Human distal lung organ and culture method and application thereof

Abstract

The invention discloses a human distal lung organoid and a culture method and application thereof, and the organoid comprises a basal cell-derived organoid which is in a solid sphere shape; the organ-like body secretes a cell source and is in a single-cavity or multi-cavity form; the respiratory tenobronchial cell-derived organoid is in a single-cavity or multi-cavity form; the AT2 cell-derived organoid is in a saccular cavity shape; the strain is obtained through culture of a culture medium, passage, cryopreservation and resuscitation, and the culture medium comprises an Advanced DMEM / F12 culture medium. A small molecule compound: A83-01; sB202190, SB202190; y-27632, Y-27632; a human recombinant protein: EGF; an FGF10 (fibroblast growth factor 10); an FGF7; r, R-pondin; a Noggin; and a supplement. The human far-end lung organ comprises airway and alveolar cells, and can be used for drug screening, tumor animal model construction, diagnostic reagent preparation, regenerative medicine, scientific research of in-vitro non-treatment purposes and the like.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a human distal lung organoid and its culture method and application. Background Art

[0002] Organoids are an exciting stage in stem cell research. Researchers have successfully developed a method to direct the differentiation of embryonic stem cells and induced pluripotent stem cells (hPSCs) into 3D lung organoids, a process similar to branching morphogenesis, which can be used to simulate fibrotic interstitial lung diseases and viral infections.

[0003] Human distal lung organoids are a three-dimensional tissue structure cultured in vitro that can mimic the physiological and pathological characteristics of the human distal lung, contain terminal bronchioles and alveoli that promote gas exchange, can be cultured in vitro for a long time, and can express molecules related to human distal lung infections such as ACE2, making them a powerful tool for studying viral infections and other lung diseases. Constructing infection models through human distal lung organoids, including animal models, helps to promote applications in the pharmaceutical industry such as precision medicine, disease models, drug development, drug sensitivity testing, and gene editing.

[0004] In the prior art, for example, patent document CN115667496A discloses a method for manufacturing organoids from lung epithelial cells or lung cancer cells, including the step of culturing a sample containing lung epithelial cells or lung cancer cells, a method for manufacturing organoids from lung epithelial cells or lung cancer cells, the culture medium contains 0-10% v / v extracellular matrix, and contains a combination of at least one selected from keratinocyte growth factor (KGF), fibroblast growth factor (FGF) 10, and hepatocyte growth factor (HGF), a bone morphogenetic protein (BMP) inhibitor, and a TGFβ inhibitor, and is substantially free of feeder cells. Patent document CN117757721A discloses a culture medium for culturing lung cancer organoids and a method for culturing lung cancer organoids. Activin A and SB431542 added to this culture medium are related to tumorigenesis and promote cell proliferation. Adding them to the lung cancer organoid culture medium can effectively generate lung cancer organoids, and can be cultured and passaged in vitro for a long time. The cultured cells can be used for targeted drug screening for lung cancer, guiding the treatment of clinical lung cancer patients with precise drug use. Patent document CN117247904A discloses lung cancer organoids and their culture method and application, as well as a method for preparing a lung cancer organoid culture medium. The lung cancer organoid culture medium includes a basal medium, a conditioned medium, and a specific additive factor, does not contain serum, and the specific additive factor includes HGF and / or VEGF. It can grow rapidly in vitro, the formed lung cancer organoids have regular shapes and uniform sizes, have the morphological characteristics of lung cancer tissues in vitro culture, and better retain the heterogeneity of lung cancer tissues.

[0005] In summary, airway and alveolar cell types exist in human distal lung organoids. Using organoids to conduct research on related biomarkers and therapeutic targets during acute lung injury and repair is of great significance for deeply understanding the pathogenesis of lung diseases and developing new treatment strategies. Summary of the Invention

[0006] In view of the above deficiencies in the prior art, the main object of the present invention is to provide a human distal lung organoid, which includes airways and alveolar cells.

[0007] Another object of the present invention is to provide a method for culturing the human distal lung organoid.

[0008] Still another object of the present invention is to provide the application of the human distal lung organoid in drug screening, constructing tumor animal models, preparing diagnostic reagents, regenerative medicine, and scientific research for non-therapeutic purposes in vitro.

[0009] To achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0010] In the first aspect of the present invention, a human distal lung organoid is provided, including basal cell-derived organoids (BCOs), which present as solid spherical shapes; secretory cell-derived organoids (SCOs), which present as single-lumen or multi-lumen morphologies; respiratory bronchiole cell-derived organoids (RASC-Org), which present as single-lumen or multi-lumen morphologies; and AT2 cell-derived organoids (AT2-Org), which present as sac-like hollow morphologies.

[0011] Preferably, the BCOs express the basal cell markers KRT5 and PDPN.

[0012] Preferably, the SCOs express the secretory cell markers SFTPB and SCGB1A1.

[0013] Preferably, the RASC-Org express the markers SFTPB and SCGB3A2 related to respiratory bronchioles.

[0014] Preferably, the AT2-Org express the AT2 cell marker SFTPC.

