Construction method and application of mouse airway organoid inflammation model

Abstract

The invention discloses a construction method and application of a mouse airway organoid inflammation model, and relates to the technical field of biology. The construction method comprises the following steps: (1) taking a mouse airway tissue, digesting with digestive juice, and centrifuging to obtain a cell suspension; (2) centrifuging the cell suspension, discarding the supernatant, adding matrigel, uniformly mixing, incubating, and culturing by using an airway organoid complete medium; (3) adding a cell recovery solution into the cells cultured in the step (2), incubating, degumming, centrifuging, discarding supernatant, adding matrigel, uniformly mixing, incubating, and adding an airway organ complete culture medium for continuous culture; and (4) adding 1-20 [mu] g / ml of LPS into the airway organoid complete culture medium, and incubating. The AO constructed by the invention covers four main cell types of cilia cells, goblet cells, basal cells and rod-like cells, causes epithelial injury in the AO and expression of AO inflammatory factors, and can provide reference for development of CRD new drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a mouse airway organoid inflammation model and an application thereof. Background Art

[0002] Chronic respiratory disease (CRD), such as asthma, allergic rhinitis, pharyngitis, tracheitis, and chronic obstructive pulmonary disease, is one of the most common non-communicable diseases. CRD is characterized by a long course, repeated attacks, high morbidity and mortality.

[0003] Although the triggering factors and pathological mechanisms of different respiratory diseases are slightly different, they all manifest as epithelial damage or dysfunction, and tissue inflammation. Therefore, studying the relationship between airway inflammation and epithelial cell function is crucial to understanding the pathophysiology of CRD and developing effective treatments. Traditional in vivo models and 2D cell culture have limitations in simulating the complex structure and cell interactions of the airways. With the development of stem cell biology and 3D culture technology, in vitro culture of organoids of various tissues such as brain, liver, intestine, and pancreas has been achieved. This technological breakthrough provides a powerful tool for simulating CRD in a more physiologically relevant environment and studying the basic mechanisms of inflammation.

[0004] Organoids are three-dimensional structures derived from stem cells that can self-assemble into miniaturized organs while retaining key structural and functional characteristics. Several methods for constructing mammalian airway organoids (AO) have been explored in the prior art, including isolating exfoliated epithelial cells from nasal biopsy samples, using iPS cells to induce differentiation, isolating epithelial cells from lung tissue to construct AO, etc.

[0005] Chinese patent CN117025511A discloses a method for establishing an airway organoid model of COPD patients. The establishment method described in the patent mainly uses tracheal tissue from surgical specimens of COPD patients. The main steps are: obtaining tracheal tissue from surgical specimens of early lung cancer patients with COPD, shearing to obtain tissue fragments, mixing tissue cleaning solution + 100 mg / ml protease + 1 mg / ml deoxyribonuclease to form a mixed solution, and adding the above tissue fragments to 10 times the volume of the mixed solution to obtain mixed tissue. Scrape the cells on the surface of the trachea, centrifuge to obtain a precipitate, add tissue digestion solution and incubate at 37°C for 60 minutes, stop digestion, filter and collect the cell suspension, centrifuge, discard the supernatant, and obtain cell precipitate A; resuspend the cell precipitate in expansion medium for expansion culture, use trypsin to digest the cells into a single cell suspension, centrifuge, discard the supernatant, and form cell precipitate B; resuspend the above cell precipitate B with airway organoid culture medium to obtain a cell suspension; add 80μl of cell suspension to each well of the matrix gel-coated 3D culture plate for culture; add 100μl of cell disaggregation solution to each well, separate the organoid from the matrix gel, transfer to a centrifuge tube, centrifuge to make the organoid aggregate at the bottom of the centrifuge tube, and the collected airway organoid is set as a model. However, the sample used in this patent is the airway tissue of COPD patients, and the model constructed is unique.

[0006] There are few studies on the use of mouse airway tissue to construct airway organoid inflammation models in the prior art. The present invention isolates mouse airway tissue epithelial cells, constructs AO through 3D culture, and stimulates with LPS. The purpose is to establish an organoid model for evaluating airway epithelial cells and inflammatory changes, and provide a reference for the development of new CRD drugs. Summary of the invention

[0007] The purpose of the present invention is to provide a method for constructing a mouse airway organoid inflammation model and its application.

