of immune checkpoint inhibitors-related interstitial pneumonia for anti-PD1 tumor immunotherapy[J]. Immunobiology,2025, 230(2): 152884.) co-cultures lung organoids with peripheral blood immune cells and immune checkpoint inhibitors to construct an interstitial pneumonia model, which provides a reference for in vitro research on CIP. However, this model focuses on the level of interstitial lung disease and fibrosis, and does not evaluate the acute lung injury and functional impairment of CIP. Meanwhile, this method still has the following limitations: (1) When lung organoids and peripheral blood immune cells come from different individuals, allogeneic cell co-culture may introduce non-specific immune activation, thereby affecting the model's judgment of the source of lung injury. (2) Peripheral blood immune cells mainly reflect the peripheral circulating immune status and are difficult to fully represent the local immune microenvironment of the lungs in CIP patients. (3) Simply relying on ICIs drugs to induce healthy tissues in vitro is difficult to stably reproduce the pathological state of clinical CIP patients, and the induction efficiency and phenotypic stability still need to be improved. This inherent defect limits the application value of the model.

[0005] Bronchoalveolar lavage fluid (BALF) is a lung fluid sample obtained through bronchoalveolar lavage, which directly reflects the local microenvironment of the alveoli. BALF contains cellular components, inflammatory mediators, chemokines, antibodies, complement, surfactant, and other soluble protein components. LINPENG Z et al. pointed out that BALF from patients with CIP exhibits characteristic changes associated with local lung immune activation and inflammatory damage, and may be closer to the true pathological environment of the CIP site than peripheral blood samples (LINPENG Z, JING Y, YAXIAN Q, et al. Bronchoalveolar lavage fluid in immune checkpoint inhibitor-related pneumonitis: from pathophysiological window to aprecision diagnostic tool[J]. Frontiers in Immunology, 2026, 17: 1788186.). Therefore, BALF has potential value as a source for inducing an in vitro model of CIP. However, untreated BALF has a complex composition and may contain mucus, cell debris, proteases, microbial contaminants, and other factors that affect the stability of in vitro culture, making it unsuitable for direct culture. Therefore, how to properly process BALF samples and establish a stable, reproducible, interventional, and quantifiable in vitro pathological model of CIP to provide an experimental platform for CIP pathogenesis research, toxicity assessment, drug evaluation, and clinical translation remains a technical problem that urgently needs to be solved in this field.

[0006] In summary, current technologies still lack an in vitro model that can simultaneously reflect the characteristics of human lung epithelial tissue and the local inflammatory microenvironment of the lungs in CIP patients. Establishing such a model is of great significance for studying the pathogenesis of CIP, evaluating immunotherapy-related lung toxicity, screening interventional drugs, and promoting the development of personalized treatment strategies. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a method for constructing an in vitro model of immune checkpoint inhibitor-associated pneumonia (CIP) based on human lung organoids. This invention utilizes human lung organoid culture, combined with bronchoalveolar lavage fluid (BALF) from CIP patients, to successfully construct an in vitro pathological model of CIP, laying the foundation for studying the pathological mechanisms of CIP and evaluating related drugs.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing an in vitro model of immune checkpoint inhibitor-associated pneumonia based on human lung organoids. The method involves co-culturing bronchoalveolar lavage fluid from CIP patients with lung organoids to obtain an in vitro model of immune checkpoint inhibitor-associated pneumonia.

[0009] Preferably, the bronchoalveolar lavage fluid is the supernatant that has been filtered and centrifuged to remove impurities and cells.

[0010] Preferably, the supernatant also includes Primocin and / or a protease inhibitor.

[0011] Preferably, the final concentration of the bronchoalveolar lavage fluid is 5% to 50%.

[0012] Preferably, the lung organoids include whole lung organoids or alveolar organoids.

