Construction method of mouse airway organ-like model

Abstract

The invention discloses a construction method of a mouse airway organoid model, and relates to the technical field of animal models, and the technical key points are as follows: mouse airway epithelial cells are extracted, and stable passage of airway organoid of premature and full-month newborn mice is established; meanwhile, morphological examination, immunofluorescence, quantitative real-time PCR (Polymerase Chain Reaction), transcriptome sequencing, protein microarray analysis and other methods are used for detecting the characteristics of airway organs of full-term and premature mice and the reaction difference condition of the airway organs to respiratory syncytial virus (RSV) infection, and an excellent in-vitro model is provided for researching respiratory system diseases and airway development.

Description

Technical Field

[0001] The present invention relates to the technical field of animal models, and in particular to a method for constructing a mouse airway organoid model. Background Art

[0002] Due to improvements in perinatal care, the number of preterm infants has been increasing, accounting for approximately 11% of births worldwide. Despite this, surviving preterm infants often face considerable respiratory challenges, including bronchopulmonary dysplasia, exacerbated symptoms following infection, increased rates and duration of hospitalization, and negative effects on growth and development. Prematurity disrupts the natural course of respiratory development, leading to adverse respiratory outcomes that persist into childhood. Although the underlying mechanisms leading to these adverse respiratory outcomes are not fully understood, intrinsic abnormalities within the airway epithelium may play a role.

[0003] Recent studies on the airway epithelium have focused on disease and infection models, while only a few studies have examined airway epithelial development. There is no consensus on the differences in functional cells (including goblet cells and club cells) in the airways of preterm and term infants and their association with susceptibility of preterm infants to respiratory diseases. However, an imbalance in secretory cells has been implicated in many diseases, including pneumonia, airway obstruction, and progressive interstitial lung disease, which may be an underlying cause of the vulnerability of the immature airways.

[0004] To uncover differences and underlying mechanisms in the premature airway, experimental models have evolved from autopsy samples of deceased infants in the 1990s to modern animal models such as mice, rats, sheep, and monkeys. However, autopsy samples from the 1990s differ significantly from contemporary infants, and their availability is limited. Obtaining human samples from newborns via bronchoscopic biopsy is challenging due to ethical issues. Large animals such as sheep and monkeys require high breeding costs and extended culture times. Direct use of small animals, such as premature mice, is difficult to maintain once separated from their mothers, making it impossible to track continuous physical changes after experimental interventions.

[0005] To this end, the present invention aims to provide a method for constructing a mouse airway organoid model to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems and provide a method for constructing a mouse airway organoid model. By improving the culture conditions, the survival rate and status of mouse airway organoids are improved, providing a reliable in vitro model for the study of airway development and respiratory diseases in premature and full-term mice, which helps to deeply understand the developmental differences and immune response mechanisms of the neonatal airway.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a method for constructing a mouse airway organoid model, the method comprising the following steps:

[0009] S1. Preparation of mouse airway samples: mice were raised in a specific pathogen free (SPF) environment. When the gestational age of the female mice reached 16.5 days, the female mice were delivered by caesarean section. The mouse pups were taken out, and their weight and length were recorded. After the mouse pups were anesthetized, their airway tissues were dissected to obtain mouse airway samples.

[0010] S2. Sample tissue processing and airway organoid culture: After the mouse airway samples were rinsed twice with cold DPBS, they were immersed in digestion solution and digested on a shaker at 37°C for 1 hour. The cell suspension was blown and filtered using a laboratory pipette tip. The filtered solution was centrifuged in a centrifuge for 3 minutes. The cell pellet was taken and washed twice with AdDF+++ and embedded in Matrigel matrix gel.

[0011] A suspension containing matrix gel and airway cells was seeded in a single well of a well plate. After solidification at 37°C for 15 minutes, airway organoid culture medium was added to each well and incubated under standard culture conditions. When the diameter of the airway organoid reached 100-200 μm, the mouse airway organoid was passaged to complete the airway organoid culture.

[0012] S3. Statistical analysis: Each experiment was repeated at least three times. All data are expressed as mean ± SD. All statistical analyses were performed using GraphPadPrism software (version 9). A normality test was performed to determine whether all statistical data conformed to a normal distribution. The differences between the two groups were compared using the t-test. For comparisons between two or more groups, a one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was used. When the data were not normally distributed, the Mann-Whitney-Wilcoxon test was used instead of the t-test to compare the differences between the groups. For comparisons between three or more groups, a one-way analysis of variance (ANOVA) was used for statistical comparisons. Significant differences between groups are indicated by *p<0.05 (the statistical significance level was set at p<0.05).

