An iPS-driven nasopharyngeal epithelial organoid and a preparation method and application thereof

Abstract

The application provides an iPS-driven nasopharyngeal epithelial organoid and a preparation method and application thereof. The preparation method comprises the following steps: differentiating humanized iPS cells into endoderm cells; wrapping the endoderm cells with Matrigel and then placing the endoderm cells in DMEM / F-12 culture medium containing Dorsomorphin and SB431542 for culture to obtain foregut organoids; culturing the foregut organoids in DMEM / F-12 culture medium containing FGF10 protein, CHIR-99021, retinoic acid and BMP4 protein to obtain airway epithelial progenitor cell organoids; and culturing the airway epithelial progenitor cell organoids in DMEM / F-12 culture medium containing FGF10 protein, retinoic acid and BMP4 protein to obtain nasopharyngeal epithelial organoids. The iPS cells are continuously differentiated in multiple stages by using culture medium containing different inducers, and the nasopharyngeal epithelial organoids are mass-produced.

Description

Technical Field

[0001] This application relates to the field of organoid culture technology, specifically to an iPS-driven nasopharyngeal epithelial organoid, its preparation method, and its application. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a cancer originating from the nasopharyngeal epithelium. It commonly occurs in the pharyngeal recess behind the torus of the Eustachian tube. Epstein-Barr virus (EBV) infection is considered a key cause of NPC progression. However, research on NPC is currently challenging, primarily due to the lack of suitable experimental biological models of EBV infection.

[0003] First, the nasal cavity structure of rodent models, such as mice and rats, differs significantly from that of humans. Furthermore, it is generally believed that the occurrence of NPC (nasopharyngeal carcinoma) is related to the immune microenvironment, and the immune system of rodents differs from that of humans; therefore, highly pathogenic NPC factors such as EBV have very low infectivity in mice and related biological samples. Second, while primate models, such as rhesus monkeys, have been shown to be infected with EBV in previous studies and may serve as potential animal models that meet the requirements of the NPC immune microenvironment, the high experimental costs and time required make them unsuitable for research in the field of nasopharyngeal carcinoma. Finally, obtaining humanized nasopharyngeal tissue is more difficult than obtaining tissue from other organs due to the unique structure of the nasopharynx. Nasopharyngeal sampling causes significant pain for patients, and the volume of a single sample of humanized nasopharyngeal tissue is rarely larger than 2 mm. 3 It is rich in highly differentiated pseudostratified columnar epithelium, which has almost no reproductive and regenerative capacity and is difficult to expand in large quantities. Therefore, how to obtain or prepare experimental biological models of EBV infection, such as nasopharyngeal epithelial organoids, has become a key focus of research in this field. Summary of the Invention

[0004] In view of this, in order to solve at least one of the above technical problems, embodiments of this application provide a method for preparing iPS-driven nasopharyngeal epithelial organoids.

[0005] In addition, this application also provides a nasopharyngeal epithelial organoid prepared by the aforementioned preparation method and a technique for infecting the nasopharyngeal epithelial organoid with EBV virus.

[0006] In a first aspect, embodiments of this application provide a method for preparing an iPS-driven nasopharyngeal epithelial organoid, the method comprising:

[0007] Humanized iPS cells were directed to differentiate into endoderm cells;

[0008] The endoderm cells were encapsulated with Matrigel and cultured in DMEM / F-12 medium A, which contained Dorsomorphin and SB431542, to obtain foregut organoids.

[0009] The foregut organoids were cultured in DMEM / F-12 medium B, which contains FGF10 protein, CHIR-99021, retinoic acid, and BMP4 protein, to obtain airway epithelial progenitor cell organoids; and

[0010] The airway epithelial progenitor cell organoids were cultured in DMEM / F-12 medium C, which contains FGF10 protein, retinoic acid and BMP4 protein, to obtain the nasopharyngeal epithelial organoids.

[0011] In some possible embodiments, the endoderm cell organoids are detected as SOX17 positive by immunofluorescence.

[0012] In some possible embodiments, the content of Dorsomorphin in the DMEM / F-12 medium A is 1.5 μM to 4 μM, and the content of SB431542 is 5 μM to 20 μM.

[0013] In some possible embodiments, the DMEM / F-12 medium B contains 5 ng / mL to 20 ng / mL of FGF10 protein, 1.5 μM to 4 μM of CHIR-99021, 1 μM to 10 μM of retinoic acid, and 5 ng / mL to 20 ng / mL of BMP4 protein.

[0014] In some possible embodiments, the content of FGF10 protein in the DMEM / F-12 medium C is 5 ng / mL to 20 ng / mL, the content of retinoic acid is 1 μM to 10 μM, and the content of BMP4 protein is 5 ng / mL to 20 ng / mL.

[0015] In some possible embodiments, the step of encapsulating the endoderm cells with Matrigel and culturing them in DMEM / F-12 medium A includes:

[0016] The endoderm cells were suspended in Matrigel and incubated at 37°C and 5% CO2 for 30-60 minutes.

