Tracheal epithelial cell and application thereof

Abstract

The invention relates to the technical field of cell culture, and discloses a tracheal epithelial cell and application thereof, the cell is a guinea pig tracheal epithelial immortalized cell, and the preservation number is CGMCC No. 46763. In the invention, the cell has good susceptibility to respiratory pathogens, and has excellent transfection efficiency. The cells can be used for separating or culturing respiratory pathogens, preparing respiratory pathogen vaccines, screening and / or preparing medicines for preventing and / or treating diseases caused by the respiratory pathogens, serving as host cells for researching the respiratory pathogens, detecting the respiratory pathogens for a non-diagnostic purpose and constructing organoids. The method has high scientific research value and practical application potential.

Description

Tracheal epithelial cells and their applications Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically, to tracheal epithelial cells and their applications. Background Technology

[0002] Tracheal epithelial cells (especially guinea pig tracheal epithelial cells) are important in vitro models for studying respiratory diseases. Due to their readily available source and physiological characteristics similar to human respiratory epithelium, they are widely used in exploring the mechanisms and screening drugs for respiratory diseases such as asthma and chronic obstructive pulmonary disease. These cells can mimic airway inflammatory responses, mucus secretion, and epithelial barrier function, providing a reliable experimental basis for related research.

[0003] In existing technologies, research using guinea pig tracheal epithelial cells often faces challenges such as demanding cell culture conditions, difficulties in passage, low levels of viral infection, and low transfection efficiency in transgenic experiments, which limit their application in large-scale experiments and long-term studies. In published articles, Kajon et al. validated the propagation of guinea pig adenovirus (GPAdV) in various types of immortalized guinea pig cell lines (commercially available cells such as colorectal cancer (GPC-16, catalog number #CCL-242), fetal fibroblasts (104-C1, catalog number #CRL-1405), and lung fibroblasts (JH4C1, catalog number #CCL-158), as well as the guinea pig tracheal epithelial cell line GPTEC-T prepared in this study). All of these experiments supported viral infection and early propagation. However, cytopathic effects could not be observed in any of the four cell types until 5-14 days after infection, indicating a significantly low infection efficiency (Kajon, AE et al. Isolation and initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines. F1000Research8, 1597, doi:10.12688 / f1000research.20135.2 (2019)). Another experiment used adenovirus Ad5 with an MOI (multiple of infection) of 10 to infect airway epithelial cells, and the maximum infection rate was only 20% (Singhera, GK et al. Apoptosis of viral-infected airway epithelial cells limit viral production and is altered by corticosteroid exposure. Respiratoryresearch 7, 78, doi:10.1186 / 1465-9921-7-78 (2006)).The remaining studies were all in vivo infection experiments in guinea pigs, such as inoculation of guinea pigs with adenovirus, parainfluenza type III (Folkerts, G. et al. Virus-induced Changes in Airway Responsiveness, Morphology, and Histamine Levels in Guinea Pigs. American Review of Respiratory Disease 147, 1569-1577, doi:10.1164 / ajrccm / 147.6_Pt_1.1569 (1993)), and bovine parainfluenza virus type III (Han, Y. et al. Isolation and Identification of a Genotype C Bovine Parainfluenza Virus Type 3 and Its Pathogenicity in Albino Guinea Pigs. Transboundary and emerging diseases 2023, 8854528, doi:10.1155 / 2023 / 8854528). (2023).) Airway tissue lesions can be observed 3-4 days after infection (Folkerts, G., Verheyen, A. & Nijkamp, ​​FP Viral infection in guinea pigs induces a sustained non-specific airway hyperresponsiveness and morphological changes of the respiratory tract. European journal of pharmacology 228, 121-130, doi:10.1016 / 0926-6917(92)90021-4 (1992)). Therefore, there is an urgent need to develop novel cell lines that combine culture stability, high viral susceptibility, and transgenic compatibility to provide more efficient in vitro tools for respiratory-related research. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and to provide tracheal epithelial cells and their applications.

[0005] To achieve the above objectives, the first aspect of the present invention provides a tracheal epithelial cell line, which is an immortalized guinea pig tracheal epithelial cell, with the accession number CGMCC NO.46763.

[0006] Secondly, the present invention provides the application of the cells in the isolation of respiratory pathogens.