[0008] To achieve the above object, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a method for constructing a mouse airway organoid inflammation model, comprising the following steps:

[0010] (1) Obtain mouse airway tissue, digest it with digestive fluid, and centrifuge it to obtain a cell suspension;

[0011] (2) After centrifugation of the cell suspension, the supernatant was discarded, Matrigel was added and mixed, and the cells were incubated and cultured using complete airway organoid medium;

[0012] (3) adding cell recovery solution to the cells cultured in step (2) to incubate and remove gel, centrifuge, discard the supernatant, add matrix gel, mix, incubate, and add airway organoid complete culture medium to continue culturing;

[0013] (4) Add 1-20 μg / ml LPS to the complete medium of airway organoids and incubate;

[0014] The airway complete culture medium includes Advanced DMEM / F12, 1.5-2.5mM / ml Glutamax, 8-12mM / ml HEPES, 1-3% 1x PS, 1-3% 1x N2, 1-3% 1x B27, 1-2mM / ml N-acetylcysteine, 40-55ng / ml EGF, 80-120ng / ml Noggin, 400-600ng / ml R-spondin and 8-12μM Y27632.

[0015] Furthermore, the airway complete culture medium includes Advanced DMEM / F12, 2mM / ml Glutamax, 10mM / ml HEPES, 1% 1x PS, 1% 1x N2, 2% 1x B27, 1mM / ml N-acetylcysteine, 50ng / ml EGF, 100ng / ml Noggin, 500ng / ml R-spondin and 10μM Y27632.

[0016] Specifically, the mice mentioned in step (1) include mice and rats.

[0017] Specifically, in step (1), the mouse airway tissue is washed and then digested, and the washing solution includes 1x L-glutamine, 10mM HEPES and 1% double antibody in 1x Advance DMEM / F12.

[0018] In step (1), the digestion solution is collagenase type I, and the concentration of collagenase type I is 1-5 mg / ml;

[0019] Specifically, the concentration of collagenase type I in step (1) is 2 mg / ml.

[0020] Specifically, the digestion conditions in step (1) are 37° C. and digestion for 1-3 h;

[0021] Furthermore, the digestion conditions in step (1) are 37° C. and digestion for 2 hours.

[0022] Specifically, in step (1), the mass volume ratio of mouse airway tissue to digestive fluid is (8-12):1.

[0023] Furthermore, in step (1), the mass volume ratio of the mouse airway tissue to the digestive fluid is 10:1.

[0024] According to some embodiments of the present invention, in step (1), the mass volume ratio of mouse airway tissue to digestive fluid is 10 mg:1 ml.

[0025] Specifically, the centrifugation condition in step (1) is 300-500g for 3-5min; further, the centrifugation condition in step (1) is 400g for 3min.

[0026] Specifically, step (2) further includes the step of resuspending the cell suspension using matrix gel before culturing, and the volume ratio of matrix gel to cell suspension is (1-5):1;

[0027] Furthermore, step (2) further includes the step of resuspending the cell suspension using matrix gel before culturing, and the volume ratio of matrix gel to cell suspension is 2:1.

[0028] Specifically, the culture conditions in step (2) are 5% CO2, 37°C, and culture for 7-9 days.

[0029] Specifically, during the culturing process in step (2), the culture medium is replaced every two days.

[0030] Specifically, the concentration of the cell recovery solution in step (3) is 1X the original solution.

[0031] Specifically, the incubation conditions after adding the cell recovery solution in step (3) are 2-4°C and incubation for 10-30 minutes;

[0032] Furthermore, the incubation conditions after adding the cell recovery solution in step (3) are 4° C. and incubation for 30 minutes.

[0033] Specifically, the centrifugation conditions in step (3) are 4-6°C, 200-500g, and centrifugation for 4-10 min;

[0034] Furthermore, the centrifugation conditions in step (3) are 4°C, 300g, and centrifugation for 5 minutes.

[0035] Specifically, in step (3), the volume ratio of matrix gel to cell suspension is (1-5):1;

[0036] Furthermore, in step (3), the volume ratio of matrix gel to cell suspension is 2:1.

[0037] Specifically, the incubation condition after adding the matrix gel in step (3) is 37° C., and the incubation time is 15-20 min;

[0038] Furthermore, in step (3), the incubation condition after adding the matrix gel is 37° C., and the incubation time is 20 min.

[0039] Specifically, after adding the complete culture medium of the airway organoids in step (3), the culture conditions are continued at 37° C. and 5% CO 2 for 48 hours.

[0040] Specifically, the incubation time in step (4) is 12-24 hours.

[0041] Specifically, the concentration of LPS in step (4) is 1-20 μg / ml, including 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 11 μg / ml, 12 μg / ml, 13 μg / ml, 14 μg / ml, 15 μg / ml, 16 μg / ml, 17 μg / ml, 18 μg / ml, 19 μg / ml, 20 μg / ml, etc. Other point values ​​within this numerical range can be selected.

[0042] Furthermore, the concentration of LPS in step (4) is 1, 5, 10, or 20 μg / ml.