[0015] In the second aspect of the present invention, a human distal lung organoid culture medium is also provided, which includes components with the following contents (final concentrations):

[0016] Basal medium: Advanced DMEM / F12 medium;

[0017] Small molecule compounds: A83-01, 0.5 uM; SB202190, 0.5 uM; Y-27632, 10 uM;

[0018] Human recombinant proteins: EGF, 50 ng / mL; FGF10, 100 ng / mL; FGF7, 25 ng / mL; R-spondin, 500 ng / mL; Noggin, 100 ng / mL;

[0019] Supplements: Nicotinamide, 5 mM; B-27, 1×; Antibotic-Antimycotic, 100×; HEPES, 10 mM; GlutaMAX, 2 mM; N-Acetyl-L-Cysteine, 1.25 mM.

[0020] The third aspect of the present invention further provides a method for culturing the human distal lung organoids, comprising the following steps:

[0021] (i) The lung tissue is preserved in Advanced DMEM / F12 medium containing 1× Anti-Anti and placed on ice for standby;

[0022] (ii) First, the lung tissue is minced with sterile scissors, and then the tissue is resuspended with the digestive solution. The lung tissue is enzymatically digested at a constant temperature of 37 °C at a rotation speed of 110 rpm for 40 minutes. The lung tissue is pipetted every 10 minutes using a 1 mL pipette tip, and the front end of the pipette tip is cut off with sterile scissors;

[0023] (iii) The cells obtained after digestion are filtered through a 100 μm cell strainer and rinsed with DMEM / F12 medium containing 10% FBS;

[0024] (iv) The cells are centrifuged at 300×g for 5 minutes, the supernatant is removed, and the cells are lysed with red blood cell lysate for 3 minutes; if there is residual red blood cell, this step is repeated until there is no visible residual red blood cell in the cells;

[0025] (v) First, the cells are washed with 1× DPBS, then resuspended with Matrigel, and inoculated into a 24- or 48-well culture plate treated with tissue culture. The human distal lung organoid medium is as described above, and the human distal lung organoids are obtained.

[0026] Preferably, the lung tissue is derived from a patient with a lung nodule who has undergone lobectomy, and the tissue 1 cm from the outermost edge of the lung lobe is collected.

[0027] Preferably, it further comprises the step of treating the human distal lung organoids with 3 μM CHIR99021 or 100 ng / mL recombinant NRG1 protein.

[0028] Preferably, it further comprises the steps of passage, cryopreservation and resuscitation of the human distal lung organoids.

[0029] More preferably, the passage process of the human distal lung organoids includes the following steps:

[0030] (1) Culture the human distal lung organoids in a 24- or 48-well culture plate for 10 - 14 days; wash the culture wells with 500 μL of PBS and aspirate; add 300 μL of TypLE-EDTA to the wells, scrape and suspend the Matrigel with a 1000 μL pipette tip, and gently pipette 6 - 10 times;

[0031] (2) Incubate the 24- or 48-well culture plate in a cell culture incubator, pipette 6 - 10 times every 5 minutes; add 600 - 800 μL of washing medium, dissociate the human distal lung organoids by gently pipetting, and retain cell clusters with more than 100 cells for routine passage;

[0032] (3) Transfer the sphere suspension to a 15 mL centrifuge tube, add 5 mL of washing medium; centrifuge at 300 g for 5 minutes at room temperature; aspirate the supernatant, leaving 100 μL; add 500 μL - 1 mL of washing medium to resuspend the cells; transfer the cells to a 1.5 mL tube with a dilution ratio of 1:4 - 8; centrifuge at 300 g for 5 minutes at room temperature; completely aspirate the supernatant, first aspirate with a vacuum pump, leaving 100 μL, and then completely aspirate the remaining supernatant with a 200 μL or 10 μL pipette;

[0033] (4) Place the tube on ice, resuspend the spheres with 30 μL of Matrigel per well, gently pipette until the cell clusters are completely dispersed; place the centrifuge tube and the 24- or 48-well culture plate on ice, pipette 30 μL of the cell-Matrigel suspension to the center of each well with a pipette, and then spread it with the pipette tip;

[0034] (5) Incubate the 24- or 48-well culture plate in a cell culture incubator to polymerize the Matrigel, invert the culture plate to prevent the cells from sinking to the surface of the well plate; add 500 μL of medium to the wells of the 24- or 48-well culture plate and culture for 2 - 3 days, changing the medium at least once every 2 days to obtain human distal lung organoids.

[0035] More preferably, the cryopreservation process of the human distal lung organoids includes: culturing the human distal lung organoids in a culture plate for 10 - 14 days, scraping and suspending the Matrigel in the medium by gentle pipetting, transferring the suspension to a 1.5 mL tube or a 15 mL centrifuge tube, centrifuging at 300 g for 5 minutes at room temperature, aspirating the supernatant, leaving 100 μL, and then gently resuspending the human distal lung organoids in 500 μL of cryopreservation solution per well, transferring the cell suspension to a cryotube, placing the cryotube in a programmable freezing container, freezing the programmable freezing container at -80 °C using an ultra-low temperature freezer, and storing the cryotube in a liquid nitrogen tank.

