Preparation method of mouse nasopharyngeal epithelial cell line with long-term stable passage

Abstract

The invention provides a preparation method of a long-term stable passage mouse nasopharyngeal epithelial cell line, which comprises the following steps: firstly, acquiring a mouse nasopharyngeal tissue, and extracting and separating primary mouse nasopharyngeal epithelial cells; purifying the nasopharyngeal epithelial cell line through time digestion; an NEC culture medium added with growth factors such as Wnt3a, R-spondin, Noggin and the like is used for culturing primary mouse nasopharyngeal epithelial cells for a long time. According to the method provided by the invention, the nasopharyngeal epithelial cells of the mouse can be stably proliferated for a long time, high proliferative activity is kept, and EMT transformation tendency is avoided. The invention provides a valuable cell tool for researching nasopharyngeal epithelium related diseases and formulating treatment strategies.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for preparing a mouse nasopharyngeal epithelial cell line with long-term stable passage. Background Art

[0002] The nasopharynx, a key component of the upper respiratory tract, is located at the junction of the Eustachian tube, oropharynx, and trachea. The nasopharynx is at the forefront of pathogen exposure and antigen sampling, and plays a vital role in maintaining respiratory health. Nasopharyngeal diseases cover a range of diseases from chronic inflammatory diseases to tuberculosis, viral infections, and even malignant tumors. Respiratory infections are an important cause of human morbidity and mortality. Common respiratory viruses, such as adenovirus, influenza virus, parainfluenza virus, rhinovirus, respiratory syncytial virus, seasonal viruses, and the recently emerged new coronavirus, mainly infect the nasal epithelium and spread in the nasopharynx to cause respiratory infections. Nasopharyngeal sampling is unanimously recognized as a highly sensitive means of detecting these viruses. In vitro models of respiratory viral infection are important tools for elucidating the mechanisms of viral infection and replication and evaluating antiviral strategies.

[0003] Epstein-Barr virus (EBV) is the first oncogenic virus discovered in humans, and approximately 95% of the world's population are asymptomatic carriers. EBV infection is closely associated with a variety of malignant tumors, among which nasopharyngeal carcinoma and EBV-related gastric cancer are the two most common EBV-related epithelial malignancies, accounting for 80% of the total cases. Nasopharyngeal carcinoma is a typical EBV-related malignancy originating from the nasopharynx, and more than 90% of patients with undifferentiated nasopharyngeal carcinoma are EBV serum positive. EBV infection is closely related to the occurrence and progression of nasopharyngeal carcinoma and is recognized as an important pathogenic factor. In-depth research on the mechanism by which EBV affects the occurrence and development of nasopharyngeal carcinoma will provide new therapeutic directions for the treatment of nasopharyngeal carcinoma. However, there is currently a lack of suitable cell models to study nasopharyngeal-related diseases such as nasopharyngeal carcinoma. We urgently need to develop a simple and effective method to achieve long-term culture and robust expansion of primary nasopharyngeal cells.

[0004] Currently, cell immortalization technology has been widely used in long-term in vitro studies. However, studies have shown that the immortalization process itself may lead to genomic instability, thereby changing cell characteristics and making them different from normal cells. For example, the oxidase activity of immortalized rat hepatocytes is significantly higher than that of primary cells. So far, there is no mouse nasopharyngeal epithelial cell line that can be cultured for a long time. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a method for preparing a mouse nasopharyngeal epithelial cell line with long-term stable passage. The present invention prepares mouse nasopharyngeal epithelial cells from mouse nasopharyngeal tissue, culture them using a specific culture medium, and can still stably proliferate after multiple passages. It has been identified that the mouse nasopharyngeal epithelial cell line prepared by the present invention is indeed of epithelial origin. The present invention provides a valuable cell tool for studying nasopharyngeal epithelial-related diseases.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for preparing a mouse nasopharyngeal epithelial cell line with long-term stable passage, comprising:

[0008] Step S1: Rinse the mouse nasopharyngeal epithelial tissue 3 to 5 times with 1×PBS saline containing 1% penicillin / streptomycin to remove blood clots, and then place the rinsed tissue in a centrifuge tube and transport it to a clean bench for subsequent operations;

[0009] Step S2: In a clean bench, cut the nasopharyngeal epithelial tissue into 1 mm 3 Add digestion solution to the fragments of different sizes and incubate for 1 to 2 hours;

[0010] Step S3: After the incubation is completed, DMEM / F12 medium containing 10% fetal bovine serum (FBS) is added and gently pipetted to terminate the digestion, filtered, centrifuged, and the obtained cell pellet is resuspended in NEC (nasopharyngeal epithelial cell) medium, inoculated in a culture dish, and placed in an incubator at 37°C and 5% CO2 for culture;

[0011] Step S4: When the cell density reaches 80-90%, the cells are passaged, and the area containing non-epithelial cell clusters (fibroblasts) is identified under a microscope and marked at the bottom of the culture dish. The old culture medium is aspirated, and the cells are rinsed with PBS for 1-2 times. A 0.25% trypsin solution is added and gently shaken to cover the cell surface, and the cells are placed in a 37°C, 5% CO2 incubator for digestion for 3-5 minutes. Under an inverted microscope, after the fibroblasts are observed to be round and partially separated, a serum-containing culture medium is added to terminate the digestion, and the cells are resuspended after centrifugation and added with NEC culture medium, and the cells are pipetted into the culture dish and then fresh NEC culture medium is added to continue culturing in the culture dish;

[0012] Step S5: Repeat step S4 3 to 5 times to obtain a purified nasopharyngeal epithelial cell population.

[0013] Furthermore, in step S1, the mouse nasopharyngeal epithelial tissue is from one-week-old female C57BL / 6 mice.

[0014] Furthermore, in step S2, the composition of the digestive fluid is 200 U / mL type IV collagenase; and the volume ratio of the nasopharyngeal epithelial tissue fragments to the digestive fluid is 1:5.

[0015] Furthermore, in step S2, the cells are incubated on a shaker in a cell culture incubator; and the cells are gently blown several times with a Pasteur pipette every 15 to 20 minutes to completely separate the single cells.

[0016] Further, in step S3, the composition of the NEC culture medium is: 1% HEPES, conditioned medium containing 10% Wnt-3A, R-spondin 3 and Noggin, 1.25mM N-acetylcysteine, 10mM nicotinamide, 1% glutamine, 1% penicillin / streptomycin, 5μM Y-27632, 500nM A83-01, 1μM SB202190, 5ng / mL FGF-7, 20ng / mL FGF-10, 2% B-27, 20μg / mL gentamicin, 50ng / mL amphotericin B; the rest is Advanced DMEM / F12 culture medium.

[0017] Furthermore, the preparation method of the conditioned medium containing 10% Wnt-3A, R-spondin 3 and Noggin is as follows:

[0018] (a) 2*10 7 Each L-WRN cell was inoculated in a T75 culture flask and 25-30 mL of DMEM / F12 medium containing 10% FBS was added;

[0019] (b) After 60-72 h of culture, the first batch of conditioned medium was collected and centrifuged at 2000 g for 5 min. The supernatant was transferred to a 50 mL centrifuge tube.

[0020] In the heart tube, temporarily store at 4°C, add fresh medium to the culture bottle and continue culturing;

[0021] (c) Repeat the above step (b) twice to obtain 90 mL of conditioned medium.

[0022] Furthermore, in step S3, the NEC culture medium is replaced every 2 to 3 days.

[0023] Furthermore, in step S3, after terminating the digestion, the cell suspension was filtered using a 70 μm cell mesh, and then centrifuged at 300 g for 5 min.

[0024] Further, in step S3, 2×10 5 The cells were seeded at a density of 6 cm 2 In a culture dish.