[0007] Thirdly, the present invention provides the use of the cells in the preparation of respiratory pathogen vaccines.

[0008] Fourthly, the present invention provides the use of said cells in screening and / or preparing medicaments for the prevention and / or treatment of diseases caused by respiratory pathogens.

[0009] Fifthly, the present invention provides the use of said cells in culturing respiratory pathogens.

[0010] In a sixth aspect, the present invention provides the use of said cells as host cells for studying respiratory pathogens.

[0011] In a seventh aspect, the present invention provides the use of said cells in the detection of respiratory pathogens for non-diagnostic purposes.

[0012] In this invention, the respiratory pathogen can be at least one of influenza virus, novel coronavirus (SARS-CoV-2), respiratory syncytial virus, rhinovirus, parainfluenza virus, and adenovirus, preferably influenza virus H1 and / or influenza virus H5.

[0013] More preferably, the cells exhibit a faster infection response, higher infection efficiency, and more pronounced infection characteristics to the H1 influenza virus.

[0014] Eighthly, the present invention provides the application of said cells in the construction of organoids.

[0015] In this invention, the tracheal epithelial cells are immortalized cells.

[0016] In this invention, the tracheal epithelial cells are derived from guinea pigs (Cavia porcellus).

[0017] Influenza viruses with high aerosol transmissibility exhibit higher infection efficiency in the tracheal epithelial cells provided in this invention, indicating that these cells have good susceptibility to respiratory pathogens and can more realistically simulate the viral infection process. In this invention, susceptibility specifically refers to the characteristic that cells can be successfully infected by the target pathogen within a short period of time (e.g., 24 hours). Simultaneously, these cells possess excellent transfection efficiency, facilitating molecular biology operations such as gene knockout and gene overexpression, providing a powerful experimental platform for in-depth research on the infection mechanisms, transmission characteristics, and interactions with host cells of respiratory pathogens such as influenza viruses.

[0018] The tracheal epithelial cells of this invention exhibit significant advantages in pathogen isolation and amplification, effectively supporting the culture and identification of respiratory pathogens such as influenza virus, and providing a reliable cell matrix for vaccine research and production. Furthermore, they have broad application prospects in molecular mechanism research, antiviral drug screening, and pathogen pathogenicity assessment, helping to reveal key regulatory factors and potential therapeutic targets of pathogen infection.

[0019] The establishment of tracheal epithelial cells in this invention not only helps improve the efficiency and accuracy of respiratory pathogen research, but also provides important technical support for pathogen early warning identification, risk assessment and prevention and control strategy formulation, and has high scientific research value and practical application potential.

[0020] The cells provided in this invention are immortalized guinea pig tracheal epithelial cells, deposited on December 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46763. The immortalized guinea pig tracheal epithelial cells provided in this invention are named GTE1. Attached Figure Description

[0021] Figure 1 shows the results of the transfection experiment of immortalized tracheal epithelial cells of the present invention; Figure 2 shows the results of the passage test of immortalized tracheal epithelial cells of the present invention; Figure 3 shows the relative expression level of viral nucleoprotein (NP) in immortalized tracheal epithelial cells of the present invention after viral infection; Figure 4 shows the relative expression level of viral M gene mRNA in immortalized tracheal epithelial cells of the present invention after viral infection; Figure 5 shows the results of immunofluorescence detection of immortalized tracheal epithelial cells of the present invention after viral infection. Detailed Implementation

[0022] The present invention will be described in detail below through embodiments.

[0023] In the following examples, influenza virus H1 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with strain number A / California / 04 / 2009 (H1N1); the Genbank accession number of H1N1 is GCA_038510525.1; influenza virus H5 was provided by the Academy of Military Medical Sciences of the Chinese People's Liberation Army, with strain number A / chicken / Hebei / 3399 / 2017 (H5N6); the HA and NA gene sequences of H5N6 correspond to Genbank accession numbers PX884831 and PX884830, respectively.

[0024] Example 1 This example illustrates the culture of primary cells.