[0036] More preferably, the resuscitation process of the human distal lung organoids includes: incubating the frozen cryotube in a 37°C water bath for rapid thawing, transferring it to a laminar flow hood, transferring the human distal lung organoids into a pre-warmed 15 mL centrifuge tube, adding 9 mL of washing medium, centrifuging at 300 g for 5 minutes, and culturing the resuscitated human distal lung organoids in the wells of a culture plate.

[0037] The present invention also provides the application of the human distal lung organoids, including their application in drug screening, constructing tumor animal models, preparing diagnostic reagents, regenerative medicine, and scientific research for non-therapeutic purposes in vitro.

[0038] Preferably, the human distal lung organoids serve as a screening model for biomarkers and therapeutic targets during acute lung injury and repair.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows: The human distal lung organoids cultured in the present invention include airway and alveolar cells, and can be used in aspects such as drug screening, constructing tumor animal models, preparing diagnostic reagents, regenerative medicine, and scientific research for non-therapeutic purposes in vitro. Especially as a screening model for biomarkers and therapeutic targets during acute lung injury and repair, it can more comprehensively reproduce various epithelial cell types and physiological states of the real human distal bronchiole, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Immunofluorescence staining identification picture of distal lung organoids in the example; the distal lung organoids contain multiple cell types, including airway and alveolar cells; BCO presents as a solid sphere and expresses basal cell markers KRT5 (green) and PDPN (red), SCO and RASC-Org present as single-lumen and multi-lumen morphologies respectively, and AT2-Org presents as a cystic cavity-like organoid; scale bar: 50 μm.

[0041] Figure 2 Flow cytometry analysis diagram of cells obtained by digesting human distal lung tissue in the example, with the red circle indicating the NGFR+ positive population.

[0042] Figure 3 Result of a large proportion of basal cells being produced by human distal lung organoids cultured in airway medium in the example.

[0043] Figure 4 Flow cytometry analysis diagram of human distal lung organoids in the example.

[0044] Figure 5 Situation of abnormal basal cells expressing pro-inflammatory and profibrotic related genes in the example.

[0045] Figure 6 Schematic flow chart of comparing the contributions of different cell types in the distal lung to CD66c+ abnormal basal cells in the example.

[0046] Figure 7 Verification of the ability of CD66c+ secretory cells to form abnormal basal cells in the example; (A) Flow sorting protocol for CD66c+ secretory cells; (B) Bright-field images and immunofluorescence staining images of organoids formed by CD66c+ secretory cells; (C) Flow analysis diagram of organoids formed by CD66c+ secretory cells.

[0047] Figure 8 Immunofluorescence staining images of serial sections of organoids in the example.

[0048] Figure 9 Verification of the ability of CD66c- basal cells to form abnormal basal cells in the example; (A) Flow sorting protocol for CD66c- basal cells; (B) Bright-field images and immunofluorescence staining images of organoids formed by CD66c- basal cells; Scale bar: 50 μm.

[0049] Figure 10 Bright-field image (left) of AT2 cells cultured in airway medium and immunofluorescence staining image (right) of serial sections of the same organoid in the example.

[0050] Figure 11 Cultivation of distal lung organoids in the example under culture conditions with or without CHIR; (A) Morphological observation and immunofluorescence staining of distal lung organoids cultured under culture conditions with or without CHIR; (B) Flow analysis diagram of digested distal lung organoids cultured under culture conditions with or without CHIR.

[0051] Figure 12 Cultivation of sorted CD66c+ secretory cells in the example under culture conditions with or without CHIR; (A) Morphological observation and immunofluorescence staining of organoids cultured under culture conditions with or without CHIR; (B) Flow analysis diagram of digested organoids cultured under culture conditions with or without CHIR.

[0052] Figure 13 Bright-field image (left) and immunofluorescence staining image (right) of organoids formed by sorted CD66c- basal cells cultured under culture conditions with or without CHIR in the example.

[0053] Figure 14 Bright-field image (left) and immunofluorescence staining image (right) of organoids formed by sorted AT2 cells cultured under culture conditions with or without CHIR in the example.

[0054] Figure 15 The mechanism by which CHIR maintains AT2 cell and secretory cell characteristics by regulating ERBB4 signaling in the examples; (A) RNA-seq and ATAC-seq analyses of untreated and CHIR-treated DLOs are shown respectively; (B) Principal component analysis (PCA) of untreated and CHIR-treated DLOs; (C) Volcano plot comparing differentially expressed genes (DEGs) between CHIR-treated and untreated DLOs; (D) Determination of the mRNA expression levels of basal cell marker genes based on FPKM values obtained from RNA sequencing data; (E) KEGG pathway enrichment analysis of differentially expressed genes in human DLOs treated with and without CHIR; Red bar graphs represent upregulated pathways, and blue bar graphs represent downregulated pathways.