[0013] The airway organoid culture medium includes AdDF+++, 1×B27, 5mM nicotinamide, 1.25mM N-acetylcysteine, 500ng / mL R-Spondin1, 25ng / mL recombinant human FGF7, 100ng / mL recombinant human FGF10, 100ng / mL recombinant human Noggin, 5μM Y27632, 500nM SB202190 and 500nM A-8301; the airway organoid culture medium is replaced every 4 days or when the airway organoid culture medium turns yellow. The digestion solution consists of 400U / mL collagenase I, 0.25mg / mL protease E, 10μM Y27632 and 10U / ml DNAse I.

[0014] Compared with the prior art, this solution has the following beneficial effects:

[0015] 1. The immature airways simulated by mice in the present invention can be cultured for a long time and maintain their functions, and are suitable for pathogen intervention research, especially showing good application prospects in the study of RSV infection, providing more research platforms for studying respiratory diseases in premature infants, and can be used for dynamic monitoring of airway development, pathogen response and drug screening, etc.;

[0016] 2. The present invention improves the survival rate and status of mouse airway organoids by improving culture conditions, providing a reliable in vitro model for the study of airway development and respiratory diseases in premature and full-term mice, and helping to gain a deeper understanding of the developmental differences and immune response mechanisms of the neonatal airway. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : is a histological cell morphology diagram of mouse airway organoids in an embodiment of the present invention, wherein A is a schematic diagram of mouse airway organoid culture; B is the weight and length of newborn mice; C is HE staining of airway organoids of newborn mice in the full-term group (P0) and the premature group (E16.5) (Bar: 50um); D is immunofluorescence (IF) staining (Bar: 50um) (KRT5 is a basal cell marker, Ace-tublin is a ciliated cell marker, and CC10 is a club cell marker);

[0018] Figure 2: is a schematic diagram of the morphology and staining of mouse airway organoids in an embodiment of the present invention, and the relative expression of related gene markers, wherein A is a bright field view of premature group (E16.5) and full-term organoids (P0) (Bar: 200um); B is IF staining of premature and full-term organoids (Bar: 50um); C is the relative expression of airway markers at different birth dates; D is the relative expression of airway markers in premature group (E16.5) and full-term group (P10), (KRT5 is a basal cell marker, FOXJ1 is a ciliated cell marker, CC10 is a club cell marker, and MUC5AC is a secretory cell marker);

[0019] Figure 3 : is the transcriptomic analysis of mouse airway organoids in stable and infected states in the embodiment of the present invention; wherein A is the DEGs of normal organoids (E16.5vsE18.5); B is the DEGs of infected organoids (E16.5_RSV vs E18.5_RSV); C is the KEGG pathway enrichment bubble map of E16.5 and E18.5 normal organoids; D is the KEGG pathway enrichment bubble map of E16.5 RSV infected organoids and E18.5RSV infected organoids; E is the organoid volcano map (E16.5 normal organoids and E18.5 normal organoids); F is the DEGs volcano map (E16.5_RSV and E18.5_RSV infected organoids);

[0020] Figure 4 Schematic diagram, bright field view, IF staining, relative and absolute expression diagram of RSV infection of airway organoids of premature group (E16.5) and full-term group (P0) mice in the embodiment of the present invention, wherein A is a schematic diagram of organoid virus infection experiment (; B is a bright field view of RSV-infected organoid (Bar: 200um); C is IF staining of RSV-infected organoid (Bar: 50um); D is the expression of airway markers after RSV infection of organoid; E is the ddPCR result of RSV copy number in organoid at different time points; F is the expression of inflammatory factors in RSV-infected organoid;

[0021] Figure 5 Schematic diagram of the relative expression of airway markers in steady-state and RSV-infected mouse airway organoids (E15.5 and E18.5) in the embodiments of the present invention; A is the relative expression of airway marker genes in E15.5 (premature) and E18.5 (full-term) mouse airway organoids; B is the relative level of RSV RNA in E15.5 and E18.5 airway organoids 48 hours after infection;

[0022] Figure 6 It is a schematic diagram of the relative expression of inflammatory factors in airway organoids of E16.5 and E18.5 mice infected with RSV in an embodiment of the present invention. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] Example:

[0026] 1. Animal Ethics and Model Preparation

[0027] The animal experiments were approved by the Experimental Animal Management and Ethics Committee of West China Second Hospital, Sichuan University (approval number 2023-021). All animal procedures were in compliance with the regulations of the Animal Care and Use Committee of Sichuan University.