[0025] Furthermore, in step S4, the fibroblast area is carefully blown with a 200 μL pipette tip, taking care to avoid touching the epithelial cell area, the fibroblasts are blown down and then sucked out and discarded, and then the area is rinsed 1 to 2 mL of PBS 1 to 2 times.

[0026] Furthermore, the method further comprises step S6: freezing the nasopharyngeal epithelial cell population obtained in step S5 for long-term storage by the following steps: digesting the cells when they grow to a density of 80-90%, terminating the digestion, centrifuging, removing the supernatant and resuspending the cells in freezing solution, transferring the cells into freezing tubes, marking the names, generations and freezing dates of the frozen cells, and storing the cells in a liquid nitrogen tank for a long term.

[0027] The beneficial effects of the present invention are embodied in:

[0028] The present invention provides a simplified and effective method for culturing primary mouse nasopharyngeal epithelial cells. First, mouse nasopharyngeal tissue is obtained to extract and separate primary mouse nasopharyngeal epithelial cells; the nasopharyngeal epithelial cell line is purified by time digestion difference; and the primary mouse nasopharyngeal epithelial cells are cultured for a long time using NEC culture medium supplemented with growth factors such as Wnt3a, R-spondin, and Noggin. The present invention uses a specific culture medium that activates the Wnt3a and R-spondin pathways to enable mouse nasopharyngeal epithelial cells to proliferate stably for a long time and maintain high proliferation activity. The NEC culture medium has a selective advantage over stably expanded cytokeratin 5 and EMA-positive epithelial cells, rather than α-SMA-labeled fibroblasts, and prevents EMT transformation. This invention provides a valuable cell tool for studying the pathogenesis and progression of nasopharyngeal epithelial-related diseases such as respiratory virus infection, EBV infection, and nasopharyngeal carcinoma, as well as formulating treatment strategies.

[0029] 1. Successfully isolated, purified and cultured a mouse nasopharyngeal epithelial cell line that can be cultured for a long time, filling the gap in the acquisition and culture technology of mouse nasopharyngeal epithelial cells;

[0030] 2. The present invention can stably culture mouse nasopharyngeal epithelial cells for a long time under sterile conditions, greatly reducing the risk of contamination;

[0031] 3. This technology is simple and convenient to sample, has low cost and low operating difficulty. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0033] Figure 1It is a schematic diagram of nasopharyngeal sampling of C57BL / 6 mice in Example 1 of the present invention and a flow chart of extraction, separation and purification of primary mouse nasopharyngeal epithelial cells.

[0034] Figure 2 It is a cell morphology picture of mouse nasopharyngeal epithelial cells of different generations, P3, P10 and P20, under a microscope in Example 1 of the present invention.

[0035] Figure 3 These are the results of measuring cell viability of mouse nasopharyngeal epithelial cells of different generations in Example 2 of the present invention.

[0036] Figure 4 These are the results of measuring the proliferation capacity of mouse nasopharyngeal epithelial cells of different generations in Example 2 of the present invention.

[0037] Figure 5 These are the identification results of epithelial and EMT markers of mouse nasopharyngeal epithelial cells of different generations in Example 3 of the present invention. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with specific implementation methods. The following specific embodiments are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0039] Example 1

[0040] This embodiment is a method for preparing mouse nasopharyngeal epithelial cells that are cultured stably for a long time, and the preparation method comprises the following steps:

[0041] The sampling location and extraction, separation and purification methods of mouse nasopharyngeal epithelial cells are as follows: Figure 1 :

[0042] (1) Euthanize one-week-old female C57BL / 6 mice under anesthesia. Use sterile ophthalmic scissors to cut the mandible along the midline between the two lower incisors to fully expose the oral cavity. Under a stereomicroscope, carefully dissect the nasopharyngeal tissue. After peeling off the nasopharyngeal tissue, rinse it 3 to 5 times with ice-cold 1× PBS saline containing 1% penicillin / streptomycin to remove blood clots.