[0055] Figure 16 NRG1 inhibits the transdifferentiation of secretory cells into basal-like cells in the examples; (A) Schematic diagram of isolating secretory cells from human DLOs and then treating these cells with recombinant NRG1 protein; (B) Flow cytometry analysis of digested organoids derived from secretory cells, comparing organoids treated with NRG1 for 14 days with organoids not treated with NRG1; (C) Bright-field and immunofluorescence images of organoids derived from secretory cells, showing the effects of NRG1 treatment and non-treatment; (D) RT-qPCR analysis of organoids derived from secretory cells treated with and without NRG1; (E) Volcano plot showing DEGs in organoids derived from secretory cells treated with and without NRG1. Detailed implementation manners

[0056] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the examples and the drawings. Obviously, the described examples are some, but not all, of the examples of the present invention. All other examples obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0057] The following examples propose a method for culturing human distal lung organoids, including the steps of culturing, subculturing, cryopreserving, and resuscitating human distal lung organoids; specifically, it includes the following steps:

[0058] (I) Culturing of lung organoids:

[0059] Human lung specimens are stored in Advanced DMEM / F12 medium containing 1× Anti-Anti and placed on ice for subsequent processing.

[0060] (1) Use sterile scissors to cut the lung tissue into pieces, resuspend the tissue with digestive fluid, and enzymatically digest the tissue at a constant temperature of 37°C for 40 minutes at a rotation speed of 110 rpm. Pipette the lung tissue every 10 minutes with a 1 mL pipette tip, and cut the front end of the pipette tip with sterile scissors.

[0061] (2) After digestion is completed, the cells are filtered through a 100 μm cell strainer and rinsed with DMEM / F12 medium containing 10% FBS.

[0062] (3) Centrifuge the cells at 300×g for 5 minutes, remove the supernatant, and lyse red blood cells with red blood cell lysate for 3 minutes. If significant red blood cell residues remain, repeat this step until no visible red blood cells remain in the cells.

[0063] (4) Wash the cells with 1×DPBS, then resuspend them with Matrigel and seed them onto a 24-well plate treated with tissue culture. The culture medium is shown in Table 1. To explore the effects of growth factors or compounds, the organoids are treated with 3 μM CHIR99021 or 100 ng / mL recombinant NRG1 protein.

[0064] (II) Subculture of lung organoids:

[0065] (1) Culture the organoids in a 24- or 48-well culture plate for 10 - 14 days. This is the optimal subculture interval for maintaining lung organoids, and the interval may vary slightly depending on the initial density of the lung organoids.

[0066] (2) Wash the culture wells with 500 μL of PBS, then aspirate and discard.

[0067] (3) Add 300 μL of TypLE-EDTA to the wells, scrape and suspend the Matrigel with a 1000 μL pipette tip, and gently pipette 6 - 10 times.

[0068] (4) Incubate the culture plate in a cell culture incubator and pipette 6 - 10 times every 5 minutes. Avoid over-digestion to prevent affecting cell viability. Determine the optimal incubation time based on the size of the organoids and observe the digestion effect under a microscope.

[0069] (5) Add 600 - 800 μL of washing medium and dissociate the organoids by gently pipetting. Avoid over-pipetting to prevent affecting cell viability. Retain larger cell clusters (>100 cells) for routine subculture.

[0070] (6) Transfer the sphere suspension to a 15 mL centrifuge tube and add 5 mL of washing medium.

[0071] (7) Centrifuge at 300 g for 5 minutes at room temperature.

[0072] (8) Aspirate and discard the supernatant, leaving approximately 100 μL.

[0073] (9) Add 500 μL - 1 mL of washing medium and resuspend the cells.

[0074] (10) Transfer the cells to a 1.5 mL tube. The dilution ratio is determined according to the cell density and growth rate, and the dilution ratio of lung organoids is between 1:4 and 1:8.

[0075] (11) Centrifuge at 300 g for 5 minutes at room temperature.

[0076] (12) Completely aspirate and discard the supernatant. First, aspirate and discard with a vacuum pump, leaving approximately 100 μL, and then completely aspirate and discard the remaining supernatant with a 200 μL or 10 μL pipette.

[0077] (13) Place the tube on ice. Resuspend the spheres with Matrigel (30 μL per well). Pipette gently until the cell clumps are completely dispersed. Avoid generating air bubbles in the Matrigel. Avoid touching the bottom of the centrifuge tube with your hand.

[0078] (14) Place the centrifuge tube and 24-well culture plate on ice. Pipette 30 μL of the cell-Matrigel suspension into the center of each well, and then carefully spread it with the pipette tip.

[0079] (15) Place the culture plate in the cell culture incubator to incubate and polymerize the Matrigel. Invert the culture plate to prevent the cells from sinking to the surface of the well plate.

[0080] (16) Add 500 μL of medium to the wells and change the medium at least once every 2 days.

[0081] (17) Culture the organoids in a 24-well culture plate for 2 - 3 days. Ensure that most of the cells in the organoids are in the growth phase to maximize the survival rate after passage. Do not overculture the organoids (i.e., the medium turns yellow); ensure that most of the organoids are spherical, with few dead cells and debris.

[0082] (III) Cryopreservation of lung organoids:

[0083] (1) Culture the organoids in a 24-well culture plate for 10 - 14 days.

[0084] (2) Scrape and suspend the Matrigel in the medium by gentle pipetting.

[0085] (3) Transfer the suspension to a 1.5 mL tube (up to 2 wells) or a 15 mL centrifuge tube (> 2 wells).

[0086] (4) Centrifuge at 300 g for 5 minutes at room temperature.

[0087] (5) Discard the supernatant, leaving approximately 100 μL, and then gently resuspend the organoids in 500 μL of cryopreservation medium per well.