[0025] Two healthy SPF (specific pathogen-free) female Hartley guinea pigs (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing approximately 300-350 g, were selected. Tracheal tissue was aseptically harvested after cervical dislocation. The tracheal tissue was minced and seeded into cell culture dishes, then cultured in primary culture medium containing DMEM / F12 (GIBCO, 21331020), 1% epithelial cell culture additive (Zhejiang Meisen Cell Technology Co., Ltd., CTCC-009-613-2), and 1% penicillin / streptomycin (GIBCO, 15140148). To screen for and remove contaminating fibroblast populations in the primary cell preparation, the primary cells were passaged and purified for two consecutive generations using serum-free medium. Once the epithelial cell purity met experimental requirements, subsequent functional verification experiments were conducted.

[0026] Example 21. Cell validation of bacterial, fungal, and mycoplasma detection to ensure cell freedom from microbial contamination. Specific steps are as follows: (1) Bacterial and fungal contamination detection culture method: Take 100 μL of cell culture medium and spread it on LB solid medium plates (Solepro, L1015) and PDA solid medium plates (Solepro, P8931); at the same time, set up a blank control by spreading 100 μL of sterile PBS on the same medium plates. Invert the plates after spreading, place the LB plates in a 37℃ constant temperature incubator for 24 h, and the PDA plates in a 28℃ constant temperature incubator for 48 h. Observe whether colonies grow on the plates.

[0027] Microscopic observation: Observe whether there are granular, filamentous, or moving small dots; and observe whether the culture medium is turbid, yellow, or contains flocculent or membrane-like substances.

[0028] (2) Mycoplasma contamination detection qPCR method: Take 2 μL of cell culture medium and prepare the reaction system according to the instructions of the mycoplasma qPCR detection kit (Beyotime, C0303S). Set the qPCR reaction program as follows: pre-denaturation 95℃ 2 min; denaturation 95℃ 15 s; annealing / extension (60℃) 20 s, for a total of 40 cycles. If the test sample does not have a typical S-type amplification curve in the FAM channel or the Ct value is >35, and the positive control test result is positive and the negative control test result is negative, the result is judged to be mycoplasma negative.

[0029] 2. Cell transduction (1) The day before the experiment, target cells were seeded into a six-well plate. The number of wells was seeded according to the number of experimental groups, and at least one well was reserved as a negative control. The cell quantity per well was 2-3 × 10⁶ cells. 5 Each cell.

[0030] (2) When the cell count reaches 60-70%, digest the cells in one well and count them. Using MOI=100, substitute the virus titer and cell count into the formula to calculate the volume (μL) of virus solution to be taken. The formula is as follows: MOI = titer × volume × 10 -3 / Cell count (3) Except for the negative control wells, remove the culture medium from the wells, add 1 mL of fresh culture medium, and place in an incubator for later use.

[0031] (4) 1×10 lentivirus 8 TU / mL (Suzhou Haixing Biotechnology Co., Ltd., HX-SV40T-LV1) and polybrene (Suzhou Haixing Biotechnology Co., Ltd., GUOR-R001) were dissolved at 4℃, and polybrene was added to each experimental group to make a final concentration of 5 μg / mL.

[0032] (5) Prepare the necessary consumables and reagents in the biosafety cabinet, add the required virus solution to each well, shake gently to mix, and place in a 37°C, 5% CO2 incubator for incubation.

[0033] (6) Observe cell morphology 6 h after transfection. If there is a toxic reaction, change the medium. If there is no abnormality, the time can be extended to a maximum of 24 h for medium change.

[0034] (7) Observe cell fluorescence 24 h after transfection. Take one bright field image and one fluorescence image of each group of cells in the same field of view. The results of the cell transfection experiment are shown in Figure 1. It can be seen that the cells of the present invention have extremely high transfection efficiency.

[0035] (8) When the cell count reached 90% or higher, the six-well cells were passaged and seeded in 6 cm culture dishes. By comparing the proliferation of the primary cells with that of the primary cells, when the primary cells stopped proliferating while the immortalized cells could continue to proliferate, the expression level of SV40T was detected by qPCR.

[0036] (9) The expression levels of the target gene (SV40-T) were analyzed by qPCR in primary tracheal epithelial cells and recombinant cell lines transfected with lentivirus (CGMCC No. 46763). A qPCR detection kit (Novizan, Q712-02 / 03) was used, and the qPCR reaction was performed according to the kit instructions. The primer sequences used are shown in Table 1; the qPCR reaction program is shown in Table 2; and the detection results are shown in Table 3.