[0013] Preferably, the method for preparing the whole lung organoid includes the following steps: (1) Clean lung tissue, remove impurities, cut into pieces, add tissue digestion solution, digest for 0.5-1.5 h, add digestion termination solution, grind and filter, centrifuge, discard supernatant, add red blood cell lysis solution, incubate for 5-15 min, add washing solution to terminate lysis, centrifuge, discard supernatant, add DMEM / F12 medium, filter, and obtain cells; (2) The cells were suspended in a mixture of culture medium and matrix gel and allowed to stand. Lung organoid culture medium containing Y-27632 was added and cultured to obtain whole lung organoids.

[0014] Preferably, the method for preparing alveolar organoids includes the following steps: obtaining cells using the method in step (1), resuspending the cells in blocking antibody, incubating for 5-15 min, adding magnetic beads, incubating for 20-40 min, washing with MACS buffer, centrifuging, discarding the supernatant, adding MACS buffer, loading the sample onto a MACS sorting column, eluting, removing the sorting column, washing with MACS buffer, collecting the epithelial cell components, centrifuging, resuspending in MACS buffer, centrifuging, collecting the precipitate, adding MACS buffer and IgM magnetic beads to the precipitate, incubating for 20-40 min, adding MACS buffer, centrifuging, collecting the precipitate, adding MACS buffer to the precipitate, adding it to an MS column, washing with MACS buffer 1-5 times, removing the MS column, adding MACS buffer to collect the effluent, obtaining alveolar epithelial cells, seeding the alveolar epithelial cells in a matrix gel for culture, adding alveolar organoid culture medium containing Y-27632 for culture, and obtaining alveolar organoids.

[0015] Preferably, the initial inoculation quantity of the lung organoids is 500 to 4000 cells.

[0016] Preferably, the co-culture time is 1 to 10 days.

[0017] This invention also provides the application of the in vitro model of immune checkpoint inhibitor-associated pneumonia obtained according to the described construction method in screening drugs for the treatment of CIP.

[0018] Compared with existing technologies, the present invention has the following beneficial effects: The present invention filters and centrifuges BALF directly derived from CIP patients to obtain a supernatant free of mucus, impurities, and cells, effectively removing cellular interference. Primocin is added to the supernatant to inhibit mycoplasma, fungal, and bacterial contamination, while protease inhibitors are added to reduce protein degradation. After filtration and sterilization, BALF supernatant is obtained for co-culturing with lung / alveolar organoids. Co-culturing this supernatant with lung / alveolar organoids successfully constructs a stable in vitro model of immune checkpoint inhibitor-associated pneumonia, laying the foundation for studying the pathological mechanism of CIP and evaluating related drugs. Attached Figure Description

[0019] Figure 1 To assess the effect of BALF on lung organoid activity, the study included... This indicates that P < 0.05; Figure 2 The figures show the stability of the CIP in vitro pathological model, where a is the donor stability figure and b is the batch stability figure. Figure 3 The dose-response relationship between cell seeding density and ATP luminescence signal intensity is shown, where This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001; Figure 4 The effect of co-culture days on cell viability; Figure 5 The effect of co-culture days on cell damage; Figure 6 The effect of BALF concentration on cell viability, among which This indicates that P < 0.05. This indicates that P < 0.01; Figure 7 The effect of BALF concentration on cell damage, among which This indicates that P < 0.05; Figure 8 The effect of BALF on the activity of lung organoids was shown by staining for live and dead cells, with the scale bar at 500 μm; Figure 9Immunofluorescence staining to show the effect of CIP BALF co-culture on lung organoid proliferation capacity, with a scale bar of 100 μm; Figure 10 The expression of genes related to inflammation and injury response in the CIP in vitro pathological model, among which This indicates that P < 0.05; Figure 11 The expression of genes related to lung epithelial cell function and lineage markers in the CIP in vitro pathological model, among which This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001. Detailed Implementation

[0020] This invention provides a method for constructing an in vitro model of immune checkpoint inhibitor-associated pneumonia based on human lung organoids. The method involves co-culturing bronchoalveolar lavage fluid from CIP patients with lung organoids to obtain an in vitro model of immune checkpoint inhibitor-associated pneumonia.