[0088] (6) Transfer the cell suspension to a cryotube. Place the cryotube in a programmable freezing container and use an ultralow temperature freezer to freeze the programmable freezing container at -80 °C. As soon as possible, store the cryotube in a liquid nitrogen tank.

[0089] (IV) Recovery of lung organoids:

[0090] (1) Incubate the frozen cryotube in a 37 °C water bath for rapid thawing. When there is only a little ice crystal left in the cryotube, transfer it to the laminar flow hood for operation.

[0091] (2) Transfer the organoids to a pre-warmed 15 mL centrifuge tube containing 9 mL of washing medium.

[0092] (3) After centrifuging at 300 × g for 5 minutes, culture the recovered organoids in 2 - 4 wells of a 24-well culture plate.

[0093] Example 1

[0094] I. Method

[0095] The samples for organoid culture were obtained from patients with lung nodules who underwent lobectomy. Take 1 cm of tissue from the outermost edge of the lung lobe, digest it, and then use the optimized human distal lung organoid medium (Table 1) for organoid culture.

[0096] Table 1

[0097]

[0098] Human lung specimens were stored in Advanced DMEM / F12 medium containing 1× Anti-Anti and placed on ice for subsequent processing.

[0099] (1) Use sterile scissors to cut the lung tissue into small pieces, resuspend the tissue with the prepared digestion solution, and perform enzymatic digestion of the tissue at a constant temperature of 37 °C at a rotation speed of 110 rpm for 40 minutes. Pipette the lung tissue every 10 minutes with a 1 mL pipette tip, and cut the front end of the pipette tip with sterile scissors.

[0100] (2) After digestion, filter the cells through a 100 μm cell strainer and wash with DMEM / F12 medium containing 10% FBS.

[0101] (3) Centrifuge the cells at 300 × g for 5 minutes, remove the supernatant, and lyse red blood cells with red blood cell lysis buffer for 3 minutes. If there is significant red blood cell residue, repeat this step until there is no visible red blood cell residue in the cells.

[0102] (4) The cells were washed with 1×DPBS and then resuspended in Matrigel and seeded onto tissue culture-treated 24-well plates. The culture medium is shown in Table 1.

[0103] In addition, to explore the effects of growth factors or compounds, the organoids were treated with 3 μM CHIR99021 or 100 ng / mL recombinant NRG1 protein.

[0104] II. Results

[0105] 1.1 Culture of human distal lung organoids

[0106] Identified by immunofluorescence staining, it was found that human distal lung organoids contained organoids of multiple airway cell types, including BCO and SCO. Morphologically, these two types of organoids showed significant differences. BCO presented a dense and cavity-free structure, with a shape similar to a solid ball. Compared with other organoids, this type of organoid had a larger diameter and a relatively faster growth rate. Further identification found that this type of organoid expressed specific markers KRT5 and PDPN of basal cells. While SCO showed the characteristic of neatly arranged epithelial cells, with single or multiple lumen structures inside. Similarly, this type of organoid had a larger diameter and expressed specific markers SFTPB and SCGB1A1 of secretory cells. In addition to BCO and SCO, a part of RASC-Org was also included in this culture system. Further identification found that these organoids expressed specific markers related to respiratory bronchioles such as SFTPB and SCGB3A2, and the expression of these markers confirmed the origin of these organoids from respiratory bronchioles. In addition, AT2-Org was also included in this system, and the cell origin of these organoids was confirmed by the expression of specific marker SFTPC of AT2 cells ( Figure 1 ).

[0107] Based on the above results, it was found that all the organoids grew well and could be passaged for a long time. Immunofluorescence detection found that there were multiple types of lung epithelial cells in these distal lung organoids, and this cell heterogeneity could be maintained stably during the passage process. This stability and reproducibility made distal lung organoids a powerful tool for studying the biology of lung epithelial cells and disease mechanisms.

[0108] 1.2 A large number of CD66c+ abnormal basal cells appeared during organoid culture

[0109] To confirm whether the cell types and proportions obtained from tissue digestion are consistent with those of the cultured organoids, flow cytometry analysis was performed on the cells obtained from primary tissue digestion. In flow cytometry analysis, three antibodies, HT2-280, NGFR, and CD66c, were selected as markers, corresponding to the surface antigens of AT2, basal cells, and secretory cells, respectively. The analysis results showed that the cells obtained from primary tissue digestion indeed contained a certain proportion of secretory cells and AT2 cells, and the proportion of basal cells in the cells obtained from primary tissue digestion was relatively small ( Figure 2 ). However, after culturing the cells obtained from tissue digestion into organoids in airway medium, a large proportion of BCOs appeared in the system. The results of immunofluorescence staining and flow cytometry analysis both showed an increase in the proportion of basal cells ( Figure 3 ), indicating that a certain type of progenitor cell may have undergone extensive transdifferentiation into KRT5+ basal cells.

[0110] Flow cytometry analysis of cultured human distal lung organoids showed a relatively high proportion of basal cells with high expression of CD66c ( Figure 4 ). To confirm whether these CD66c+ basal cells are abnormal basal cells, the CD66c+ abnormal basal cells and secretory cells were sorted and subjected to qPCR analysis. It was found that the expression of fibrosis-promoting and inflammation-promoting related genes increased in the cultured abnormal basal cells ( Figure 5 ), indicating that the cultured organoids may have cell compositions and functional characteristics different from those of primary tissues.