[0037] Table 1

[0038] Table 2

[0039] Note: Fluorescence signal collection was performed in the second program segment (60℃ annealing and extension).

[0040] Table 3

[0041] As shown in Table 3, the cells of this invention can successfully express SV40-T, that is, the immortalized guinea pig tracheal epithelial cell line has been successfully constructed.

[0042] 3. Continuous passage culture, expansion and quality inspection (1) Primary cell passage: Passage continuously at a ratio of 1:3 until P3 cells show obvious aging and proliferation almost stops, then terminate the experiment; the primary cells are derived from guinea pig tracheal epithelial tissue.

[0043] (2) Cell passage (CGMCC NO. 46763): Cells were cultured in a medium containing DMEM / F12 + 10% FBS (fetal bovine serum, GIBCO, 10099141C) + 1% epithelial cell culture additive + 1% penicillin / streptomycin. Cells were passaged continuously at a 1:3 ratio to P5. Cell morphology was normal and proliferation was rapid. One plate of cells was selected for further passage, and the remaining four plates were cryopreserved for future use. Cells were passaged continuously at a 1:3 ratio to P15. Cell morphology was normal and proliferation was stable. These cells were then cryopreserved for seed culture.

[0044] (3) Cell thawing and passage: P15 cells were thawed and seeded in T25 culture flasks and passaged continuously to P60 at a ratio of 1:3. The cells had normal morphology and stable proliferation. (4) Cell expansion and cryopreservation: Cells were seeded in 10 cm culture dishes to reach 90% cryopreservation. Three tubes were cryopreserved per dish and stored in liquid nitrogen.

[0045] (5) Quality inspection: Culture method and microscopic observation: bacteria and fungi negative; qPCR method: mycoplasma negative.

[0046] The results are shown in Figure 2 and Table 4.

[0047] Table 4

[0048]

[0049] As can be seen from Table 4, the cells of the present invention can be stably and continuously passaged.

[0050] 4. Cell proliferation detection (1) P20 and P60 cells were administered at a concentration of 1×10⁻⁶. 5(1) 10 cells / well were seeded into 10 wells of a 6-well plate; (2) 24 h later, the cells in 2 wells were digested and the number of cells was counted (3 samples were taken from each well); (3) For 5 consecutive days, the cells in 2 wells were digested and the number of cells was counted at the same time each day (3 samples were taken from each well).

[0051] The results are shown in Table 5.

[0052] Table 5

[0053] As can be seen from Table 5, the cells of the present invention still exhibit good proliferation characteristics after 60 passages.

[0054] 5. Experimental methods for determining viral infection and viral proliferation levels: (1) Virus incubation: Immortalized guinea pig tracheal epithelial cells (CGMCC NO. 46763) were collected, the cell supernatant was discarded, and influenza virus H1 or H5 was diluted to a final MOI of 0.01 using serum-free OPTIMEM medium (GIBCO, 31985070). The diluted virus solution was added to the cell wells of a six-well plate at a volume of 500 μL per well and incubated at 37℃ for 1 h. After incubation, the virus supernatant was discarded, and then 2 mL of maintenance medium (0.5% FBS + DMEM / F12 + 1% epithelial cell culture additive + 1% penicillin / streptomycin) was added to each well and the plate was returned to the incubator for 60 h of further culture.

[0055] (2) WB Sampling and Testing Methods a. Discard the supernatant from each well of the virus-infected cells from step (1), add 200 μL of prepared cell protein lysis buffer (Beyotime, P0013C and P1065), place on ice for 10 min, then collect into a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 10 min at 4℃, and add the supernatant to a new pre-cooled 1.5 mL centrifuge tube; add 5×SDS-PAGE loading buffer (Yisheng, 20315ES05) to the supernatant, boil at 100℃ for 10 min to denature, ensuring that the virus particles rupture and release proteins. Centrifuge at 12000 r / min for 1 min, and use the supernatant for WB detection.