[0021] In this invention, bronchoalveolar lavage fluid from CIP patients is preferably filtered through a 40-100 μm cell sieve to remove sputum and large particulate impurities, yielding a filtrate. The filtrate is then centrifuged at 600-1000g for 5-15 min at 2-6°C to obtain a supernatant and cell pellet. The supernatant is used for co-culture. The preferred particle size of the cell sieve is 60-80 μm, more preferably 70 μm. The preferred centrifugation temperature is 3-5°C, more preferably 4°C. The preferred centrifugation speed is 700-900g, more preferably 800g. The preferred centrifugation time is 8-12 min, more preferably 10 min.

[0022] In this invention, preferably, 100 μg / mL Primocin is added to the supernatant to inhibit mycoplasma, fungal and bacterial contamination, and the protease inhibitor is added to BALF at a ratio of protease inhibitor: supernatant = 1:200 to reduce protein degradation. Then, the supernatant is sterilized by filtration using a 0.22 μm filter membrane to obtain bronchoalveolar lavage fluid for co-culture with lung organoids.

[0023] In this invention, the final concentration of the bronchoalveolar lavage fluid is 5% to 50%, preferably 10% to 30%, and more preferably 25%.

[0024] In this invention, the lung organoids include whole lung organoids or alveolar organoids.

[0025] In this invention, the method for preparing the whole lung organoid includes the following steps: (1) Clean lung tissue, remove impurities, cut into pieces, add tissue digestion solution, digest for 0.5-1.5 h, add digestion termination solution, grind and filter, centrifuge, discard supernatant, add red blood cell lysis solution, incubate for 5-15 min, add washing solution to terminate lysis, centrifuge, discard supernatant, add DMEM / F12 medium, filter, and obtain cells; (2) The cells were suspended in a mixture of culture medium and matrix gel and allowed to stand. Lung organoid culture medium containing Y-27632 was added and cultured to obtain whole lung organoids.

[0026] In this invention, preferably, 1g of surgically removed normal tissue from the distal lung is collected, repeatedly washed in pre-cooled PBS containing penicillin-streptomycin antibiotics to remove pleura, trachea, arteries, and veins, and then the remaining tissue is cut into pieces to approximately 1-2mm. 3 The cells were transferred to centrifuge tubes containing 10 mL of tissue digestion solution and digested at 37°C and 10 rpm for 0.5–1.5 h. After digestion, an equal volume of digestion termination solution (PBS + 5 mM EDTA + 1% penicillin antibody + 20% FBS) was added to terminate the digestion. Subsequently, the cells were ground and filtered twice through a 100 μm cell filter, and the filtrate was collected and centrifuged at 300 g for 10 min at 4°C. The supernatant was discarded, and the cell pellet was resuspended in 5 mL of erythrocyte lysis buffer and incubated at room temperature (25°C) in the dark for 10 min. Lysis was terminated by adding 5 times the volume of erythrocyte lysis buffer. The cells were centrifuged at 300 g at 4°C for 10 min, the supernatant was discarded, and the cell pellet was resuspended in 10 mL of DMEM / F12 medium. After filtration through a 70 μm cell filter, cell counting was performed. The tissue digestion solution includes DMEM-F12, Primocin at a final concentration of 100 μg / mL, Dispase I at a final concentration of 50 U / mL, Collagenase D at a final concentration of 625 μg / mL, and DNase I at a final concentration of 200 μg / mL. The digestion time is preferably 0.75~1.25 h, more preferably 1 h, and the incubation time is preferably 8~12 min, more preferably 10 min.

[0027] In this invention, preferably, the obtained cells are resuspended at a density of 1000 cells / μL in a mixture of culture medium and matrix gel, seeded into 24-well plates, and incubated at 37°C for approximately 10 minutes to allow the matrix gel to solidify. Then, 500 μL of lung organoid culture medium containing 10 μM Y-27632 is added to each well, and the plates are cultured at 37°C and 5% CO2. The medium is changed every 4 days, and the plates are passaged approximately every 14 days to obtain whole lung organoids.