[0028] C57 / BL mice were housed in a specific pathogen free (SPF) environment with supplementary food and water sources and a 12-hour light-dark cycle. When the gestation of the female mice reached 16.5 days, the female mice were anesthetized and delivered by cesarean section. After the mouse pups were removed, their weight and length were recorded, and the airway tissue of the mouse pups was dissected from the thyroid cartilage to the tracheal bifurcation after anesthesia.

[0029] 2. Tissue Processing and Airway Organoid Culture

[0030] After obtaining the mouse airway samples, the tissues were rinsed twice with cold DPBS and transferred to 1 mL of digestion solution containing 400 U / mL collagenase I (Sigma, St. Louis, MO, USA, 9001-12-1), 0.25 mg / mL proteinase E (Sigma, P5147), 10 μM Y27632 (Selleck, Houston, TX, USA, S6390) and 10 U / ml DNAse I (Sigma, 10104159001) and digested on a shaker at 37°C for 1 hour. After enzyme digestion, the cell suspension was pipetted through a 1-mL pipette tip (laboratory pipette tip), filtered with a 40 um filter, and centrifuged at 200 × g for 3 minutes; the supernatant was discarded. The cell pellet was washed twice with AdDF+++ and embedded in Matrigel (Corning, New York, NY, USA, 356231).

[0031] A suspension containing 30 μL of Matrigel and 5000 airway cells was seeded in a single well of a 24-well plate. After solidification at 37°C for 15 minutes, 500 μL of pre-prepared airway organoid culture medium was added to each well and incubated under standard culture conditions (37°C, 5% CO2). Mouse airway organoid culture medium included AdDF+++, 1×B27 (Gibco, Grand Island, NY, USA, 0080085SA), 5 mM nicotinamide (Sigma, N0636), 1.25 mM N-acetylcysteine ​​(Sigma, A0737), 500 ng / mL R-Spondin1 (R&D, Minneapolis, MN, USA, 4645), 25 ng / mL recombinant human FGF7 (PeproTech, NJ, USA, 450-61), 100 ng / mL recombinant human FGF10, 100 ng / mL recombinant human Noggin (R&D, 6057), 5 μM Y27632 (CST, Boston, MA, USA, 13624), 500 nM SB202190 (Selleck, S1077) and 500 nM A-8301 (Selleck, S8301). The medium was changed every 4 days or when the medium turned yellow.

[0032] Mouse airway organoids were passaged when the organoid diameter reached 100-200 μm. All organoids and Matrigel were suspended in AdDF+++ buffer. After centrifugation at 200×g for 3 minutes, the organoid pellet was suspended with 1 mL TripLE 1× (Gibco, A1217701), incubated at 37°C for 5 minutes, and evenly blown with a 1-mL pipette tip (laboratory pipette tip). After washing twice with AdDF+++, the cell pellet was filtered with a 40 μm filter, suspended in Matrigel, and re-seeded in another well. Single cells were seeded in each well of a 24-well plate at a density of 5,000 cells per well.

[0033] 3. RNA Preparation and qRT-PCR

[0034] Total RNA was extracted from airway organoids using RNAprep Pure Micro Kit (Tiangen, Beijing, CA, China, DP420). cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland, 04897030001) according to the manufacturer's protocol. qRT-PCR was performed using SYBR green (Promega, Madison, WI, USA, A600A) and Bio-Rad system. Gene expression was quantified using the ΔΔCt method and normalized to GAPDH. The primers used are listed in Table 1.

[0035] Table 1 Primer list

[0036]

[0037] 4. Droplet Digital PCR

[0038] All droplet digital PCR (ddPCR) procedures followed the manufacturer's instructions for the QX200 Droplet Digital PCR System, using dUTP-free probe Supermix (Bio-Rad, Hercules, CA, USA). The final reaction volume was 20 μL, including the following components: 10 μL 2× dUTP-free probe Supermix (Bio-Rad); 2 μL cDNA generated using 100 ng RNA of the target sample; 0.5 μL RSV-NF; 0.5 μL RSV-NR; 0.5 μL RSV-NP; and 6.5 μL nuclease-free water. The 20 μL mixture was then converted into droplets using a QX200 droplet generator (Bio-Rad). The droplet partition samples were transferred to a 96-well plate, sealed, and subjected to the following cycling protocol in a T100 thermal cycler (Bio-Rad): 95°C for 10 min (DNA polymerase activation); 40 cycles of 94°C for 30 s (denaturation) and 58°C for 1 min (annealing); 98°C for 10 min; and a final hold at 4°C. The cycled 96-well plate was then read using a QX200 reader (Bio-Rad) on both the FAM and HEX channels.

