Construction method and application of COPD (chronic obstructive pulmonary disease) source far-end airway organoid

Abstract

The invention discloses a construction method and application of a COPD (chronic obstructive pulmonary disease)-sourced far-end airway organ, the method is simple in process, and related culture can be completed only by two steps of proliferation and differentiation after tissue single cells are extracted, so that the method is easier to popularize. According to the method, the culture days are shortened, the culture of related organoid can be completed within 18 days at least, and the experiment efficiency is greatly improved. Meanwhile, experimental data show that the organoid cultured by the method disclosed by the invention is highly similar to an in-situ tissue (far-end airway) in transcriptional level, well restores the pathological characteristics of COPD in protein expression level and function level, and is suitable for in-vitro study on pathological changes and disease occurrence and development mechanisms of COPD in the far-end airway. The COPD-sourced far-end airway organ obtained by the construction method can be used for non-therapeutic purpose cilia pulsation function detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for constructing a COPD-derived distal airway organoid and its application. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a highly heterogeneous chronic respiratory disease characterized by chronic respiratory symptoms (dyspnea, cough, sputum) due to progressive, irreversible airflow obstruction caused by airway remodeling, inflammation, and / or alveolar abnormalities. Since the current gold standard for COPD diagnosis is spirometry, animal models remain the main vehicle for studying COPD. However, there are species differences between different species, and the results obtained using animal models are of limited help in studying human COPD.

[0003] For a long time, in vitro studies of COPD have mainly used various immortalized lung cell lines. Although these cell lines retain many phenotypic characteristics of the original cell types, due to their single structural components, whether they can effectively demonstrate disease-specific processes has been widely questioned by researchers. Compared with cell lines, lung organoids reproduce the three-dimensional tissue structure of the lungs, such as alveoli, airways, and their functions in vitro, and have been used to study a variety of lung diseases. Studies have shown that the distal airways are the site of early and major lesions in COPD. However, the existing methods for constructing COPD-derived airway organoids still have the following problems: (1) The process is complex, the construction cycle is long, and it is not widely used; (2) Existing COPD-derived airway organoids are mainly bronchial organoids of the proximal large airways or have no clear positioning, and are not suitable for in vitro research on COPD lesions and disease development mechanisms in the distal airways. Summary of the invention

[0004] The present invention provides a method for constructing COPD-derived distal airway organoids and their application, so as to solve the problems of the prior art having complex processes, long construction cycles, and being unsuitable for in vitro studies of COPD lesions in the distal airways and the mechanisms of disease occurrence and development.

[0005] One aspect of the present invention provides a method for constructing COPD-derived distal airway organoids, comprising: Step S1, immersing COPD-derived distal lung tissue in digestion solution, digesting at 37° C. for 30-45 min, using a mild sample buffer to terminate digestion, cutting the tissue into pieces, repeatedly blowing to obtain a single cell suspension, and obtaining a COPD-derived lung tissue single cell a after centrifugation; Step S2, filtering the single cell a with a cell mesh to collect the COPD-derived lung tissue single cell b, removing the supernatant after centrifugation, and washing with a washing solution for 1-2 times to obtain the COPD-derived lung tissue single cell c; Step S3, observe whether the single cell c has red clumps. If no red clumps are seen, directly proceed to step S4; if red clumps are seen, add 1 mL of red blood cell lysis solution to lyse at room temperature for 3 minutes, then separate at a speed of 300g, discard the supernatant, and obtain COPD-derived lung tissue single cells d; Step S4, mixing single cell c or single cell d with matrix gel and then seeding the plate, inverting the plate and letting it stand in a cell culture incubator, adding organoid expansion medium after the gel solidifies, and continuing to culture in the cell culture incubator, replacing the medium every 3-4 days to obtain the original generation COPD-derived lung organoids; Step S5, after enzymatic hydrolysis of the original COPD-derived lung organoids, mix with matrix gel and then plate them. Invert them in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture them in a cell culture incubator. Replace the organoid differentiation medium after 7-10 days of culture, and replace the medium every 3-4 days. COPD-derived distal airway organoids are obtained after 11-14 days of culture.