[0056] b. Place a 10% high-resolution precast gel (Yaxin, LK403) in an electrophoresis tank and add an appropriate amount of protein electrophoresis buffer. Add markers and protein samples to the gel wells, adjust the voltage to 80 V, and start electrophoresis. After 20 min, observe the lanes (running under the stacking gel). Once the bands have separated, adjust the voltage to 120 V and stop electrophoresis after 70 min. Cut a PVDF membrane (Millipore, IPVH00010) according to the gel size. Immerse the PVDF membrane in 100% methanol (Shanghai Test, 10010018) for 5 min and in 20% methanol for 2 min. Start the PVDF membrane transfer at a constant current of 400 mA for 30 min. Place the PVDF membrane in a container and block it with TBST (Yaxin, PS103S) containing 5% skim milk powder (Solepro, D8340) at room temperature for 1 h. After blocking, wash the membrane three times with TBST for 10 min each time.

[0057] c. Primary antibody: Place the PVDF membrane containing the target protein in a solution of influenza A virus nucleoprotein antibody (Xinbosheng, GTX636282-S) diluted 1:5000 using universal antibody dilution buffer. Place the PVDF membrane containing the internal control in a solution of actin antibody (MBL, M177-3) diluted 1:8000 using universal antibody dilution buffer. Incubate at 4°C on a shaker for 16-18 h. Wash the membrane three times with TBST for 10 min each time.

[0058] d. Secondary antibody: The PVDF membrane containing the target protein was placed in a solution of goat anti-rabbit antibody (Xinbosheng, GTX213110-01) diluted 1:5000 using a universal antibody diluent. The PVDF membrane containing the internal control was placed in a solution of goat anti-mouse antibody (Immunoway, RS0001) diluted 1:14000 using a universal antibody diluent. The membranes were incubated at room temperature for 1.5 h. The membranes were washed three times with TBST for 10 min each time. A developing solution (Pulley, P1010) was prepared to develop the PVDF membranes.

[0059] (3) qPCR sampling and testing method a. After discarding the supernatant from each well of the virus-infected cells in step (1), extract RNA using an RNA extraction kit (TransGold, ER111) according to the kit instructions. After determining the RNA concentration using NanoDrop, store at -80℃.

[0060] b. Using a reverse transcription kit (TAKARA, RR047), follow the kit instructions to reverse transcribe the RNA extracted in step a to obtain cDNA.

[0061] c. qPCR reaction: A qPCR detection kit (TransGen, AQ621) was used, and the qPCR reaction was performed according to the kit instructions. The upstream primer sequence for the M gene was 5'-CTTCTAACCGAGGTCGAAACG-3' (SEQ ID NO: 5), and the downstream primer sequence for the M gene was 5'-CTTTAGCCACTCCATGAGAGC-3' (SEQ ID NO: 6). The upstream primer sequence for the internal reference gene (GAPDH) was 5'-AACTTCGGCATTGTGGAGGG-3' (SEQ ID NO: 7), and the downstream primer sequence for the internal reference gene was 5'-GGATGCGGGGATGATGTTCT-3' (SEQ ID NO: 8). The qPCR reaction was performed according to the reaction conditions in Table 6.

[0062] Table 6

[0063] Note: Fluorescence signal collection was performed in the second program segment (56℃ annealing and extension).

[0064] (4) Immunofluorescence experiment: After removing the supernatant from each well of the virus-infected cells from step (1), add 500 μL of pre-cooled 4% paraformaldehyde fixative (Solepro, P1110) and let stand at room temperature for 30 min. After fixation, discard the 4% paraformaldehyde and wash with PBS 3 times, 5 min each time. Add 1 mL of 1% Triton X-100 solution to the washed cells and permeate at room temperature for 30 min. After permeation, wash with PBS 3 times, 5 min each time. After washing, add 1 mL of 3% BSA and incubate at 37℃ for 30 min. After incubation, wash with PBS 3 times, 5 min each time. Add 200 µL of primary antibody (Xinbosheng, GTX636675 and GTX631830, diluted 1:200 and 1:300 respectively) and incubate overnight at 4℃. Wash with PBST 3 times on a shaker, 5 min each time. Then, add 200 µL of secondary antibody (positive energy, AF594, 1:200 dilution; Jackson ImmunoResearch, 115-545-003, 1:200) and incubate at 37°C in the dark for 2 h. Wash the cells 5 times with PBST on a shaker for 5 min each time. After washing, add 20 µL of DAPI staining solution (Solepro, S2110) and incubate at room temperature for 10 min.