[0039] 5. Section IF and Whole-Mount IF

[0040] Airway organoids were harvested and fixed with 4% formaldehyde overnight at 4°C. For IF sections, fixed organoids were dehydrated, paraffin-embedded and sectioned. For whole-tissue embedding IF, fixed organoids were permeabilized with 0.5% Triton X-100 and blocked with 5% bovine serum albumin (BSA) for 1 hour. Organoids were then incubated with primary antibodies, including ace-tubulin (SantaCruz, Santa Cruz, CA, USA, sc-23950), Krt5 (Abcam, CB, Waltham, MA, USA, ab52635), p63 (Santa Cruz, sc-25268) and SCGB1A1 (CC10) (Santa Cruz, sc-365992), overnight at 4°C. The next day, the organoids were washed three times and incubated for 1 hour with Hoechst 33342 (Invitrogen, Life Technologies, Carlsbad, CA, USA, H3570) and secondary antibodies labeled with Alexa Fluor 488 (Invitrogen, A11001) or Alexa Fluor 594 (Invitrogen, A11012). Finally, the organoids were washed three times and blocked with anti-fluorescence quenching blocking film (YEASEN, Shanghai, CA, China, 36307ES08). Images were acquired using a laser scanning confocal microscope (Olympus, Waltham, MA, USA).

[0041] 6. Respiratory syncytial virus (RSV) infection

[0042] RSVA2 (ATCC, Manassas, VA, USA, VR-1540) was obtained from Chongqing Medical University and cultured in HEp-2 cells (ATCC). Virus titer was determined based on previous studies. Airway organoids were prepared at a density of 5000 cells per well in 24-well plates. After washing with cold AdDF+++, one well of organoid was incubated with incubation solution (200 μL organoid medium plus 2 μL virus, 1 × 10 8 pfu / mL) were infected in a single 48-well plate. After incubation for 6 h at 37°C with 5% CO2, infected organoids were washed with AdDF+++ and seeded as above.

[0043] 7. Statistical Analysis

[0044] Each experiment was repeated at least three times. All data are expressed as mean ± SD. All statistical analyses were performed using GraphPad Prism software (version 9). A normality test was performed to determine whether all statistical data conformed to a normal distribution. The differences between the two groups were compared using the t-test. For comparisons between two or more groups, one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was used. When the data were not normally distributed, the Mann-Whitney-Wilcoxon test was used instead of the t-test to compare the differences between the groups. For comparisons between three or more groups, one-way analysis of variance (ANOVA) was used for statistical comparisons. Significant differences between groups are indicated by *p<0.05 (the statistical significance level was set at p<0.05).

[0045] 8. Results

[0046] 8.1 Selecting Mouse Organoid Models to Study the Preterm and Term Airway

[0047] To obtain preterm and full-term mouse samples, E16.5 pregnant mice were delivered by cesarean section after anesthetizing the mice, and their pups were used as the preterm group; while P0 pups were collected after natural vaginal delivery as the full-term group. E16.5 is a time point commonly used in studies related to immature mice, which is reported to be equivalent to 16-26 weeks of human lung development. Cesarean sections were performed to minimize the effects of traditional drugs that induce preterm birth in mouse models, including lipopolysaccharide (LPS) and Poly(I:C). These drugs can induce inflammatory responses in both the mother and the fetus. Cesarean sections were performed on E18.5 mice, and pup samples 4 days after natural vaginal delivery of mice (P4) were collected, and their weight and length were recorded, and it was observed that both groups of data increased with increasing gestational age ( Figure 1 A, 1B).

[0048] We selected E15.5 mouse pups to examine the airways and found that they exhibited hyperproliferation and differentiation of epithelial cells compared to organoid cultures of E18.5 mouse airways ( Figure 5 A). It was observed that RSV infection severity in E15.5 mice was lower than that in E18.5 mouse airway organoids ( Figure 5 B) We hypothesize that this paradox may be attributed to the incomplete development of cell surface receptors for RSV, resulting in lower levels of viral entry in the airways of E15.5 mice. Therefore, E15.5 may not be the optimal age to study airway development in preterm mice, either under steady-state or infection conditions.