[0043] Place the rinsed tissue in a centrifuge tube and transfer it to the clean bench for subsequent operations.

[0044] (2) In a clean bench, use sterile ophthalmic scissors to cut the nasopharyngeal epithelial tissue into 1 mm 3 Add 200U / mL IV

[0045] The ratio of the volume of nasopharyngeal tissue fragments to the volume of digestion solution is 1:5, and the cells are placed on a shaker in a cell culture incubator for incubation for 1 to 2 hours. Every 15 to 20 minutes, the cells can be gently blown several times with a Pasteur pipette to completely separate the single cells.

[0046] (3) After incubation, add DMEM / F12 medium containing 10% fetal bovine serum (FBS) and gently pipette to terminate digestion, and filter through a 70 μm cell sieve. After filtration, centrifuge the cell suspension at 300 g for 5 min. Resuspend the resulting cell pellet in nasopharyngeal epithelial cell (NEC) culture medium and plate approximately 2 × 10 cells per culture dish. 5 The cells were seeded at a density of 6 cm 2 The culture dish was placed in an incubator at 37°C and 5% CO2 for culture.

[0047] Table 1 Components, reagent manufacturers and working concentrations of NEC culture medium

[0048]

[0049] The preparation method of the conditioned medium containing 10% Wnt-3A, R-spondin 3 and Noggin is as follows:

[0050] (a) 2*10 7 Each L-WRN cell was inoculated in a T75 culture flask and 25-30 mL of DMEM / F12 medium containing 10% FBS was added;

[0051] (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g for 5 min, and the supernatant was transferred to a 50 mL centrifuge tube, temporarily stored at 4°C, and fresh medium was added to the culture flask to continue the culture;

[0052] (c) Repeat step (b) twice to obtain 90 mL of conditioned medium. After mixing, filter through a 0.22 μm filter membrane, aliquot and freeze at -80°C. It can be stored for at least 3 months.

[0053] (4) When the cell density reaches 80-90%, it is passaged. The area containing non-epithelial cell clusters (fibroblasts) is identified under a microscope and marked at the bottom of the culture dish. The old culture medium is aspirated, and 1 mL of PBS is used to rinse 1-2 times. 1 mL of 0.25% trypsin solution is added and gently shaken to cover the cell surface. The cells are placed in a 37°C, 5% CO2 incubator for digestion for 3-5 minutes. Under an inverted microscope, after the fibroblasts are observed to be round and partially separated, a 200 μL pipette tip is used to carefully blow the fibroblast area, taking care to avoid touching the epithelial cell area. The fibroblasts are blown down and aspirated and discarded, and then the area is rinsed 1-2 mL of PBS 1-2 times. Digestion is terminated by adding serum-containing culture medium. After centrifugation, 1 mL of NEC culture medium is added to resuspend, pipette into the culture dish, and then 5 mL of fresh NEC culture medium is added to continue culturing in the culture dish. NEC culture medium is replaced every 2-3 days.

[0054] (5) Repeat 3 to 5 times to obtain a purified nasopharyngeal epithelial cell population.

[0055] (6) The obtained nasopharyngeal epithelial cell population was cryopreserved for long-term storage by the following steps: when the cells grew to a density of 80% to 90%, they were digested, digestion was terminated, and centrifuged. After the supernatant was removed, the cells were resuspended in cryopreservation solution, transferred into cryopreservation tubes, and the names, generations, and cryopreservation dates of the cryopreserved cells were marked. The cells were stored in a liquid nitrogen tank for a long time. After 20 generations, the cell growth was as follows: Figure 2 As shown. Figure 2 It can be seen that the morphology of mouse nasopharyngeal epithelial cells did not change significantly with the increase in the number of passages.