[0006] In the above-mentioned method for constructing COPD-derived distal airway organoids, in step S1, the digestion solution is a TrypLE digestion solution containing 10-50 mM EDTA.

[0007] In the above method for constructing COPD-derived distal airway organoids, in step S1, the mild sample buffer is a PBS buffer containing 1-3% BSA and no calcium or magnesium ions.

[0008] The above-mentioned method for constructing COPD-derived distal airway organoids, wherein, in step S2, the composition and content of the cleaning solution are: amphotericin B, 1-3ug / mL; primocin, 10-1000μg / mL; gentamicin, 100-500μg / mL.

[0009] In the above-mentioned method for constructing COPD-derived distal airway organoids, the centrifugation conditions in step S1 and step S2 are: centrifugation speed 300-500g, temperature 4°C, and centrifugation time 5-10min.

[0010] The above-mentioned method for constructing COPD-derived distal airway organoids, wherein, in step S4 and step S5, the components and contents of the organoid expansion medium are: Advanced DMEM / F12, 1X; R-Spondin 1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL.

[0011] The above-mentioned method for constructing COPD-derived distal airway organoids, wherein, in step S5, the components and contents of the organoid differentiation medium are: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL; DAPT, 10μM.

[0012] Another aspect of the present invention provides the use of COPD-derived distal airway organoids obtained by the above-mentioned construction method in the detection of ciliary beat function for non-therapeutic purposes.

[0013] Beneficial effects: (1) The process of the present invention is simple. After extracting single cells from tissues, only two steps of proliferation and differentiation are required to complete the relevant culture, making the method easier to popularize. The present invention shortens the number of culture days. It only takes 18 days at the shortest to complete the culture of relevant organoids, greatly improving the experimental efficiency. At the same time, experimental data show that the organoids cultured by the method of the present invention are highly similar to the in situ tissue (distal airway) at the transcriptional level, and better restore the pathological characteristics of COPD at the protein expression level and functional level. It is suitable for in vitro research on the lesions and disease occurrence and development mechanisms of COPD in the distal airways.

[0014] (2) The COPD-derived distal airway organoids obtained in the present invention can be used for the detection and application of drug effectiveness and potential target drugs for clinical COPD patients. In terms of clinical guidance of medication, the organoid drug screening results show consistent consistency with the treatment effects reported in existing literature, providing data support and possibilities for precision medicine for COPD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a bright field image of the Yuan-generation COPD-derived lung organoid obtained in Example 1; Figure 2 This is a bright field image of the COPD-derived distal airway organoid obtained in Example 1; Figure 3 This is a bright field image of the Yuan-generation COPD-derived lung organoid obtained in Example 2; Figure 4 This is a bright field image of the COPD-derived distal airway organoid obtained in Example 2; Figure 5 This is a bright field image of the Yuan-generation COPD-derived lung organoid obtained in Example 3; Figure 6 This is a bright field image of the COPD-derived distal airway organoid obtained in Example 3; Figure 7 This is a diagram showing the immunofluorescence staining results of COPD-derived distal airway organoids obtained in Example 1; Figure 8 The results of MUC5AC and ACCTUB immunofluorescence staining of COPD-derived distal airway organoids obtained in Example 1 and normal distal airway organoids obtained in Comparative Example 1 are shown; Fig. 9 This is a comparison result diagram of the ciliated cell ratio and goblet cell ratio of the COPD-derived distal airway organoids obtained in Examples 1-3 and the normal distal airway organoids obtained in Comparative Example 1; Fig.10 This is a comparison chart of the ciliary beat frequencies obtained in Examples 1-3 and the ciliary beat frequencies of normal distal airway organoids obtained in Comparative Example 1. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0017] Embodiment 1: A method for constructing COPD-derived distal airway organoids, comprising: Step S1, immersing COPD-derived distal lung tissue in digestion solution and digesting at 37°C for 45 minutes, wherein the COPD-derived distal lung tissue is obtained from a COPD patient with lung nodules, and the source site is tissue 2-3 cm away from the lung nodules. The digestion solution is TrypLE digestion solution containing 50mM EDTA, and the digestion is terminated using a mild sample buffer, which is a PBS buffer containing 1% BSA and no calcium or magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a single cell a of the COPD-derived lung tissue is obtained. The centrifugation conditions are: a centrifugal speed of 300g, a temperature of 4°C, and a centrifugation time of 10min.