[0049] 8.2 Preterm and term mice present different features in airway tissue

[0050] To identify specific morphological differences in the airway epithelium, hematoxylin-eosin (HE) and immunofluorescence (IF) staining were performed on airway tissue sections from preterm and term mice ( Figure 1 C, 1D). HE and IF staining of airway structures showed an increase in circumference according to the gestational age of mouse samples ( Figure 1 C, 1D). Cilia are clearly visible after HE staining ( Figure 1 White arrows in C) and IF staining ( Figure 1 D (green arrows in the lower row), all functional cells, including club cells and ciliated cells, are arranged around the airway epithelium, and KRT5+ basal cells surround the basal layer ( Figure 1 D).

[0051] Using qRT-PCR to examine classical genes and cells ( Figure 1 C, 1D). In the tissue, the expression of FOXJ1 and MUC5AC in E16.5 premature airway epithelial cells was higher than that in CC10 ( Figure 1 C). With increasing gestational age, the expression level of CC10 increased and exceeded that of FOXJ1 and MUC5AC, while they reached a relative balance in samples 4 days after birth ( Figure 2 C). Comparison of gene signatures between the E16.5 and P0 groups revealed that marker expressions of basal cells and ciliated cells were higher in the preterm group than in the term group, whereas CC10 of club cells was less expressed in the preterm group.

[0052] 8.3 Respiratory epithelial progenitor cells digested from airway tissue generate robust 3D organoid models

[0053] To establish a mouse airway model in vitro, the trachea of ​​newborn mice was dissected, the airway tissue was digested into isolated cells, and then they were embedded in Matrigel (matrigel), and differentiation medium ( Figure 2 A). Preterm and term samples were observed to form 3D hollow spheroids a few days after embedding. Although preterm samples grew faster than term samples at P0 (spheroid size reached 200um in 3 days, while term samples required 4-5 days), the expansion rate slowed down when passaged to P1 and P2, eventually becoming slower than term samples and stabilizing after P3. Other than the growth rate we observed from P0 to P2, there seemed to be no other differences under bright field ( Figure 2 B).

[0054] After successful formation of organoid spheroids, we examined the expression of classical airway marker genes in organoid cells using qRT-PCR and IF staining. Cells stained with KRT5 (basal cell marker), CC10 (club cell marker), Ace-tublin (ciliary cell marker), or MUC5AC (secretory cell marker) were localized to their corresponding in vivo locations in tracheal tissue ( Figure 2 C), which is consistent with the results in airway tissue.

[0055] 8.4 Preterm and term mouse airways display distinct transcriptome signatures in the epithelium

[0056] To investigate differences at the transcriptome level, bulk RNA sequencing was performed on preterm and term airway organoids. Transcriptome analysis of E16.5 and P0 mouse samples revealed differentially expressed genes (DEGs, padj, 0.05) between the two groups ( Figure 3 A, 3C). Compared with full-term samples, premature mice had 5481 DEGs, including 2782 upregulated genes and 2699 downregulated genes. Many of the DEGs upregulated in premature samples were related to inflammatory response, such as JAK2, JAK3, and LIFR. Genes in the PI3K-Akt signaling pathway also showed higher expression in the premature group, including Itga1, Col2a, Lama, etc. Among the epithelial differentiation signaling pathway genes, STRA6 (a retinoic acid-responsive gene) was significantly upregulated in the premature group. In contrast, the expression of Wnt5a, Wnt9a, and Wnt7b was lower than that in the full-term group, indicating lower Wnt signaling but higher epithelial cell differentiation. Gene set enrichment analysis also revealed DEGs in basal cell proliferation (p53 signaling pathway) and viral response (COVID-19) ( Figure 3 B), the latter being consistent with previously reported increased susceptibility of premature infants to SARS-CoV-2.