[0056] Example 2

[0057] This example is to identify the cell viability and proliferation ability of mouse nasopharyngeal epithelial cells:

[0058] (1) CCK-8 method was used to determine the growth activity of mouse nasopharyngeal epithelial cells of different generations: cells were cultured at 1.5×10 4 Cells were seeded at a concentration of 100 μL of cell suspension per well in a 96-well plate. Six replicates were established for each treatment group at each time point, and the cell counting kit-8 (CCK-8) was used to assess viable cell counts every day. Each well contained 90 μL of complete medium and 10 μL of CCK-8 reagent. Subsequently, the 96-well plate was incubated in an incubator at 37°C and 5% CO2 for 1 h. After incubation, the absorbance of each well at 450 nm was measured using a microplate reader. Time was plotted on the x-axis and the average optical density (OD) at 450 nm was plotted on the y-axis to construct a cell growth curve.

[0059] (2) Using an ATP kit to test the growth support performance of NEC culture medium and DMEM / F12 culture medium containing 10% FBS on mouse nasopharyngeal epithelial cells: Use a 96-well plate suitable for chemiluminescence detection to inoculate P3 mouse nasopharyngeal epithelial cells cultured with NEC culture medium and DMEM / F12 culture medium containing 10% FBS, respectively, with the inoculation number as described above. Take out the cell culture plate and equilibrate it at room temperature for 10 minutes. Then, add 100 μL CellTiter-Lumi TM Steady II luminescence detection reagent was shaken at room temperature for 2 minutes to promote cell lysis, and then incubated at room temperature (about 25°C) for 10 minutes to stabilize the luminescence signal. Finally, a multifunctional microplate reader was used for chemiluminescence detection, and the relative viability of the cells was directly calculated based on the chemiluminescence readings.

[0060] Specific test results such as Figure 3 As shown, Figure 3 (A) Proliferation curves of P3, P10, and P20 mouse nasopharyngeal epithelial cells measured by CCK-8; (B) Relative viability values ​​of P3 mouse nasopharyngeal epithelial cells cultured in NEC medium and DMEM / F12 medium.

[0061] Depend on Figure 3 It can be seen that nasopharyngeal epithelial cells of different generations (P3, P10 and P20) showed similar "S"-shaped growth curves, and the proliferation rate of nasopharyngeal epithelial cells did not decrease with the increase in the number of passages. NEC medium, rather than DMEM / F12 medium, can well maintain the growth of nasopharyngeal epithelial cells with a satisfactory proliferation rate.

[0062] (3) Determination of proliferation rate of mouse nasopharyngeal epithelial cells of different generations by IF: Place sterilized cell slides in a 24-well plate, drip mouse nasopharyngeal epithelial cell suspensions of different generations onto the slides, and then culture them in an incubator at 37°C and 5% CO2. When the cell density is about 70%, discard the culture medium, and use 4% paraformaldehyde diluted in warm PBS to drip 2-3 mm thick on the cells, and let stand at room temperature for 15 minutes. Discard the fixative and rinse in PBS three times, 3 minutes each time. Cover the cells or tissue sections with 0.5% ice-cold Triton (covering a thickness of 3-5 mm to prevent drying) and incubate for 20 minutes. Immerse in PBS three times, 3 minutes each time. Add normal goat serum to the slides and block at room temperature for 30 minutes. During the blocking period, prepare the primary antibody working solution with antibody dilution buffer. Discard the blocking solution, do not wash, add the diluted primary antibody working solution, and incubate at 4°C overnight. Secondary antibody incubation: Rinse in PBS 3 times, 3 minutes each time. Add the diluted fluorescent secondary antibody working solution of the corresponding species and incubate at room temperature in the dark for 1 hour. Rinse in PBS 3 times, 3 minutes each time. Use anti-fluorescence quenching mounting medium containing DAPI to cover the slide and incubate for 5 minutes. Take pictures, and finally observe and collect images under a fluorescence microscope or confocal microscope, and select appropriate excitation wavelengths and emission wavelengths according to different fluorescent substances.