[0028] In this invention, the method for preparing alveolar organoids includes the following steps: obtaining cells using the method in step (1), resuspending the cells in blocking antibody, incubating for 5-15 min, adding magnetic beads, incubating for 20-40 min, washing with MACS buffer, centrifuging, discarding the supernatant, adding MACS buffer, loading the sample onto a MACS sorting column, eluting, removing the sorting column, washing with MACS buffer, collecting epithelial cell components, centrifuging, resuspending in MACS buffer, centrifuging, collecting the precipitate, adding MACS buffer and IgM magnetic beads to the precipitate, incubating for 20-40 min, adding MACS buffer, centrifuging, collecting the precipitate, adding MACS buffer to the precipitate, adding to an MS column, washing with MACS buffer 1-5 times, removing the MS column, adding MACS buffer to collect the effluent, obtaining alveolar epithelial cells, seeding the alveolar epithelial cells in a matrix gel for culture, adding alveolar organoid culture medium containing Y-27632 for culture, and obtaining alveolar organoids.

[0029] In this invention, it is preferred that the cells obtained in step (1) are used at a concentration of 1×10⁻⁶. 7Resuspend the cells in 60 μL of MACS buffer, add 20 μL of FCR blocking antibody, mix well, and incubate for 10 min. Then add 20 μL of CD326 (EpCAM) magnetic beads, mix well, and incubate on ice for 30 min. Wash with 1 mL of MACS buffer, centrifuge at 300 g for 10 min at 4 °C, and discard the supernatant. Resuspend the pellet in 500 μL of MACS buffer, load it onto a MACS sorting column, elute with 500 μL of MACS buffer, remove the sorting column, wash with 1 mL of MACS buffer, and collect the epithelial cell fraction (EpCAM-positive cells). Centrifuge the EpCAM-positive cells at 300 g for 10 min at 4 °C, resuspend the pellet in 300 μL of MACS buffer, add 6 μL of mouse anti-human HTII-280 antibody, and incubate at 4 °C for 1 h. Wash with 1 mL of MACS buffer, centrifuge at 450 g for 5 min at 4 °C, and repeat the washing once. Add 80 μL of MACS buffer and 20 μL of IgM magnetic beads to the precipitate, mix well, and incubate at 4°C in the dark for 30 min. Add 1 mL of MACS buffer and centrifuge at 300 g for 10 min at 4°C. Resuspend the precipitate in 500 μL of MACS buffer, add it to an MS column, and wash three times with 500 μL of MACS buffer. Remove the MS column, add 1 mL of MACS buffer, and collect the eluent to obtain alveolar epithelial cell (AT2 cell) fraction. Count the obtained AT2 cells and seed them at a density of 1000 cells / μL in Matrigel. After the Matrigel solidifies, add 500 μL of alveolar organoid culture medium containing 10 μM Y-27632 to each well and culture at 37°C and 5% CO2. Change the medium every 4 days and passage approximately every 14 days to obtain alveolar organoids.

[0030] In this invention, the initial inoculation amount of the lung organoids is 500-4000 cells, preferably 800-4000 cells, and more preferably 1000-4000 cells.

[0031] In this invention, the co-cultivation time is 1 to 10 days, preferably 3 to 8 days, and more preferably 5 days.

[0032] This invention also provides the application of the in vitro model of immune checkpoint inhibitor-associated pneumonia obtained according to the described construction method in screening drugs for the treatment of CIP.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example

[0035] 1. Establishment of lung organoids / alveolar organoids

[0036] 1.1 Culture of whole lung organoids

[0037] One g of normal tissue from the distal lung removed during surgery was collected and repeatedly washed in pre-cooled PBS containing penicillin-streptomycin antibiotics. After removing the pleura, trachea, arteries, and veins, the remaining tissue was minced to approximately 1-2 mm. 3 The samples were transferred to centrifuge tubes containing 10 mL of tissue digestion solution (DMEM-F12, containing 100 μg / mL Primocin, 50 U / mL Dispase I, 625 μg / mL Collagenase D, and 200 μg / mL DNase I) and digested at 37°C and 10 rpm for 1 h. After digestion, an equal volume of digestion termination solution (PBS + 5 mM EDTA + 1% penicillin-dextrin antibody + 20% FBS) was added to terminate the digestion.