[0111] 2 It was found that different cell types in the distal lung were capable of generating CD66c+ abnormal basal cells

[0112] To investigate the origin of CD66c+ abnormal basal cells, the contribution of different progenitor cells to abnormal basal cells was compared. The experimental procedure was as follows: First, the distal lung tissue was digested and cultured into organoids to increase the number of epithelial cells. Then, flow cytometry was used to sort the organoids to obtain pure secretory cells, basal cells, and AT2 cells. Finally, these sorted cells were cultured into organoids, and the proportion of abnormal basal cells was compared ( Figure 6 ).

[0113] 2.1 CD66c+ secretory cells have the ability to form abnormal basal cells

[0114] CD66c is a typical surface antigen of secretory cells. CD66c+ secretory cells may undergo a dedifferentiation process and transform into CD66c+ abnormal basal cells. First, CD66c+ secretory cells were sorted from human distal lung tissue and observed in organoid culture. In flow cytometry sorting, pure CD66c+ secretory cells were successfully obtained by excluding basal cells ( Figure 7A). The CD66c+ secretory cells were cultured as organoids. Bright-field and immunofluorescence results showed that most of the formed organoids were solid spherical BCOs ( Figure 7 B). To confirm whether there are abnormal basal cells in these organoids, flow cytometry analysis was performed. The results showed that these organoids contained a large number of CD66c+ abnormal basal cells ( Figure 7 C). In addition, continuous section observation of the same organoids found that some organoids simultaneously expressed markers of basal cells and secretory cells, and the localization of these markers in the organoids was different (mainly distributed on the inner and outer sides of the organoids) ( Figure 8 ). This situation suggests that these organoids may be undergoing a dedifferentiated state, further confirming that CD66c+ secretory cells may undergo a dedifferentiation process and transform into CD66c+ abnormal basal cells.

[0115] 2.2 CD66c- basal cells have the ability to form abnormal basal cells

[0116] As the main progenitor cells of the airway, basal cells play an important role in the development and repair of the lungs. Therefore, they are also a possible source of CD66c+ abnormal basal cells. To verify this hypothesis, CD66c- basal cells were also sorted for organoid culture ( Figure 9 A). Through organoid culture, it was found that CD66c- basal cells could differentiate into CD66c+ basal cells during the culture process; immunofluorescence results showed that there was a certain proportion of CD66c+ basal cells in the organoid culture system ( Figure 9 B). Therefore, CD66c- basal cells can transform into CD66c+ basal cells during organoid culture, thus becoming another potential source of CD66c+ abnormal basal cells.

[0117] 2.3 AT2 cells have the ability to form abnormal basal cells

[0118] Studies have found that AT2 cells can respond to fibrotic signals and transform into basal cells through a transdifferentiation process. AT2 cells are also a possible source of CD66c+ abnormal basal cells. To verify this hypothesis, HT2-280+ AT2 cells were sorted for organoid culture, and it was found that AT2 could transdifferentiate into solid spherical BCOs in airway medium. After staining the continuous sections of the organoids, it was found that the same organoids simultaneously expressed CD66c and KRT5 ( Figure 10 ), indicating that AT2 cells can transdifferentiate into CD66c+ basal cells. Therefore, AT2 cells are also a possible source of CD66c+ abnormal basal cells.

[0119] 3 CHIR99021 can prevent the generation of CD66c+ abnormal basal cells

[0120] 3.1 CHIR99021 inhibits the generation of basal cells in distal lung organoids

[0121] The study found that the addition of CHIR99021, a WNT signaling pathway activator, could inhibit the transformation of AT2 cells into basal cells. First, the effect of WNT signaling on CD66c+ abnormal basal cells was explored. A large number of SCOs were observed in distal lung organoids in the culture system supplemented with CHIR99021. However, a large number of BCOs were still observed in the medium without CHIR. Immunofluorescence staining results showed that some organoids co-expressed KRT5 and SFTPB. This finding suggests that there may be a phenomenon of transdifferentiation between these progenitor cells ( Figure 11 A); Flow cytometry analysis results also showed that the proportion of basal cells in organoids cultured in the medium containing CHIR was significantly reduced ( Figure 11 B).

[0122] 3.2 CHIR99021 inhibits the transdifferentiation of secretory cells into CD66c+ basal cells

[0123] To verify whether the activation of WNT signaling can inhibit the transdifferentiation of different cell types in the distal lung into CD66c+ abnormal basal cells, pure secretory cells were isolated by flow sorting technology and cultured under culture conditions with or without CHIR. After a period of culture, it was found that the medium containing CHIR significantly inhibited the transdifferentiation of secretory cells into CD66c+ abnormal basal cells ( Figure 12 ), indicating that CHIR99021 has an inhibitory effect on the abnormal proliferation of basal cells.