[0063] (4) Determine the proliferation rate of mouse nasopharyngeal epithelial cells of different generations using a cell senescence kit: Inoculate nasopharyngeal epithelial cells of different generations cultured with NEC and P3 mouse nasopharyngeal epithelial cells cultured with DMEM / F12 medium containing 10% FBS in a six-well plate. When the density reaches 70-90%, senescence staining can be performed. Prepare the staining working solution in advance according to the proportion in the instructions. Aspirate the cell culture medium in the well plate, wash once with PBS or HBSS, add 1 mL of β-galactosidase staining fixative, and fix at room temperature for 15 minutes. Aspirate the cell fixative, wash the cells 3 times with PBS or HBSS, 3 minutes each time. Aspirate the PBS or HBSS, and add 1 mL of staining working solution to each well. Incubate overnight at 37°C in the dark. The 6-well plate can be covered with parafilm or plastic wrap to prevent evaporation. Observe and take pictures under an ordinary optical microscope the next day.

[0064] Specific test results such as Figure 4 As shown, Figure 4 In the figure, (A) Immunofluorescence staining images of Ki67 in mouse nasopharyngeal epithelial cells at passages 3, 10, and 20, with a scale bar of 100 μm; (B) Cell senescence staining images of mouse nasopharyngeal epithelial cells at passages 3 (a), 10 (b), and 20 (c) cultured in NEC medium and cell senescence staining images of mouse nasopharyngeal epithelial cells at passage 3 (d) cultured in DMEM / F12 medium, with a scale bar of 50 μm.

[0065] Depend on Figure 4 It can be seen that the Ki67 fluorescence intensity of nasopharyngeal epithelial cells at passages 3, 10, and 20 was significantly higher, but there was no significant difference between passages. In nasopharyngeal epithelial cells cultured in NEC medium, there was almost no typical senescent phenotype at passages 3, 10, and 20. However, cells cultured in ordinary DMEM / F12 medium showed a wide range of senescent cell phenotypes, characterized by enlarged cytoplasm and blue staining of SA-β-Gal, as shown by the black arrow. This shows that NEC medium can well maintain the long-term expansion of mouse nasopharyngeal epithelial cells.

[0066] Example 3

[0067] This example is immunofluorescence staining (IF) to detect the expression of epithelial and epithelial-mesenchymal transition (EMT) markers in mouse nasopharyngeal epithelial cells of different generations:

[0068] The IF experimental method is as described above, and the specific test results are as follows Figure 5 As shown, immunofluorescence detection of markers in mouse nasopharyngeal epithelial cells of different passages: (A) Cytokeratin 5; (B) EMA; (C) ɑ-SMA; (D) E-cadherin; (E) Vimentin; DAPI, cell nucleus; scale bar, 100 μm.

[0069] Depend on Figure 5 It can be seen that in the nasopharyngeal cells cultured at the 3rd, 10th and 20th generations, the expression of epithelial markers Cytokeratin 5 and EMA remained consistent, indicating that the epithelial origin characteristics of these cells have been continuously retained. And the proportion of fibroblast marker smooth muscle actin (ɑ-SMA) positive always remained at a low level, indicating that the number of fibroblasts did not increase with the increase in the number of passages. In addition, in the nasopharyngeal epithelial cells of the 3rd, 10th and 20th generations, there was always a strong expression of epithelial adhesion protein E-cadherin and negative expression of vimentin, indicating that EMT transformation did not occur in these cells.