[0043] On the other hand, the present invention provides a mouse airway organoid inflammation model, which is obtained by the above-mentioned construction method.

[0044] On the other hand, the present invention provides the use of the above-mentioned mouse airway organoid inflammation model in screening drugs for preventing and / or treating respiratory diseases.

[0045] Specifically, the respiratory diseases include but are not limited to chronic respiratory diseases.

[0046] Furthermore, the chronic respiratory diseases include but are not limited to chronic bronchitis, chronic obstructive pulmonary disease, bronchiectasis, bronchial asthma, and interstitial pneumonia.

[0047] The beneficial effects of the present invention are:

[0048] (1) The present invention separates mouse airway epithelial cells and constructs AO through 3D culture. During the culture process, AO spontaneously forms a cystic structure with clear edges. As the culture time increases, the structure gradually increases, the internal cavity thickens, and the cells begin to fall off. As the number of passages increases, the characteristics of the AO remain unchanged.

[0049] (2) The constructed AO covers four major cell types: ciliated cells, goblet cells, basal cells, and club cells;

[0050] (3) The model constructed by the present invention significantly caused epithelial damage in AO;

[0051] (4) The model constructed in the present invention significantly induced the expression of AO inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0053] Figure 1 To isolate tracheal epithelial cells, A in the figure shows the blood vessels and muscle tissues around the tracheal tissues, B shows the tracheal tissue fragments, C shows the droplet state after resuspension, and D shows the addition of AO complete medium so that the Matrigel dome is completely immersed in the medium.

[0054] Figure 2 This is a representative bright field image of AO.

[0055] Figure 3 This is a representative image of AO immunofluorescence staining, in which A is a ciliated cell, B is a goblet cell, C is a basal cell, and D is a club cell.

[0056] Figure 4 This is a representative picture of AO live-dead staining.

[0057] Figure 5 The figure shows the effect of LPS stimulation on the expression of AO inflammatory factors. Figure A shows the expression of IL-1β factor; Figure B shows the expression of IL-6 factor; Figure C shows the expression of TNF-α factor.

[0058] Figure 6 This is a graph showing the experimental results of Comparative Example 1.

[0059] Figure 7 This is the experimental result diagram of Comparative Example 2.

[0060] Figure 8 The figure shows the effect of TNF-α and IFN-γ stimulation on the expression of AO inflammatory factors in comparative example 3. Figure A is the expression diagram of IL-1β factor; Figure B is the expression diagram of IL-6 factor; Figure C is the expression diagram of TNF-α factor. DETAILED DESCRIPTION

[0061] The manufacturer and item number of the experimental materials:

[0062]

[0063]

[0064] Example 1 Construction of mouse airway organoid inflammation model

[0065] 1. Isolation of mouse airway epithelial cells

[0066] Mice were anesthetized with carbon dioxide and then killed by dislocating the neck. They were then disinfected by soaking in 75% alcohol. Then they were transferred to a biosafety cabinet, the skin was cut along the middle side of the neck, the neck muscle layer was opened, the tracheal tissue was freed, and it was cut from the bottom of the larynx to the bronchus. The tracheal tissue was rinsed with PBS until no other tissue was left and then minced. It was washed 5 times with Advance DMEM / F12 containing 1x L-glutamine, 10mM HEPES and 1% double antibody. Digested with 2mg / ml collagenase type I at 37℃ for 2h, and mixed continuously during the period. After digestion, the cells were filtered through a 70μm filter, centrifuged at 400g for 3min, and counted.

[0067] 2AO Cultivation

[0068] The obtained cells were resuspended with 50-200 μl of Matrigel, and then inoculated into 24-well plates at 40-50 μl per well, maintaining the Matrigel droplet shape. After incubation at 37°C for 20 min, each well was supplemented with 500 μl of complete airway organoid medium (Advanced DMEM / F12, Glutamax (2 mM / ml), HEPES (10 mM / ml), 1% 1x PS, 1xN2, 1x B27, N-acetylcysteine ​​(1 mM / ml), EGF (50 ng / ml), Noggin (100 ng / ml), R-spondin (500 ng / ml)) and 10 μM Y27632 after Matrigel solidified. The medium was changed every two days. The cells were passaged every 7-9 days.