[0124] 3.3 CHIR99021 inhibits the transdifferentiation of CD66c− basal cells into abnormal basal cells

[0125] Pure CD66c− basal cells were isolated by flow sorting technology and cultured under culture conditions with or without CHIR. After a period of culture, it was found that the medium containing CHIR had a significant inhibitory effect on the generation of basal cells. In the medium containing CHIR, the main cell type generated in the system was cavity-containing SCO, and immunofluorescence staining results also showed an increase in the proportion of secretory cells and a decrease in the proportion of basal cells ( Figure 13 ), indicating that the addition of CHIR inhibits the formation of basal cells and may promote the differentiation of secretory cells to some extent.

[0126] 3.4 CHIR99021 inhibits the transdifferentiation of AT2 cells into abnormal basal cells

[0127] Pure AT2 cells were successfully isolated by flow sorting technology and cultured under culture conditions with or without CHIR. After observing and recording the growth and differentiation of AT2 cells under the two culture conditions, it was found that AT2 cells cultured in the medium without CHIR underwent obvious transformation, and these cells were almost completely transformed into basal cells with obvious morphological changes. However, the medium containing CHIR could basically maintain the original cell fate of AT2 cells( Figure 14 ). These cells maintained their specific morphological and functional characteristics during the culture process, suggesting that CHIR plays an important role in maintaining the original fate of AT2 cells. The research results further support the key role of CHIR in maintaining the original cell fate and inhibiting the formation of basal cells, and at the same time suggest that it is necessary to further explore the regulatory mechanism of CHIR on basal cell differentiation and its possible impact on tissue regeneration, repair and disease development.

[0128] 4 Specific mechanism by which CHIR prevents the generation of abnormal CD66c+ basal cells

[0129] 4.1 CHIR treatment leads to upregulation of the ERBB4 signaling pathway in organoids

[0130] To study the effect of CHIR on the maintenance of AT2 cell and secretory cell characteristics, bulk RNA sequencing and ATAC-seq were performed on human lung organoids (DLOs) with or without CHIR treatment. Principal component analysis (PCA) showed that DLOs treated with CHIR clustered significantly and were similar to the control group. CHIR treatment significantly downregulated genes related to basal cells and the NOTCH signaling pathway, as reflected by a decrease in the FPKM value. Specifically, the analysis showed that CHIR treatment upregulated genes related to the mitogen-activated protein kinase (MAPK), calcium signaling, and RAS signaling pathways, and these pathways are known to regulate ERBB4 upregulation. In contrast, CHIR treatment downregulated the NOTCH pathway, which is an important regulator of basal cell characteristics( Figure 15 ).

[0131] 4.2 The NRG1-ERBB4 signal inhibits the formation of abnormal basal cells

[0132] To verify the effect of the NRG1-ERBB4 signaling pathway on the transdifferentiation of secretory cells, secretory cells were isolated and organoids were cultured, and then treated with recombinant NRG1 protein. After 14 days of treatment, most secretory cells retained their original morphological characteristics and failed to transdifferentiate into basal cells, and this result was confirmed by immunofluorescence staining. Flow cytometry analysis showed that the proportion of CD66c+ ABCs was significantly decreased after NRG1 treatment. In addition, RT-qPCR results showed that the expression of basal cell marker genes in the NRG1 treatment group was significantly reduced compared with the control group. Further research was carried out on RNA-seq of organoids derived from secretory cells with and without NRG1 treatment, and the results showed that the expression of basal cell marker genes in the NRG1-treated organoids was significantly downregulated, which was consistent with the results of the decreased expression of these marker genes shown by the FPKM values( Figure 16 ).

[0133] In summary, the present invention successfully constructs human distal lung organoids, and it is of great significance to use organoids to carry out research on related biomarkers and therapeutic targets during acute lung injury and repair.

[0134] The above are the preferred embodiments of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Any equivalent or modified implementation completed without departing from the spirit disclosed by the present invention falls within the protection scope of the present invention.

Claims

1. A human distal lung organoid, characterized in that, Comprising: Organoids derived from basal cells, presenting as solid spheres; organoids derived from secretory cells, presenting as single-lumen or multi-lumen morphology; Organoids derived from respiratory bronchiole cells, presenting as single-lumen or multi-lumen morphology; organoids derived from AT2 cells, presenting as cystic cavity-like morphology.

2. The human distal lung organoid according to claim 1, characterized in that, Comprising: The organoids derived from basal cells express basal cell markers KRT5 and PDPN; And / or the organoids derived from secretory cells express secretory cell markers SFTPB and SCGB1A1; And / or the organoids derived from respiratory bronchiole cells express markers SFTPB and SCGB3A2 related to respiratory bronchioles; And / or the organoids derived from AT2 cells express AT2 cell marker SFTPC.

3. A human distal lung organoid culture medium, characterized in that, For culturing the human distal lung organoids according to claim 1 or 2, which comprises components with the following contents: Basal medium: Advanced DMEM / F12 medium; Small molecule compounds: A83-01, 0.5 μM; SB202190, 0.5 μM; Y-27632, 10 μM; Human recombinant proteins: EGF, 50 ng / mL; FGF10, 100 ng / mL; FGF7, 25 ng / mL; R-spondin, 500 ng / mL; Noggin, 100 ng / mL; Supplements: Nicotinamide, 5 mM; B-27, 1×; Antibotic-Antimycotic, 100×; HEPES, 10 mM; GlutaMAX, 2 mM; N-Acetyl-L-Cysteine, 1.25 mM.