[0065] Experimental results: (1) Cell lesions: direct observation under a microscope.

[0066] Evaluation criteria: Using the uninfected control group as a reference, the evaluation was based on changes in cell morphology and adherence. When typical changes such as cell shrinkage, increased refractivity, loose intercellular connections, cell detachment / floating, monolayer destruction, and focal plaques or fusions were observed, it was judged as a positive cytopathic effect (CPE). The same criteria were used to interpret different treatment groups consistently.

[0067] (2) Western blot (WB) determination of viral protein (NP) expression level: After viral infection of cells, cell proteins were extracted, and the expression level of the protein was determined using viral protein-specific antibodies and Western blot (WB) detection technology. The relative protein expression level is a quantitative value obtained by correcting the gray value of the target protein (NP) band with the gray value of the internal reference protein (β-actin) band in the same sample.

[0068] (3) qPCR determination of viral M gene mRNA expression level: After viral infection of cells, cellular RNA was extracted, and qPCR was performed using specific primers targeting viral M gene mRNA. The relative expression level of viral M gene mRNA was calculated using a relative quantification method. Specifically, the target gene and internal reference gene were detected in each sample. ① The ΔCt value of the target gene and internal reference gene was calculated: ΔCt = Ct(target gene) - Ct(internal reference gene). ② The ΔΔCt value between the treatment group and the control group was calculated: ΔΔCt = ΔCt(treatment group) - ΔCt(control group). ③ The relative expression level was calculated: 2^(-ΔΔCt).

[0069] (4) Immunofluorescence: After the virus infects the cells, the cells are fixed with formaldehyde, and the viral proliferation level is determined using a virus matrix protein (M) specific antibody and immunofluorescence technology. The relative fluorescence intensity is calculated by reading the fluorescence value using ImageJ.

[0070] The results are shown in Table 7.

[0071] Table 7

[0072] As shown in Table 7, 12 h after infection, cytopathic effects were observed in tracheal epithelial cells after H1 infection, but not after H5 infection. The infection rate of H1 in tracheal epithelial cells was significantly higher than that of H5. Western blot results showed that the relative expression levels of NP protein in both groups increased significantly after 24 h of infection, and the relative expression levels of NP protein in the H1 group were significantly higher than those in the H5 group from 24 to 60 h after infection. p<0.0001 (Figure 3); qPCR results showed that 6 h after infection, the relative expression levels of M gene mRNA in groups H1 and H5 increased significantly, and 12-60 h after infection, the relative expression level of M gene mRNA in group H1 was significantly higher than that in group H5. (p<0.0001) (Figure 4); Immunofluorescence results showed that H1 could proliferate and infect tracheal epithelial cells in large quantities, significantly higher than the infection level of H5 (Figure 5). In summary, the results of cytopathic effects, qPCR, WB, and immunofluorescence were consistent, indicating that H1 has a faster, more efficient, and more obvious infective ability on tracheal epithelial cells, suggesting that these cells are more susceptible to respiratory pathogens with high aerosol transmission capacity.

[0073] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of tracheal epithelial cells, characterized in that, These cells are immortalized guinea pig tracheal epithelial cells, with the preservation number CGMCC No. 46763.

2. The use of the cells according to claim 1 in isolating respiratory pathogens.

3. The use of the cells according to claim 1 in the preparation of respiratory pathogen vaccines.

4. The use of the cells of claim 1 in screening and / or preparing medicaments for the prevention and / or treatment of diseases caused by respiratory pathogens.

5. The use of the cells according to claim 1 in culturing respiratory pathogens.

6. The use of the cell according to claim 1 as a host cell for studying respiratory pathogens.

7. The use of the cells of claim 1 in the detection of respiratory pathogens for non-diagnostic purposes.

8. The application according to any one of claims 2-7, wherein, The respiratory pathogen is at least one of influenza virus, novel coronavirus, respiratory syncytial virus, rhinovirus, parainfluenza virus, and adenovirus.

9. The application according to any one of claims 2-7, wherein, The respiratory pathogens are influenza virus H1 and / or influenza virus H5.

10. The use of the cell of claim 1 in the construction of organoids.

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