[0018] Step S2, filter the single cell a with a 70 μm cell mesh to collect the COPD-derived lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 300g, temperature 4°C, centrifugation time 10min, and wash twice with a cleaning solution to obtain the COPD-derived lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 3ug / mL; Primocin, 1000μg / mL; gentamicin, 500μg / mL.

[0019] Step S3, after observation, if no red clumps are seen in the single cell c, the single cell c is mixed with the extracellular matrix gel and plated, and then inverted in a cell culture incubator and left to stand. After the gel solidifies, the organoid expansion medium is added, and the composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100 μg / mL; Primocin, 50 μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 4 days, and culture the original COPD-derived lung organoids, such as Figure 1 shown.

[0020] Step S4, after enzymatic hydrolysis of the original COPD-derived lung organoids, mix with matrix gel and then plate them, invert them in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture in a cell culture incubator. After 7 days of culture, replace the organoid differentiation medium. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100 μg / mL; Primocin, 50 μg / mL; DAPT, 10 μM, the culture medium was replaced every 4 days, and COPD-derived distal airway organoids were obtained after 11 days of culture. Figure 2 shown.

[0021] Based on the above cultured COPD-derived distal airway organoids, ciliary beat function was tested after 3 days. Ciliary beat frequency (CBF) is an important indicator for evaluating ciliary beat function and mucociliary clearance rate, and can reflect the functional level of the pulmonary mucociliary clearance system to a certain extent. Mucociliary clearance system disorders are common in chronic respiratory diseases such as chronic obstructive pulmonary disease, primary ciliary dysfunction, cystic pulmonary fibrosis and asthma.

[0022] Embodiment 2: A method for constructing COPD-derived distal airway organoids, comprising: Step S1, immersing COPD-derived distal lung tissue in digestion solution and digesting at 37°C for 30 minutes, wherein the COPD-derived distal lung tissue is obtained from a COPD patient with lung nodules, and the source site is tissue 2-3 cm away from the lung nodules. The digestion solution is TrypLE digestion solution containing 10 mM EDTA, and the digestion is terminated using a mild sample buffer, which is a PBS buffer containing 3% BSA and no calcium or magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a single cell a of the COPD-derived lung tissue is obtained. The centrifugation conditions are: a centrifugal speed of 500g, a temperature of 4°C, and a centrifugal time of 5 minutes.

[0023] Step S2, filter the single cell a with a 100 μm cell mesh to collect the COPD-derived lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 500g, temperature 4°C, centrifugation time 5min, and wash once with a cleaning solution to obtain the COPD-derived lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 1ug / mL; Primocin, 10μg / mL; gentamicin, 100μg / mL.

[0024] Step S3: upon observation, red clumps are visible in the single cell c, and 1 mL of red blood cell lysis solution is added to lyse the cell at room temperature for 3 minutes, and then the cell is separated at a speed of 300 g, and the supernatant is discarded to obtain the COPD-derived lung tissue single cell d.

[0025] Step S4, after mixing the above single cell d with the extracellular matrix gel, plate them, invert them and let them stand in a cell culture incubator, and after the gel solidifies, add the organoid expansion medium, the composition and content of which are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500 ng / mL; FGF 7, 25 ng / mL; FGF 10, 100 ng / mL; Noggin, 100 ng / mL; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 μM; B27 supplement, 1X; N-Acetylcysteine, 1.25 mM; Nicotinamide, 5 mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 3 days, and culture the original COPD-derived lung organoids, such as Figure 3 shown.

[0026] Step S5, after enzymatic hydrolysis of the original COPD-derived lung organoids, mix with matrix gel and then plate them, invert them in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture in a cell culture incubator. After 10 days of culture, replace the organoid differentiation medium. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100 μg / mL; Primocin, 50 μg / mL; DAPT, 10 μM, the culture medium was replaced every 3 days, and COPD-derived distal airway organoids were obtained after 14 days of culture. Figure 4 shown.