[0070] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a long-term stable passage mouse nasopharyngeal epithelial cell line, comprising: Step S1: Rinse the mouse nasopharyngeal epithelial tissue 3 to 5 times with 1×PBS saline containing 1% penicillin / streptomycin to remove blood clots, and then place the rinsed tissue in a centrifuge tube and transport it to a clean bench for subsequent operations; Step S2: In a clean bench, cut the nasopharyngeal epithelial tissue into 1 mm 3 Add digestion solution to the fragments of different sizes and incubate for 1 to 2 hours; Step S3: After the incubation is completed, DMEM / F12 medium containing 10% fetal bovine serum is added and gently pipetted to terminate the digestion, filtered, centrifuged, and the obtained cell pellet is resuspended in NEC medium, inoculated in a culture dish, and placed in an incubator at 37°C and 5% CO2 for culture; Step S4: When the cell density reaches 80-90%, the cells are passaged, and the area containing non-epithelial cell clusters is identified under a microscope and marked at the bottom of the culture dish. The old culture medium is aspirated, and the cells are rinsed with PBS for 1-2 times. A 0.25% trypsin solution is added and gently shaken to cover the cell surface, and the cells are placed in a 37°C, 5% CO2 incubator for digestion for 3-5 minutes. Under an inverted microscope, after the fibroblasts are observed to be round and partially separated, a serum-containing culture medium is added to terminate the digestion, and the cells are resuspended after centrifugation and added with NEC culture medium, and the cells are pipetted into the culture dish and then fresh NEC culture medium is added to continue culturing in the culture dish; Step S5: Repeat step S4 3 to 5 times to obtain a purified nasopharyngeal epithelial cell population.

2. The preparation method according to claim 1, characterized in that: In step S1, the mouse nasopharyngeal epithelial tissue is from one-week-old female C57BL / 6 mice.

3. The preparation method according to claim 1, characterized in that: In step S2, the digestive fluid contains 200 U / mL type IV collagenase; and the volume ratio of the nasopharyngeal epithelial tissue fragments to the digestive fluid is 1:

5.

4. The preparation method according to claim 1, characterized in that: In step S3, the composition of the NEC culture medium is: 1% HEPES, conditioned medium containing 10% Wnt-3A, R-spondin 3 and Noggin, 1.25mM N-acetylcysteine, 10mM nicotinamide, 1% glutamine, 1% penicillin / streptomycin, 5μM Y-27632, 500nM A83-01, 1μM SB202190, 5ng / mL FGF-7, 20ng / mL FGF-10, 2% B-27, 20μg / mL gentamicin, 50ng / mL amphotericin B; the rest is Advanced DMEM / F12 culture medium.

5. The preparation method according to claim 4, characterized in that: The preparation method of the conditioned medium containing 10% Wnt-3A, R-spondin 3 and Noggin is as follows: (a) 2*10 7 Each L-WRN cell was inoculated in a T75 culture flask and 25-30 mL of DMEM / F12 medium containing 10% FBS was added; (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g for 5 min, and the supernatant was transferred to a 50 mL centrifuge tube, temporarily stored at 4°C, and fresh medium was added to the culture flask to continue the culture; (c) Repeat the above step (b) twice to obtain 90 mL of conditioned medium.

6. The preparation method according to claim 1, characterized in that: In step S3, the NEC culture medium is replaced every 2 to 3 days.

7. The preparation method according to claim 1, characterized in that: In step S3, after terminating the digestion, the cell suspension was filtered using a 70 μm cell mesh, and then centrifuged at 300 g for 5 min.

8. The preparation method according to claim 1, characterized in that: In step S3, 2 × 10 5 The cells were seeded at a density of 6 cm 2 In a culture dish.

9. The preparation method according to claim 1, characterized in that: In step S4, use a 200 μL pipette tip to carefully blow the fibroblast area, taking care to avoid touching the epithelial cell area, blow the fibroblasts down and then aspirate and discard them, then rinse the area 1 to 2 times with 1 to 2 mL PBS.

10. The preparation method according to any one of claims 1 to 9, characterized in that: The method further comprises step S6: freezing the nasopharyngeal epithelial cell population obtained in step S5 for long-term storage by the following steps: digesting the cells when they grow to a density of 80% to 90%, terminating the digestion, centrifuging, removing the supernatant and resuspending the cells in a freezing solution, transferring the cells into a freezing tube, marking the names, generations and freezing dates of the frozen cells, and storing the cells in a liquid nitrogen tank for a long term.

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