[0038] Subsequently, the cells were ground and filtered twice through a 100 μm cell filter, and the filtrate was collected and centrifuged at 300 g for 10 min at 4 °C. After discarding the supernatant, the cell pellet was resuspended in 5 mL of erythrocyte lysis buffer and incubated at room temperature (25 °C) in the dark for 10 min. Lysis was terminated by adding 5 times the volume of erythrocyte lysis buffer. The cells were centrifuged at 300 g at 4 °C for 10 min, the supernatant was discarded, and the cell pellet was resuspended in 10 mL of DMEM / F12 medium. After filtering through a 70 μm cell filter, cell counting was performed.

[0039] The obtained cells were resuspended at a density of 1000 cells / μL in a mixture of culture medium and matrix gel, and seeded into 24-well plates. The plates were then incubated at 37°C for approximately 10 minutes to allow the matrix gel to solidify. Subsequently, 500 μL of lung organoid culture medium containing 10 μM Y-27632 was added to each well, and the plates were incubated at 37°C with 5% CO2. The medium was changed every 4 days, and the plates were passaged approximately every 14 days.

[0040] 1.2 Culture of alveolar organoids

[0041] The pretreatment steps for alveolar organoids were the same as those for lung tissue digestion and single-cell preparation described above. The separated lung single-cell suspension was further sorted using EpCAM and HTII-280 antibodies via magnetic bead separation to enrich and obtain AT2 cells.

[0042] The specific steps are as follows: 1×10 7Resuspend the cells in 60 μL MACS buffer, add 20 μL FcR blocking antibody, mix well, and incubate for 10 min; then add 20 μL CD326 (EpCAM) magnetic beads, mix well, and incubate on ice for 30 min. Wash with 1 mL MACS buffer, centrifuge at 300 g for 10 min at 4 °C, and discard the supernatant. Resuspend the precipitate in 500 μL MACS buffer, load it onto a MACS sorting column, and elute with 500 μL MACS buffer (4 times). After removing the sorting column, wash with 1 mL MACS buffer and collect the epithelial cell fraction (EpCAM-positive cells).

[0043] EpCAM-positive cells were centrifuged at 300g for 10 min at 4°C. The pellet was resuspended in 300 μL of MACS buffer, and 6 μL of mouse anti-human HTII-280 antibody was added. The mixture was incubated at 4°C for 1 h. After washing with 1 mL of MACS buffer, the cells were centrifuged at 450g for 5 min at 4°C, and the washing was repeated once. 80 μL of MACS buffer and 20 μL of IgM magnetic beads were added to the pellet, mixed well, and incubated at 4°C in the dark for 30 min. Another 1 mL of MACS buffer was added, and the cells were centrifuged at 300g for 10 min at 4°C. The pellet was resuspended in 500 μL of MACS buffer and added to an MS column. The cells were washed three times with 500 μL of MACS buffer. After removing the MS column, 1 mL of MACS buffer was added, and the eluent was collected as the AT2 cell fraction.

[0044] After counting the obtained AT2 cells, they were seeded into a matrix gel at a density of 1000 cells / μL and cultured. After the matrix gel solidified, 500 μL of alveolar organoid culture medium containing 10 μM Y-27632 was added to each well, and the cells were cultured at 37°C and 5% CO2. The medium was changed every 4 days, and the cells were passaged approximately every 14 days.

[0045] 1.3 Passage of Lung Organoids / Alveolar Organoids

[0046] Remove the culture medium from each well, add 1 mL of Cell Recovery Solution to each well, incubate at room temperature for 1 min, then pipette to disperse the matrix gel and collect it into a centrifuge tube. Place the centrifuge tube on crushed ice and defrost at 4°C for 1 h. Then centrifuge at 4°C, 290 g for 5 min, discard the supernatant, and retain the precipitate.