[0027] Based on the above cultured COPD-derived distal airway organoids, ciliary beat function testing for non-therapeutic purposes was performed, and the steps are as follows: Organoid differentiation medium containing 0.1 μM formoterol was added to the successfully cultured COPD-derived distal airway organoids, and ciliary beat function was tested after 3 days.

[0028] Embodiment 3: A method for constructing COPD-derived distal airway organoids, comprising: Step S1, immersing COPD-derived distal lung tissue in digestion solution and digesting at 37°C for 40 minutes, wherein the COPD-derived distal lung tissue is obtained from a COPD patient with lung nodules, and the source site is tissue 2-3 cm away from the lung nodules. The digestion solution is TrypLE digestion solution containing 30 mM EDTA, and the digestion is terminated using a mild sample buffer, which is a PBS buffer containing 2% BSA and no calcium or magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a single cell a of the COPD-derived lung tissue is obtained. The centrifugation conditions are: a centrifugal speed of 400g, a temperature of 4°C, and a centrifugal time of 8 minutes.

[0029] Step S2, filter the single cell a with a 70 μm cell mesh to collect the COPD-derived lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 400g, temperature 4°C, centrifugation time 8min, and wash twice with a cleaning solution to obtain the COPD-derived lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 2ug / mL; Primocin, 500μg / mL; Gentomycin, 300μg / mL.

[0030] Step S3, after observation, if no red clumps are seen in the single cell c, the single cell c is mixed with the extracellular matrix gel and plated, and then inverted in a cell culture incubator and left to stand. After the gel solidifies, the organoid expansion medium is added, and the composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100 μg / mL; Primocin, 50 μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 3 days, and obtain the original COPD-derived lung organoids. Figure 5 shown.

[0031] Step S4, after enzymatic hydrolysis of the original COPD-derived lung organoids, mix with matrix gel and then plate them, invert them in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture in a cell culture incubator. After 8 days of culture, replace the organoid differentiation medium. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100 μg / mL; Primocin, 50 μg / mL; DAPT, 10 μM, the culture medium was replaced every 3 days, and COPD-derived distal airway organoids were obtained after 12 days of culture. Figure 6 shown.

[0032] Based on the above cultured COPD-derived distal airway organoids, ciliary beat function testing for non-therapeutic purposes was performed, and the steps are as follows: Organoid differentiation medium containing 0.1 μM theophylline was added to the successfully cultured COPD-derived distal airway organoids, and ciliary beat function was tested after 3 days.

[0033] Comparative Example 1 Method for constructing normal distal airway organoids, including: Step S1, immersing the human distal lung tissue in a digestion solution and digesting it at 37°C for 45 minutes, wherein the human distal lung tissue is obtained from a patient with lung nodules (not suffering from COPD), and the site source is tissue 2-3 cm away from the lung nodules. The digestion solution is TrypLE digestion solution containing 50mM EDTA, and the digestion is terminated using a mild sample buffer. The mild sample buffer is: PBS buffer containing 1% BSA and not containing calcium and magnesium ions. The tissue is cut into pieces, and a single cell suspension is obtained by repeated blowing. After centrifugation, a human lung tissue single cell a is obtained. The centrifugation conditions are: centrifugal speed 300g, temperature 4°C, and centrifugation time 10min.

[0034] Step S2, filter the single cell a with a 70 μm cell mesh to collect the human lung tissue single cell b, remove the supernatant after centrifugation, the centrifugation conditions are: centrifugal speed 300g, temperature 4°C, centrifugation time 10min, and wash twice with a cleaning solution to obtain the human lung tissue single cell c, the composition and content of the cleaning solution are: amphotericin B, 3ug / mL; Primocin, 1000μg / mL; gentamicin, 500μg / mL.

[0035] Step S3, after observation, if no red clumps are seen in the single cell c, the single cell c is mixed with the extracellular matrix gel and plated, and then inverted in a cell culture incubator and left to stand. After the gel solidifies, the organoid expansion medium is added, and the composition and content of the organoid expansion medium are as follows: Advanced DMEM / F12, 1X; R-Spondin 1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL, continue to culture in the cell culture incubator, replace the culture medium every 4 days, and obtain Yuan generation human lung organoids.