[0069] Passaging of 3AO

[0070] When AO is cultured for 7-9 days or black clumps appear in the center, it can be subcultured. The ratio of the number of cells in the original culture medium to the number of cells in the new culture medium is 1:3. Aspirate the waste culture medium in the well plate, add 500μl Cell recovery solution to each well, and incubate at 4℃ for 30min. Centrifuge at 4℃300g for 5min, aspirate the supernatant, add 500μl basal culture medium and repeatedly blow AO to disperse it. Centrifuge at 4℃300g for 5min, aspirate the supernatant, add Matrigel (50μl / well*number of wells inoculated = total volume of matrix gel), mix it with AO, and inoculate it into 24-well plates at 50μl / well. Incubate the culture plate in a 37℃ incubator for 20min. After the Matrigel solidifies, add 500μl complete culture medium to each well and continue culturing in the incubator.

[0071] Cryopreservation and thawing of 4AO

[0072] Organoids cultured for 4-5 days after subculturing were selected for cryopreservation. After degelling with Cell recovery solution, AO was directly resuspended in 1 ml cryopreservation solution (80% Advanced DMEM / F12+10% FBS+10% DMSO) and added to a 1 ml cryopreservation tube. After being stored at -80°C for 1 day in a programmed cooling cryopreservation box, it was transferred to liquid nitrogen for long-term storage. When reviving AO, the cryopreservation tube was removed from the liquid nitrogen, heated in a 37°C water bath, and after being dissolved, it was transferred to an EP tube and centrifuged at 300g for 5 minutes at room temperature. Complete AO inoculation according to the AO culture steps.

[0073] 5LPS intervention

[0074] The passaged AO were inoculated in a 24-well plate and cultured for 4 days. Then, 0, 1, 5, 10, and 20 μg / ml LPS were added to the complete medium and incubated in an incubator for 24 h.

[0075] 6 Statistical methods

[0076] All data are presented as mean ± standard deviation. After normality test, one-way analysis of variance was used for differences between groups. For normally distributed data, Dunnett's post hoc test was used. For non-normal distribution, Kruskal-Wallis analysis was used. All statistical graphs were generated using Graph Pad 8. *P<0.05 was considered to be significantly different.

[0077] Experimental Results

[0078] 1. Isolation of airway epithelial cells

[0079] After exposing the trachea along the middle of the neck, the blood vessels and muscle tissue surrounding the tracheal tissue are stripped away ( Figure 1 A in the figure). The trachea was cut off and transferred to a dish, and tissue fragments were obtained by mechanical separation ( Figure 1 B). After collagenase digestion, the cells were filtered. After Matrigel was resuspended, the cells were kept in a droplet state ( Figure 1 C) to maintain the 3D culture environment of epithelial cells. Add AO complete medium to make the Matrigel dome completely immersed in the medium ( Figure 1 D).

[0080] 2 Establishment of mouse AO culture system

[0081] In the 3D culture environment, primary mouse airway epithelial cells began to spontaneously form a spherical structure with clear edges within 1 day after inoculation, and there was a small cavity inside the sphere ( Figure 2A in the figure). On the 4th day of continuous culture, the spherical structure gradually increased in size. Due to cell proliferation, many new cell clusters protruded from the surface of the spherical structure. On the 5th day of culture, the spherical structure further expanded, the cavity volume gradually increased, and the tube wall became thicker than the previous day. On the 6th day of culture, it was found that the cells in the center of the cavity began to apoptosis and fall off, and appeared dark brown. When cultured continuously for 11 days, the volume of AO further increased, the boundary between the tube wall and the internal cavity was clear, and the dead cells that fell off inside the cavity made circular motions under the beating of cilia ( Figure 2 A in the figure). After subculturing, AO can spontaneously form spheres on the first day, and on the second to third day, spherical structures with cavity-like structures can be seen ( Figure 2 B).

[0082] Example 2 Construction of mouse airway organoid inflammation model

[0083] 1. Isolation of mouse airway epithelial cells

[0084] Mice were anesthetized with carbon dioxide and then killed by dislocating the neck. They were then disinfected by soaking in 75% alcohol. Then they were transferred to a biosafety cabinet, the skin was cut along the middle side of the neck, the neck muscle layer was opened, the tracheal tissue was freed, and it was cut short from the bottom of the larynx to the bronchus. The tracheal tissue was rinsed with PBS until no other tissue remained and then minced. It was washed 5 times with Advance DMEM / F12 containing 1x L-glutamine, 10mM HEPES and 1% double antibody. Digested with 4mg / ml collagenase at 37℃ for 2h, and mixed continuously during the period. After digestion, the cells were filtered through a 70μm filter, centrifuged at 400g for 3min, and counted.