4. The method for culturing human distal lung organoids according to claim 1 or 2, characterized in that, Comprising the following steps: (i) The lung tissue is preserved in Advanced DMEM / F12 medium containing 1× Anti-Anti and placed on ice for standby; (ii) First, the lung tissue is minced with sterile scissors, then the tissue is resuspended with digestive solution, and the lung tissue is enzymatically digested at a constant temperature of 37 °C at a rotation speed of 110 rpm for 40 minutes. Every 10 minutes, the lung tissue is pipetted with a 1 mL pipette tip, and the front end of the pipette tip is cut off with sterile scissors; (iii) The cells obtained after digestion are filtered through a 100 μm cell sieve and rinsed with DMEM / F12 medium containing 10% FBS; (iv) The cells are centrifuged at 300×g for 5 minutes, the supernatant is removed, and lysed with erythrocyte lysate. This step is repeated until there is no residue of visible red blood cells in the cells; (v) First, the cells are washed with 1× DPBS, then resuspended with Matrigel and seeded into a 24- or 48-well culture plate treated with tissue culture, which contains the human distal lung organoid medium according to claim 3, to obtain human distal lung organoids; also included are the subculture, cryopreservation and resuscitation steps of the human distal lung organoids.

5. The culture method of human distal lung organoids according to claim 4, characterized in that, The lung tissue is derived from a patient with a lung nodule who has undergone lobectomy, and the tissue 1 cm from the outermost edge of the lung lobe is collected.

6. The culture method of the human distal lung organoids according to claim 4, characterized in that It also includes the step of treating the human distal lung organoids with 3 μM CHIR99021 or 100 ng / mL recombinant NRG1 protein.

7. The culture method of human distal lung organoids according to claim 4, characterized in that, The passage process of the human distal lung organoids includes the following steps: (1) Cultivate the human distal lung organoids in a 24- or 48-well culture plate for 10 - 14 days; wash the culture wells with 500 μL of PBS and aspirate; add 300 μL of TypLE-EDTA to the wells, scrape and suspend Matrigel with a 1000 μL pipette tip, and gently pipette 6 - 10 times; (2) Incubate the 24- or 48-well culture plate in a cell culture incubator, and pipette 6 - 10 times every 5 minutes; add 600 - 800 μL of washing medium, dissociate the human distal lung organoids by gently pipetting, and retain cell clusters with more than 100 cells for routine passage; (3) Transfer the sphere suspension to a 15 mL centrifuge tube, add 5 mL of washing medium; centrifuge at 300 g for 5 minutes at room temperature; aspirate the supernatant, leaving 100 μL; add 500 μL - 1 mL of washing medium to resuspend the cells; transfer the cells to a 1.5 mL test tube with a dilution ratio of 1:4 - 8; centrifuge at 300 g for 5 minutes at room temperature; completely aspirate the supernatant, first aspirate with a vacuum pump, leaving 100 μL, and then completely aspirate the remaining supernatant with a 200 μL or 10 μL pipette; (4) Place the test tube on ice, resuspend the spheres with 30 μL of Matrigel per well, and gently pipette until the cell clusters are completely dispersed; place the centrifuge tube and the 24- or 48-well culture plate on ice, and pipette 30 μL of the cell-Matrigel suspension to the center of each well with a pipette, and then spread it with the pipette tip; (5) Incubate the 24- or 48-well culture plate in a cell culture incubator to polymerize Matrigel, and invert the culture plate to prevent the cells from sinking to the surface of the well plate; add 500 μL of medium to the wells of the 24- or 48-well culture plate and culture for 2 - 3 days, and change the medium at least once every 2 days to obtain human distal lung organoids.

8. The culture method of human distal lung organoids according to claim 4, characterized in that, The cryopreservation process of the human distal lung organoids includes: cultivating the human distal lung organoids in a culture plate for 10 - 14 days, scraping and suspending Matrigel in the medium by gentle pipetting, transferring the suspension to a 1.5 mL test tube or a 15 mL centrifuge tube, centrifuging at 300 g for 5 minutes at room temperature, aspirating the supernatant, leaving 100 μL, and then gently resuspending the human distal lung organoids in 500 μL of cryopreservation solution per well. Transfer the suspension to a cryotube, place the cryotube in a programmable cooling box, freeze at -80 °C, and store in a liquid nitrogen tank; And / or the recovery process of the human distal lung organoids includes: incubating the frozen cryotube in a 37 °C water bath for rapid thawing, transferring the cryotube to a laminar flow hood, transferring the human distal lung organoids to a pre-warmed 15 mL centrifuge tube, adding 9 mL of washing medium, and centrifuging at 300 g for 5 minutes to complete the recovery of the human distal lung organoids.

9. Use of the human distal lung organoids according to claim 1 or 2 in drug screening, constructing tumor animal models, preparing diagnostic reagents, regenerative medicine, and scientific research for non-therapeutic purposes in vitro.

10. The application according to claim 9, wherein The human distal lung organoids as a screening model for biomarkers and therapeutic targets during acute lung injury and repair.

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