[0057] 8.5 Different antiviral responses of term and preterm mouse organoids after viral infection

[0058] To test whether the preterm airway has a hyperactive viral response phenotype, viral responses were assessed using RSV, one of the most common pathogens in infant and young airway infections ( Figure 4 A). In bright field, infected organoids are more compact than hollow spheres under normal culture conditions ( Figure 4 B). Although there seemed to be no difference between the term and preterm groups at the same infection time under bright field vision, IF staining showed that preterm infection was more severe than term infection, showing a higher RSV signal and the number of RSV-positive cells under confocal 3D scanning ( Figure 4C). To quantify the severity of RSV infection, ddPCR was performed on infected organoids from E16.5, E18.5, P0, and P4 mouse airway tissues, and their RSV copy numbers were monitored at 6, 12, 24, and 48 hours post-infection. The results showed that mouse airway organoids were most severely affected 24 hours after infection ( Figure 4 E). Upon closer inspection, infection was found to decrease with age of the pups (in E16.5, E18.5, and P0 groups), indicating that the earlier the gestational age at birth, the more severe the infection in the pups. It is worth noting that this conclusion does not include the P4 group, which had a higher rebound in viral copy number at 24 hours after infection than the E18.5 and P0 groups ( Figure 4 D) This difference may be due to greater RSV susceptibility and mortality in infants 28 days to 6 months of age.

[0059] To investigate the differences in antiviral responses at the transcriptome level, we first performed qRT-PCR and found that premature airway organoids showed significantly higher basal cell expression levels (KRT5) after viral infection ( Figure 4 D) However, functional cell markers, including CC10 in club cells and MUC5AC in goblet cells, were expressed at lower levels than in term airway organoids ( Figure 4 E). However, there was no significant difference in the expression of FOXJ1, a marker of ciliated cells ( Figure 4 E). RNA sequencing of RSV-infected organoids revealed that preterm DEGs were enriched in many viral response signaling pathways, including HSV, Epstein-Barr virus, COVID-19, CMV, and influenza A virus ( Figure 3 DF), indicating that premature newborns can display hyperactivity against pathogenic microorganisms.

[0060] The differences in immune responses between preterm and term neonates were investigated using qRT-PCR and liquid chip immunoassays. qRT-PCR results showed that term organoids exhibited higher levels of IL-6 (p<0.05) and TNF transcription after viral infection, suggesting that in the E16.5 group, when the trachea is not fully developed, the immune response is immature ( Figure 6 A) However, when the specific concentrations of each inflammatory factor were measured, there appeared to be no statistically or biologically significant differences, suggesting that there may not be significant differences in the supernatants of organoid cultures ( Figure 6 B).

[0061] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for constructing a mouse airway organoid model, characterized by: The method comprises the following steps: S1. Preparation of mouse airway samples: Mice were raised in a specific pathogen-free environment. When the gestational age of the female mice reached 16.5 days, the female mice were given cesarean section. The mouse pups were taken out and their weight and length were recorded. After the mouse pups were anesthetized, their airway tissues were dissected to obtain mouse airway samples. S2. Sample tissue processing and airway organoid culture: After the mouse airway samples were rinsed twice with cold DPBS, they were immersed in digestion solution and digested on a shaker at 37°C for 1 hour. The cell suspension was blown and filtered using a laboratory pipette tip. The filtered solution was centrifuged in a centrifuge for 3 minutes. The cell pellet was taken and washed twice with AdDF+++ and embedded in Matrigel matrix gel. A suspension containing matrix gel and airway cells was seeded in a single well of a well plate. After solidification at 37°C for 15 minutes, airway organoid culture medium was added to each well and incubated under standard culture conditions. When the diameter of the airway organoid reached 100-200 μm, the mouse airway organoid was passaged to complete the airway organoid culture. S3. Statistical analysis: Each experiment was repeated three times or more; All data are expressed as mean ± SD; All statistical analyses were performed using GraphPad Prism software; The differences between the two groups were compared using t-test; For comparisons between two or more groups, one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was used; When data were not normally distributed, the Mann-Whitney-Wilcoxon test was used instead of the t test to compare differences between groups; For comparisons among three or more groups, one-way analysis of variance (ANOVA) was used for statistical comparisons, and significant differences between groups were indicated by *p < 0.

05.

2. The method for constructing a mouse airway organoid model according to claim 1, characterized in that: The airway organoid culture medium includes AdDF+++, 1×B27, 5mM nicotinamide, 1.25mM N-acetylcysteine, 500ng / mL R-Spondin1, 25ng / mL recombinant human FGF7, 100ng / mL recombinant human FGF10, 100ng / mL recombinant human Noggin, 5μM Y27632, 500nM SB202190 and 500nM A-8301; Replace the airway organoid medium every 4 days or when the airway organoid medium turns yellow.

3. The method for constructing a mouse airway organoid model according to claim 1, characterized in that: The digestion solution consisted of 400 U / mL collagenase I, 0.25 mg / mL proteinase E, 10 μM Y27632 and 10 U / ml DNAse I.

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