[0085] 2AO Cultivation

[0086] The obtained cells were resuspended with 50-200 μl Matrigel, and 40-50 μl was inoculated into 24-well plates per well, maintaining the Matrigel droplet shape. The cells were incubated at 37°C for 20 min. After the Matrigel solidified, 500 μl of airway organoid complete medium (Advanced DMEM / F12, Glutamax (1.5 mM / ml), HEPES (8 mM / ml), 1% 1x PS, 1% 1x N2, 2% 1x B27, N-acetylcysteine ​​(1.5 mM / ml), EGF (45 ng / ml), Noggin (80 ng / ml), R-spondin (450 ng / ml)) was added to each well, and 8 μM Y27632 was added. The culture medium was changed every two days. The cells were passaged every 7-9 days.

[0087] Passaging of 3AO

[0088] When AO is cultured for 7-9 days or when black masses appear in the center, it can be subcultured, and each well is subcultured at a ratio of 1:3. Aspirate the waste culture medium in the well plate, add 500μl Cell recovery solution to each well, and incubate at 4℃ for 30min. Centrifuge at 4℃300g for 5min, and aspirate the supernatant. Add 500μl basal culture medium and blow AO repeatedly to disperse it. Centrifuge at 4℃300g for 5min, and aspirate the supernatant. Add a certain volume of Matrigel (50μl / well*number of wells inoculated=total volume of matrix gel), mix it thoroughly with AO, and inoculate it into a 24-well plate, with 50μl cell suspension in each well. Incubate the culture plate in a 37℃ incubator for 20min. After the Matrigel solidifies, add 500μl complete culture medium to each well and place it in the incubator for continued culture.

[0089] 4AO cryopreservation and thawing

[0090] Organoids cultured for 4-5 days after subculturing were selected for cryopreservation. After degelling with Cell recovery solution, AO was directly resuspended in 1 ml cryopreservation solution (80% Advanced DMEM / F12+10% FBS+10% DMSO) and added to a 1 ml cryopreservation tube. After being stored at -80°C for 1 day in a programmed cooling cryopreservation box, it was transferred to liquid nitrogen for long-term storage. When reviving AO, the cryopreservation tube was removed from the liquid nitrogen, heated in a 37°C water bath, and after being dissolved, it was transferred to an EP tube and centrifuged at 300g for 5 minutes at room temperature. Complete AO inoculation according to the AO culture steps.

[0091] 5LPS intervention

[0092] The passaged AO were inoculated in a 24-well plate. After culturing for 4 days, 0, 1, 5, 10, and 20 μg / ml LPS were added to the complete medium and incubated in an incubator for 24 h.

[0093] 6 Statistical methods

[0094] All data are presented as mean ± standard deviation. After normality test, one-way analysis of variance was used for differences between groups. For normally distributed data, Dunnett's post hoc test was used. For non-normal distribution, Kruskal-Wallis analysis was used. All statistical graphs were generated using Graph Pad 8. *P<0.05 was considered to be significantly different.

[0095] Experimental Results

[0096] 1. Isolation of airway epithelial cells

[0097] After exposing the trachea along the middle of the neck, the blood vessels and muscle tissue surrounding the tracheal tissue are stripped away ( Figure 1 A in the figure). The trachea was cut off and transferred to a dish, and tissue fragments were obtained by mechanical separation ( Figure 1 B). After collagenase digestion, the cells were filtered. After Matrigel was resuspended, the cells were kept in a droplet state ( Figure 1 C) to maintain the 3D culture environment of epithelial cells. Add AO complete medium to make the Matrigel dome completely immersed in the medium ( Figure 1 D).

[0098] 2 Establishment of mouse AO culture system

[0099] In the 3D culture environment, primary mouse airway epithelial cells began to spontaneously form a spherical structure with clear edges within 1 day after inoculation, and there was a small cavity inside the sphere ( Figure 2 A in the figure). On the 4th day of continuous culture, the spherical structure gradually increased in size. Due to cell proliferation, many new cell clusters protruded from the surface of the spherical structure. On the 5th day of culture, the spherical structure further expanded, the cavity volume gradually increased, and the tube wall became thicker than the previous day. On the 6th day of culture, it was found that the cells in the center of the cavity began to apoptosis and fall off, and appeared dark brown. When cultured continuously for 11 days, the volume of AO further increased, the boundary between the tube wall and the internal cavity was clear, and the dead cells that fell off inside the cavity made circular motions under the beating of cilia ( Figure 2 A in the figure). After subculturing, AO can spontaneously form spheres on the first day, and on the second to third day, spherical structures with cavity-like structures can be seen ( Figure 2 B).