[0036] Step S4, after enzymatic hydrolysis of the Yuan Dynasty human lung organoids, mix with matrix gel and then plate them, turn them upside down in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture in a cell culture incubator. After 7 days of culture, replace the organoid differentiation medium. The composition and content of the organoid differentiation medium are as follows: Advanced DMEM / F12, 1X; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL; DAPT, 10μM, the culture medium was replaced every 4 days, and normal distal airway organoids were obtained after 11 days of culture.

[0037] Based on the normal distal airway organoids cultured as described above, ciliary beat function was tested after 3 days.

[0038] Experiment 1 The COPD-derived distal airway organoids obtained in Example 1 were subjected to immunofluorescence staining. The results are as follows: Figure 7 shown.

[0039] Figure 7The organoids shown in the figure contain four main types of airway cells (ACCTUB: ciliated cell marker, MUC5AC: goblet cell marker, CC10: club cell marker, P63: basal cell marker) and SCGB3A2-positive secretory cells unique to the distal airways, indicating that the COPD-derived distal airway organoids have the four main airway cell types and secretory cells unique to the distal airways, indicating that the organoids are distal airway organoids.

[0040] Experiment 2 Based on the COPD-derived distal airway organoids obtained in the above three examples, the Pearson coefficient correlation analysis of the TOP200 genes of the four main types of airway cells and the corresponding COPD patient-derived distal airway tissues is shown in Table 1: Table 1

[0041] In Table 1, COPD organoids refer to COPD-derived distal airway organoids in the present invention, COPD tissues refer to COPD patient-derived distal airway tissues, and R is the correlation coefficient value. As can be seen from Table 1, COPD-derived distal airway organoids and corresponding COPD patient-derived distal airway tissues are highly similar in these four cell types.

[0042] Experiment 3 The COPD-derived distal airway organoids obtained in Example 1 and the normal distal airway organoids obtained in Comparative Example 1 were subjected to MUC5AC and ACCTUB immunofluorescence staining. The results are as follows: Figure 8 The COPD-derived distal airway organoids obtained in Examples 1-3 and the normal distal airway organoids obtained in Comparative Example 1 were compared in terms of ciliated cell ratio and goblet cell ratio. The results are shown in Fig. 9 shown. Figure 8 and Fig. 9 In the figure, Normal-organoids represents normal distal airway organoids, and COPD-organoids represents COPD-derived distal airway organoids.

[0043] from Figure 8 and Fig. 9 It can be seen that compared with normal distal airway organoids, the expression of ACCTUB in COPD-derived distal airway organoids was significantly reduced, and the expression of MUC5AC was significantly increased, indicating that COPD-derived distal airway organoids have the typical pathological characteristics of COPD, with a decrease in ciliated cells and an increase in goblet cells.

[0044] Experiment 5 The ciliary beat frequencies obtained in Examples 1-3 and the ciliary beat frequencies of the normal distal airway organoids obtained in Comparative Example 1 were compared and analyzed. The results are as follows: Fig.10 shown. Fig.10 In the figure, A represents normal distal airway organoids, B represents Example 1, C represents Example 2, and D represents Example 3.

[0045] from Fig.10 It can be seen that the COPD-derived distal airway organoids obtained in the present invention have ciliary dysfunction, a COPD disease characteristic, and the CBF level can be improved after the administration of formoterol (suggesting that the effect of drug intervention on ciliary function can be observed in the organoid), which is consistent with the trend reported in the existing literature that formoterol has an agonistic effect on the ciliary function of COPD patients. However, no study has reported whether theophylline has an effect on CBF. In the present invention, theophylline administration has no obvious effect on the CBF level of COPD-derived distal airway organoids.

[0046] In summary, the method for constructing and using COPD-derived distal airway organoids provided by the present invention has the following beneficial effects: (1) The process of the present invention is simple. After extracting single cells from tissues, only two steps of proliferation and differentiation are required to complete the relevant culture, making the method easier to popularize. The present invention shortens the number of culture days. It only takes 18 days at the shortest to complete the culture of relevant organoids, greatly improving the experimental efficiency. At the same time, experimental data show that the organoids cultured by the method of the present invention are highly similar to the in situ tissue (distal airway) at the transcriptional level, and better restore the pathological characteristics of COPD at the protein expression level and functional level. It is suitable for in vitro research on the lesions and disease occurrence and development mechanisms of COPD in the distal airways.