[0017] After the Matrigel solidifies, forming Matrigel microspheres, these Matrigel microspheres are transferred to a cell culture plate.

[0018] Remove excess liquid, add DMEM / F-12 medium A containing Dorsomorphin and SB431542, and incubate at 37°C and 5% CO2 for 48 hours; and

[0019] The foregut organoids were obtained by repeating the steps of transferring the Matrigel microspheres to cell culture plates and incubating them in the DMEM / F-12 medium A multiple times.

[0020] In some possible embodiments, the step of culturing the foregut organoid in DMEM / F-12 medium B includes:

[0021] Remove excess fluid from the foregut organoids and add DMEM / F-12 medium B to the foregut organoids. Incubate at 37°C and 5% CO2 for 48 hours. Repeat this process multiple times to obtain the airway epithelial progenitor cell organoids.

[0022] In some possible embodiments, the step of culturing the airway epithelial progenitor cell organoids in DMEM / F-12 medium C includes:

[0023] Remove excess fluid from the airway epithelial progenitor cell organoids, add DMEM / F-12 medium C containing FGF10 protein, retinoic acid and BMP4 protein to the airway epithelial progenitor cell organoids, and incubate at 37°C and 5% CO2 for 48 h; repeat multiple times to obtain the nasopharyngeal epithelial organoids.

[0024] Thirdly, this application also provides a preparation system for performing the aforementioned iPS-driven nasopharyngeal epithelial organoid preparation method, the preparation system comprising a kit including DMEM / F-12 culture medium A, DMEM / F-12 culture medium B, and DMEM / F-12 culture medium C.

[0025] The DMEM / F-12 medium A contains 1.5 μM to 4 μM Dorsomorphin and 5 μM to 20 μM SB431542; the DMEM / F-12 medium B contains 5 ng / mL to 20 ng / mL FGF10 protein, 1.5 μM to 4 μM CHIR-99021, 1 μM to 10 μM retinoic acid and 5 ng / mL to 20 ng / mL BMP4 protein; and the DMEM / F-12 medium C contains 5 ng / mL to 20 ng / mL FGF10 protein, 1 μM to 10 μM retinoic acid and 5 ng / mL to 20 ng / mL BMP4 protein.

[0026] Thirdly, this application also provides a nasopharyngeal epithelial organoid, which is prepared using the aforementioned iPS-driven nasopharyngeal epithelial organoid preparation method.

[0027] Fourthly, this application also provides an EBV virus infection model technology, including infecting organoids with EBV virus, wherein the organoids are the aforementioned nasopharyngeal epithelial organoids; and

[0028] Fluorescence detection was performed on the nasopharyngeal epithelial organoids that were not infected with EBV and those that were infected with EBV. The FoxA1 factor expression level in the EBV-infected nasopharyngeal epithelial organoids was lower than that in the uninfected EBV-infected nasopharyngeal epithelial organoids.

[0029] Compared to existing technologies, the method for preparing iPS-driven nasopharyngeal epithelial organoids provided in this application first involves the directed differentiation of humanized iPS cells into endoderm cells. After encapsulation with Matrigel, the endoderm cells undergo multi-stage continuous differentiation using DMEM / F-12 medium containing different combinations of inducing agents, ultimately forming nasopharyngeal epithelial organoids. This method allows for the large-scale, reproducible, and uniform production of nasopharyngeal epithelial organoids, effectively reducing preparation costs and shortening the preparation cycle. Furthermore, the prepared nasopharyngeal epithelial organoids can be efficiently infected by highly pathogenic NPC factors such as EBV, serving as an ideal experimental biological model for EBV infection and providing an important experimental tool for related research. This application also provides a method for in vitro infection of nasopharyngeal epithelial organoids with EBV virus, which can effectively support research on nasopharyngeal-related diseases. Attached Figure Description

[0030] Figure 1 This is a schematic flowchart illustrating a method for preparing an iPS-driven nasopharyngeal epithelial organoid according to an embodiment of this application.

[0031] Figure 2This diagram illustrates the process of endodermal cells continuously aggregating to form microspheres during the preparation of iPS nasopharyngeal epithelial organoids according to an embodiment of this application. Figure 2 Figure a shows the morphology of iPS cells in the early stage of induced transformation into endoderm cells. Figure 2 Figure b shows the morphology of iPS cells in the late stage of induced transformation into endoderm cells.

[0032] Figure 3 Endoderm cells provided in one embodiment of this application are shown to express the endoderm marker factor SOX17 under a fluorescence microscope.

[0033] Figure 4 A nasopharyngeal epithelial organoid provided in one embodiment of this application expresses the ciliary marker protein SMO under a fluorescence microscope, as indicated by the red arrow. Figure 4 Figure a shows the fluorescence detection results of immature nasopharyngeal epithelial organoids. Figure 4 Figure b in the figure shows the fluorescence detection results of mature nasopharyngeal epithelial organoids.