[0100] Example 3 Construction of mouse airway organoid inflammation model

[0101] 1. Isolation of mouse airway epithelial cells

[0102] Mice were anesthetized with carbon dioxide and then killed by dislocating the neck. They were then disinfected by soaking in 75% alcohol. Then they were transferred to a biosafety cabinet, the skin was cut along the middle side of the neck, the neck muscle layer was opened, the tracheal tissue was freed, and it was cut short from the bottom of the larynx to the bronchus. The tracheal tissue was rinsed with PBS until no other tissue remained and then minced. It was washed 5 times with 1xAdvance DMEM / F12 containing 1x L-glutamine, 10MM HEPES and 1x double antibody. Digested with 5mg / ml collagenase at 37℃ for 2h, and mixed continuously during the period. After digestion, the cells were filtered through a 70μm filter, centrifuged at 400g for 3min, and counted.

[0103] 2AO Cultivation

[0104] The obtained cells were resuspended with 50-200 μl Matrigel, and then inoculated into a 24-well plate at 40-50 μl per well, maintaining the Matrigel droplet morphology. The cells were incubated at 37°C for 20 min. After the Matrigel solidified, each well was supplemented with 500 μl of airway organoid complete medium (Advanced DMEM / F12, Glutamax (2.5 mM / ml), HEPES (12 mM / ml), 1% 1x PS, 1% 1x N2, 2% 1x B27, N-acetylcysteine ​​(2 mM / ml), EGF (55 ng / ml), Noggin (110 ng / ml), R-spondin (600 ng / ml), and 12 μM Y27632. The culture medium was replaced every two days. The cells were passaged every 7-9 days.

[0105] Passaging of 3AO

[0106] When AO is cultured for 7-9 days or when black clumps appear in the center, it can be subcultured at a ratio of 1:3 per well. Aspirate the spent culture medium in the well plate, add 500μl Cell recovery solution to each well, and incubate at 4℃ for 30min. Centrifuge at 4℃300g for 5min, and aspirate the supernatant. Add 500μl basal culture medium and blow AO repeatedly to disperse it. Centrifuge at 4℃300g for 5min, and aspirate the supernatant. Add a certain proportion of Matrigel, mix it thoroughly with AO, and inoculate it into a 24-well plate, with 50μl cell suspension per well. Incubate the culture plate in a 37℃ incubator for 20min. After Matrigel solidifies, add 500μl complete culture medium to each well and place it in the incubator for continued culture.

[0107] Cryopreservation and thawing of 4AO

[0108] Organoids cultured for 4-5 days after subculturing were selected for cryopreservation. After degelling with Cell recovery solution, AO was directly resuspended in 1 ml cryopreservation solution (80% Advanced DMEM / F12+10% FBS+10% DMSO) and added to a 1 ml cryopreservation tube. After being stored at -80°C for 1 day in a programmed cooling cryopreservation box, it was transferred to liquid nitrogen for long-term storage. When reviving AO, the cryopreservation tube was removed from the liquid nitrogen, heated in a 37°C water bath, and after being dissolved, it was transferred to an EP tube and centrifuged at 300g for 5 minutes at room temperature. Complete AO inoculation according to the AO culture steps.

[0109] 5LPS intervention

[0110] The passaged AO were inoculated in a 24-well plate and cultured for 4 days. Then, 0, 1, 5, 10, and 20 μg / ml LPS were added to the complete medium and incubated in an incubator for 24 h.

[0111] 6 Statistical methods

[0112] All data are presented as mean ± standard deviation. After normality test, one-way analysis of variance was used for differences between groups. For normally distributed data, Dunnett's post hoc test was used. For non-normal distribution, Kruskal-Wallis analysis was used. All statistical graphs were generated using Graph Pad 8. *P<0.05 was considered to be significantly different.

[0113] Experimental Results

[0114] 1. Isolation of airway epithelial cells

[0115] After exposing the trachea along the middle of the neck, the blood vessels and muscle tissue surrounding the tracheal tissue are stripped away ( Figure 1 A in the figure). The trachea was cut off and transferred to a dish, and tissue fragments were obtained by mechanical separation ( Figure 1 B). After collagenase digestion, the cells were filtered. After Matrigel was resuspended, the cells were kept in a droplet state ( Figure 1 C) to maintain the 3D culture environment of epithelial cells. Add AO complete medium to make the Matrigel dome completely immersed in the medium ( Figure 1 D).

[0116] 2 Establishment of mouse AO culture system

[0117] In the 3D culture environment, primary mouse airway epithelial cells began to spontaneously form a spherical structure with clear edges within 1 day after inoculation, and there was a small cavity inside the sphere ( Figure 2 A in the figure). On the 4th day of continuous culture, the spherical structure gradually increased in size. Due to cell proliferation, many new cell clusters protruded from the surface of the spherical structure. On the 5th day of culture, the spherical structure further expanded, the cavity volume gradually increased, and the tube wall became thicker than the previous day. On the 6th day of culture, it was found that the cells in the center of the cavity began to apoptosis and fall off, and appeared dark brown. When cultured continuously for 11 days, the volume of AO further increased, the boundary between the tube wall and the internal cavity was clear, and the dead cells that fell off inside the cavity made circular motions under the beating of cilia ( Figure 2 A in the figure). After subculturing, AO can spontaneously form spheres on the first day, and on the second to third day, spherical structures with cavity-like structures can be seen ( Figure 2 B).