[0047] (2) The COPD-derived distal airway organoids obtained in the present invention can be used for the detection and application of drug effectiveness and potential target drugs for clinical COPD patients. In terms of clinical guidance of medication, the organoid drug screening results show consistent consistency with the treatment effects reported in existing literature, providing data support and possibilities for precision medicine for COPD patients.

[0048] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing COPD-derived distal airway organoids, characterized in that: include: Step S1, immersing COPD-derived distal lung tissue in digestion solution, digesting at 37° C. for 30-45 min, using a mild sample buffer to terminate digestion, cutting the tissue into pieces, repeatedly blowing to obtain a single cell suspension, and obtaining a COPD-derived lung tissue single cell a after centrifugation; Step S2, filtering the single cell a with a cell mesh to collect the COPD-derived lung tissue single cell b, removing the supernatant after centrifugation, and washing with a washing solution for 1-2 times to obtain the COPD-derived lung tissue single cell c; Step S3, observe whether the single cell c has red clumps. If no red clumps are seen, directly proceed to step S4; if red clumps are seen, add 1 mL of red blood cell lysis solution to lyse at room temperature for 3 minutes, then separate at a speed of 300g, discard the supernatant, and obtain COPD-derived lung tissue single cells d; Step S4, mixing single cell c or single cell d with matrix gel and then seeding the plate, inverting the plate and letting it stand in a cell culture incubator, adding organoid expansion medium after the gel solidifies, and continuing to culture in the cell culture incubator, replacing the medium every 3-4 days to obtain the original generation COPD-derived lung organoids; Step S5, after enzymatic hydrolysis of the original COPD-derived lung organoids, mix with matrix gel and then plate them. Invert them in a cell culture incubator and let them stand. After the gel solidifies, add organoid expansion medium and continue to culture them in a cell culture incubator. Replace the organoid differentiation medium after 7-10 days of culture, and replace the medium every 3-4 days. COPD-derived distal airway organoids are obtained after 11-14 days of culture.

2. The method for constructing COPD-derived distal airway organoids according to claim 1, characterized in that: In step S1, the digestion solution is TrypLE digestion solution containing 10-50 mM EDTA.

3. The method for constructing COPD-derived distal airway organoids according to claim 1, characterized in that: In step S1, the mild sample buffer is a PBS buffer containing 1-3% BSA and no calcium or magnesium ions.

4. The method for constructing COPD-derived distal airway organoids according to claim 1, characterized in that: In step S2, the composition and content of the cleaning solution are: amphotericin B, 1-3ug / mL; primocin, 10-1000μg / mL; gentamicin, 100-500μg / mL.

5. The method for constructing COPD-derived distal airway organoids according to claim 1, characterized in that: The centrifugal conditions in step S1 and step S2 are: centrifugal speed 300-500g, temperature 4°C, and centrifugal time 5-10min.

6. The method for constructing COPD-derived distal airway organoids according to claim 1, characterized in that: In step S4 and step S5, the composition and content of the organoid expansion medium are: Advanced DMEM / F12, 1X; R-Spondin 1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax 100x, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Primocin, 50μg / mL.

7. The method for constructing COPD-derived distal airway organoids according to claim 1, characterized in that: In step S5, the composition and content of the organoid differentiation medium are: Advanced DMEM / F12, 1X; Noggin, 100 ng / mL; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 μM; B27 supplement, 1X; N-Acetylcysteine, 1.25 mM; Nicotinamide, 5 mM; GlutaMax 100x, 1X; HEPES, 10 mM; Penicillin / Streptomycin, 100 μg / mL; Primocin, 50 μg / mL; DAPT, 10 μM.

8. Use of COPD-derived distal airway organoids obtained by the construction method according to any one of claims 1 to 7 in the detection of ciliary beat function for non-therapeutic purposes.

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