[0047] Add 0.5 mL of TrypLE to each well of the pellet, mix thoroughly by pipetting, and digest at 37°C for 10 min. Stop digestion by adding 10% FBS-DMEM / F12 medium, count cells, and centrifuge again at 4°C, 290 g for 5 min, discarding the supernatant. Resuspend the resulting cell pellet at a density of 1000 cells / μL in a mixture of medium and matrix gel, seed in 24-well plates, and incubate at 37°C for 10 min to allow the matrix gel to solidify. Then, add organoid culture medium containing 10 μM Y-27632 and continue culturing.

[0048] 2. Processing of BALF Collections

[0049] BALF samples were collected from CIP patients (Peking University First Hospital). After collection, the samples were transported on ice and processed within 2 hours. Fresh BALF samples were filtered through a 70 μm cell sieve to remove sputum and larger particulate impurities. The filtered BALF was centrifuged at 4°C and 800g for 10 min to separate the supernatant and cell pellet. The BALF supernatant was used for subsequent co-culture experiments.

[0050] To reduce the impact of microbial contamination and protein degradation on model stability, 100 μg / mL Primocin was added to the BALF supernatant to inhibit mycoplasma, fungal, and bacterial contamination. Protease inhibitors were added to the BALF at a ratio of 1:200 (protease inhibitor: supernatant = 1:200) to reduce protein degradation. The BALF supernatant was then sterilely filtered through a 0.22 μm membrane, aliquoted, and stored. Before use, the protein concentration in the BALF supernatant was quantified using the BCA method to facilitate batch-to-batch standardization.

[0051] The control group BALF was derived from BALF samples from non-CIP tumor patients and was pretreated under the same conditions.

[0052] 3. Establishment and optimization of the lung organoid-BALF co-culture model

[0053] After passaged lung / alveolar organoids were cultured for 3-4 days until growth stabilized, pretreated BALF supernatant was added to the culture system in a specific ratio. The experiment included a CIP BALF group (BALF supernatant from CIP patients), a Control BALF group (BALF supernatant from control patients), and a Control group (PBS with an equal volume of BALF supernatant). ATP activity assays and LDH cytotoxicity assays were used to quantitatively assess the cell viability of the co-cultured organoids. The stability of the model was validated using different CIP BALF (n>3), organoids from different donor sources (n=3), and different batches of organoids (P3, P4, P7). Results are shown in [Figure number missing]. Figures 1-2 .

[0054] Depend on Figures 1-2 It can be seen that, compared with the control group, organoids treated with CIP BALF showed decreased cell viability. Figure 1 Furthermore, the model obtained using the method of this invention is stable between donors and batches. Figure 2 ).

[0055] To determine stable and reproducible co-culture conditions, suitable for CIP in vitro pathological simulation was obtained by adjusting the BALF concentration, incubation time, and organoid seeding density. Co-culture time: 1 day, 3 days, 5 days, and 7 days; final BALF concentration: 0%, 5%, 25%, and 50%; initial organoid seeding density: 1000, 2000, and 4000 cells per well. The cell viability and damage level of organoids under different conditions were quantitatively assessed using an ATP activity assay kit and an LDH cytotoxicity assay kit. Results are shown in [Figure number missing]. Figures 3-7 .

[0056] Depend on Figures 3-7 It can be seen that when the final concentration of BALF is 25%, the co-culture time is 5 days, and the number of cells seeded per well is not less than 1000, the decline in organoid viability is relatively stable and the difference is obvious. Therefore, it can be used as an optimization condition for co-culture.