[0118] Comparative Example 1

[0119] The only difference from Example 1 is the type of digestive solution, and the rest are the same. The digestive solution used in Comparative Example 1 is a combination of collagenase II and collagenase IV, and the mass ratio of collagenase II to collagenase IV is 1:1.

[0120] Comparative Example 2

[0121] The only difference from Example 1 is that the concentrations of the components of the airway organoid complete medium are different (airway organoid complete medium (Advanced DMEM / F12, Glutamax (1mM / ml), HEPES (10mM / ml), 1% 1x PS, 1xN2, 1xB27, N-acetylcysteine ​​(0.5mM / ml), EGF (25ng / ml), Noggin (50ng / ml), R-spondin (300ng / ml), and 5μM Y27632 is added), and the rest are the same.

[0122] Comparative Example 3

[0123] The only difference from Example 1 is that other inducers (TNF-α, IFN-γ) are used to construct the airway organoid inflammation model, and the concentration of the inducer is 0.01-100 μg / ml, and the rest is the same.

[0124] Experimental Example 1 Immunofluorescence staining of AO

[0125] Airway epithelial cells are mainly composed of four types of cells: ciliated cells (ACCTUB + or FOXJ1 + ), goblet cells (MUC5AC + ), basal cells (P63 + ), rod-shaped cells (CC10 + ). To further verify whether the constructed AO covers these four major cell types, immunofluorescence staining was performed.

[0126] After the airway organoids (AO) were inoculated in a 24-well plate, they were cultured in a 5% CO2, 37°C incubator for 48 hours. 500μl of Cell recovery solution was added to each well, and the matrix gel was dissolved at 4°C for 30 minutes. The AO was collected in an EP tube and centrifuged at 4°C 800rpm for 3 minutes, and the supernatant was discarded. 500μl of 4% PFA was added for fixation for 20 minutes. After washing with PBS, 0.1% Triton-100 was added and incubated for 20 minutes to break the membrane. After washing with PBS, 5% BSA was incubated for 1 hour for blocking. Then the primary antibody was diluted in a certain proportion and incubated at 4°C overnight. After washing with 0.1% Triton-100, the secondary antibody was added and incubated at room temperature for 2 hours. After incubation with DAPI for 10 minutes, laser confocal scanning was performed to take pictures.

[0127] The experimental results are as follows Figure 3 As shown. The results showed that AO contains ciliated cells ( Figure 3 A), goblet cells ( Figure 3 B), basal cells ( Figure 3 C in), rod-shaped cells ( Figure 3 D).

[0128] Experimental Example 2AO Live-Dead Staining

[0129] After degelling with Cell recovery solution, AO was collected, PI (2 μg / ml) and calcein (2 μg / ml) were added, and incubated at 37°C for 30 min. After washing 3 times with PBS, anti-fluorescence quencher was added to resuspend AO and then dripped onto the slide. Laser confocal imaging was used.

[0130] Experimental Results

[0131] After AO was stimulated with 0, 1, 5, 10, and 20 μg / ml LPS for 24 h, we used calcein and PI to label live and dead cells in AO. Figure 4 As shown, no obvious cell death was observed in the normal control group (0 μg / ml LPS) in AO. As the concentration increased, 10 and 20 μg / ml LPS significantly caused cell death in AO. This indicates that LPS stimulation caused epithelial damage in AO.

[0132] Experimental Example 3 QPCR Detection

[0133] In order to evaluate the changes in the expression of inflammatory-related factors in AO after LPS intervention, we collected AO samples, extracted RNA using the RNAeasy Animal RNA Extraction Kit (purchased from Beyotime, catalog number R0027), and performed qPCR using BeyoFast SYBR GreenqPCR Mix to detect the expression levels of target genes.

[0134] The QPCR assay consists of the following steps:

[0135] 1. Reverse transcription: Use a reverse transcription kit (Novozyme) to reverse transcribe the extracted RNA into cDNA.

[0136] 2. Prepare qPCR reaction system (take 20μl system as an example):

[0137] Table 1

[0138] Reagent name volume 2x Taq Pro Universal SYBR qPCR Master Mix 10μl Primer1 (10 μM) 0.4μl Primer2 (10 μM) 0.4μl Template cDNA 2μl <![CDATA[ddH2O]]> To 20μl

[0139] 3. qPCR reaction program settings: Stage 1 (pre-denaturation) 95°C 30s, Stage 2 (cycling reaction) 95°C 3-10s 60°C 10-30s, Stage 3 (melting curve) 95°C 15s 60°C 60s 95°C 15s.