[0057] 4. Characterization and detection of lung organoid-BALF co-culture model

[0058] After establishing the co-culture model, the model was characterized by indicators such as cell activity, cell death, proliferation capacity and related gene expression. (1) Cell activity detection: Calcein AM / PI live and dead cell staining was used to detect the cell activity of lung organoids. ATP activity detection and LDH release detection could also be combined to quantitatively analyze the overall viability and degree of damage of organoids. (2) Cell proliferation capacity detection: Immunofluorescence staining was used to detect the level of the proliferation marker Ki67, which reflects the proliferation capacity of stem / progenitor cells in organoids. (3) Damage and function-related gene detection: RT-qPCR was used to detect the expression of genes such as MUC-1, CRP, SFTPC, and STFPA. Among them, MUC1 and CRP are lung injury-related genes, KL-6 (MUC1) and CRP are both clinical CIP serological markers, and SFTPC and SFTPA can reflect alveolar function. The results are shown in […]. Figures 8-11 .

[0059] Depend on Figures 8-11 It can be seen that, compared with the control group, organoids treated with CIP BALF showed an increase in dead cells. Figure 8 Ki67 expression decreased ( Figure 9 ), MUC1 ( Figure 10Elevated CRP expression and SFTPA and SFTPC ( Figure 11 The expression of these characteristics was reduced. These results indicate that the co-culture system provided by this invention can simulate the damage and functional changes in lung epithelial tissue caused by the CIP-related inflammatory microenvironment, and can be used for CIP pathological mechanism research and related drug evaluation.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an in vitro model of immune checkpoint inhibitor-associated pneumonia based on human lung organoids, characterized in that, The construction method is as follows: bronchoalveolar lavage fluid from CIP patients is co-cultured with lung organoids to obtain an in vitro model of immune checkpoint inhibitor-associated pneumonia.

2. The construction method according to claim 1, characterized in that, The bronchoalveolar lavage fluid is the supernatant that has been filtered and centrifuged to remove impurities and cells.

3. The construction method according to claim 2, characterized in that, The supernatant also includes Primocin and / or protease inhibitors.

4. The construction method according to claim 3, characterized in that, The final concentration of the bronchoalveolar lavage fluid is 5% to 50%.

5. The construction method according to claim 1, characterized in that, The lung organoids include whole lung organoids or alveolar organoids.

6. The construction method according to claim 5, characterized in that, The method for preparing the whole lung organoid includes the following steps: (1) Clean lung tissue, remove impurities, cut into pieces, add tissue digestion solution, digest for 0.5-1.5 h, add digestion termination solution, grind and filter, centrifuge, discard supernatant, add red blood cell lysis solution, incubate for 5-15 min, add washing solution to terminate lysis, centrifuge, discard supernatant, add DMEM / F12 medium, filter, and obtain cells; (2) The cells were suspended in a mixture of culture medium and matrix gel and allowed to stand. Lung organoid culture medium containing Y-27632 was added and cultured to obtain whole lung organoids.

7. The construction method according to claim 6, characterized in that, The method for preparing alveolar organoids includes the following steps: obtaining cells using the method of step (1) of claim 6, resuspending the cells in blocking antibody, incubating for 5-15 min, adding magnetic beads, incubating for 20-40 min, washing with MACS buffer, centrifuging, discarding the supernatant, adding MACS buffer, loading onto a MACS sorting column, eluting, removing the sorting column, washing with MACS buffer, collecting epithelial cell components, centrifuging, resuspending in MACS buffer, centrifuging, collecting the precipitate, adding MACS buffer and IgM magnetic beads to the precipitate, incubating for 20-40 min, adding MACS buffer, centrifuging, collecting the precipitate, adding MACS buffer to the precipitate, adding to an MS column, washing with MACS buffer 1-5 times, removing the MS column, adding MACS buffer to collect the effluent, obtaining alveolar epithelial cells, seeding the alveolar epithelial cells in a matrix gel for culture, adding alveolar organoid culture medium containing Y-27632 for culture, and obtaining alveolar organoids.

8. The construction method according to claim 1, characterized in that, The initial inoculation quantity of the lung organoids is 500-4000 cells.

9. The construction method according to claim 1, characterized in that, The co-culture time is 1 to 10 days.

10. The application of the in vitro model of immune checkpoint inhibitor-associated pneumonia obtained by the construction method according to any one of claims 1 to 9 in screening drugs for the treatment of CIP.

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