[0140] 4. After the required detection gene is prepared according to the above reaction system, perform the plate operation, accurately add the reaction solution into 8 tubes in a 96-well plate, set up 2 replicate wells, and repeat the experiment 3 times.

[0141] All primer information is shown in Table 2.

[0142] Table 2 Primer sequences

[0143]

[0144] Experimental Results

[0145] After AO was stimulated with different concentrations of LPS for 24 hours, the expression changes of inflammatory factors in AO were detected. Compared with the normal control group, 10μg / ml LPS stimulation significantly upregulated the expression of inflammatory factors IL-1β, IL-6 and TNF-α. When stimulated with 20μg / ml LPS, the expression of TNF-α was significantly upregulated ( Figure 5 ). This suggests that LPS stimulation activates the inflammatory response of AO.

[0146] The results of immunofluorescence identification of airway organoids in Example 1 are as follows Figure 3 shown.

[0147] The results of live-dead immunofluorescence staining in Example 2 are as follows Figure 4 shown.

[0148] The results of qPCR inflammatory factor detection in Example 3 are as follows Figure 5 shown.

[0149] Experimental results of Comparative Examples 1-3:

[0150] Comparative Example 1 Digestive fluid is different

[0151] The cells cannot be completely digested and cannot grow normally to form organoids. Figure 6 shown.

[0152] Comparative Example 2 Different culture medium concentrations

[0153] The concentration of the culture medium changes and cannot meet the growth requirements of the organoids. The cells are in poor condition and gradually die. Figure 7 shown.

[0154] Comparative Example 3 Modeling reagents are different

[0155] qPCR inflammatory factor detection results Figure 8 As shown, there was no significant difference in the levels of inflammatory factors between the control group and the control group (P>0.05), and the inflammatory model was not successfully established.

Claims

1. A method for constructing a mouse airway organoid inflammation model, characterized in that: The following steps are involved: (1) Obtain mouse airway tissue, digest it with digestive fluid, and centrifuge it to obtain a cell suspension; (2) After centrifugation of the cell suspension, the supernatant was discarded, Matrigel was added and mixed, and the cells were incubated and cultured using complete airway organoid medium; (3) adding cell recovery solution to the cells cultured in step (2) to incubate and remove gel, centrifuge, discard the supernatant, add matrix gel, mix, incubate, and add airway organoid complete culture medium to continue culturing; (4) Add 1-20 μg / ml LPS to the complete medium of airway organoids and incubate; The airway complete culture medium includes Advanced DMEM / F12, 1.5-2.5mM / ml Glutamax, 8-12mM / ml HEPES, 1-3% 1x PS, 1-3% 1x N2, 1-3% 1x B27, 1-2mM / ml N-acetylcysteine, 40-55ng / ml EGF, 80-120ng / ml Noggin, 400-600ng / ml R-spondin and 8-12μM Y27632.

2. The construction method according to claim 1, characterized in that: The digestion solution in step (1) is collagenase type I, and the concentration of collagenase type I is 1-5 mg / ml.

3. The construction method according to claim 2, characterized in that: The concentration of collagenase type I described in step (1) is 2 mg / ml.

4. The construction method according to claim 1, characterized in that: The complete culture medium of airway organoids includes Advanced DMEM / F12, 2mM / ml Glutamax, 10mM / ml HEPES, 1% 1x PS, 1% 1x N2, 2% 1x B27, 1mM / ml N-acetylcysteine, 50ng / ml EGF, 100ng / ml Noggin, 500ng / ml R-spondin and 10μM Y27632.

5. The construction method according to claim 1, characterized in that: After adding the cell recovery solution in step (3), the incubation condition is 4-6° C. for 20-40 minutes.

6. The construction method according to claim 1, characterized in that: In step (3), the volume ratio of matrix gel to cell suspension is (1-5):

1.

7. The construction method according to claim 1, characterized in that: The incubation time in step (4) is 12-24 hours.

8. The construction method according to claim 7, characterized in that: The concentration of LPS in step (4) is 1, 5, 10 or 20 μg / ml.

9. A mouse airway organoid inflammation model, characterized in that: The mouse airway organoid inflammation model is obtained by the construction method described in any one of claims 1-8.

10. Use of the mouse airway organoid inflammation model constructed by the construction method according to any one of claims 1 to 8 in screening drugs for preventing and / or treating respiratory diseases.

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