[0034] Figure 5 A nasopharyngeal epithelial organoid under a bright-field microscope at 20x magnification, as provided in one embodiment of this application, wherein... Figure 5 Figure a shows a nasopharyngeal epithelial organoid with relatively long cilia. Figure 5 Figure b shows a nasopharyngeal epithelial organoid with relatively dense cilia.

[0035] Figure 6 This is a schematic diagram of EBV infection of nasopharyngeal organoids provided in an embodiment of this application.

[0036] Figure 7 The expression and morphology of EBV-infected nasopharyngeal organoid-related factors are provided in one embodiment of this application, wherein, Figure 7 Figure a shows the Merge image, which is an image of the superimposed fluorescence signal channels at 405nm, 488nm, 594nm, and 647nm. Figure 7 Figure b shows the representation and morphology of the DAPI signal. Figure 7 Figure c shows the expression and morphology of FoxA1. Figure 7 The d-figure shows the expression and morphology of SMO.

[0037] Figure 8 The expression and morphology of EBV-infected nasopharyngeal organoid-related factors are provided in one embodiment of this application, wherein, Figure 8 Figure a shows the Merge image, which is an image of the superimposed fluorescence signal channels at 405nm, 488nm, 594nm, and 647nm. Figure 8 Figure b shows the representation and morphology of the DAPI signal. Figure 8 Figure c shows the expression and morphology of FoxA1. Figure 8 The d-figure shows the expression and morphology of SMO. Detailed Implementation

[0038] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Material description:

[0041] In this embodiment, the iPS-specific culture medium includes mTeSR from Stem Cell. TM Plus reagent (item number: 100-0276);

[0042] In this application, the endoderm KIT kit includes the Stem Cell Endoderm KIT Kit (catalog number: 05110).

[0043] Unless otherwise specified, Matrigel in this application includes Matrigel (catalog number: 354230) manufactured by Corning Incorporated.

[0044] In this application, the DMEM / F-12 medium includes DMEM / F-12 medium (catalog number: 11320033) manufactured by Thermo Fisher Scientific.

[0045] In this application, Transwell includes Transwell (catalog number: 3460) cell culture plates with a pore size of 0.4 μm manufactured by Corning Incorporated.

[0046] Please see Figures 1 to 6 This application provides a method for preparing an iPS-driven nasopharyngeal epithelial organoid. The method specifically includes the following steps:

[0047] Step S1: Direct the differentiation of humanized iPS cells into endoderm cells.

[0048] Please see Figure 2 and Figure 3 Specifically, it includes the following steps:

[0049] Step S11: Pre-treat the humanized iPS cells.

[0050] Humanized iPS cells were first resuspended in iPS-specific medium containing Y-27632 to obtain an iPS cell suspension. This suspension was then incubated in 96-well plates at 37°C with 5% CO2 for 24 hours. The cells at the bottom of the plate appeared as spherical cells, approximately 5 μm to 20 μm in diameter, surrounded by numerous diffusely distributed cells. Y-27632 is a small molecule inhibitor of the ROCK signaling pathway, which can inhibit apoptosis and induce selective differentiation of iPS cells into mesenchymal lineages through epithelial-mesenchymal transition-like regulation.

[0051] In some embodiments, the concentration of iPS cells in the iPS cell suspension can be 0.8 × 10⁻⁶. 6 / mL~1.6×10 6 / mL.

[0052] In some embodiments, the volume of iPS cell suspension added to a single well in a 96-well plate is 50 μL, and an equal volume of Duchenne phosphate-buffered saline (D-PBS) is added to adjacent wells to maintain consistent vapor pressure across different culture environments. Throughout step S1, i.e., the entire endoderm cell harvesting stage, the volume of D-PBS must be kept consistent with the volume of the cell suspension to maintain consistent vapor pressure.

[0053] Step S12: The pretreated iPS cells are induced in the first step.

[0054] After incubating the iPS cell suspension for 24 hours, the supernatant in the wells was completely aspirated, and the iPS cell suspension was incubated for induction. Reagent A from the endoderm cell KIT kit was added to the iPS cell suspension, taking care to avoid breaking up the cell spheroids during the process. Incubation was carried out at 37°C and 5% CO2 for 24 hours. This day was designated as the first day of organoid induction. During this process, the criteria for evaluating whether cell spheroids could continue induction were as follows: it was acceptable to aspirate and discard loosely bound cells, but if the main body of the cell spheroid was broken up, the cells in that well needed to be completely discarded.

[0055] In some embodiments, the amount of reagent A added to each well of iPS cells is 50 μL to 200 μL.

[0056] Step S13: The iPS cells induced in the first step are induced in the second step to obtain endoderm cells.

[0057] After incubating the cell spheroids with reagent A for 24 hours, completely aspirate the supernatant from the wells. The criteria for evaluating whether the cell spheroids can continue induction can refer to the criteria used when adding reagent A in step S12. Add reagent B to the endoderm cell KIT. Record this day as the second day of organoid induction. Repeat this step until the sixth day to obtain iPS-driven endoderm cells. Please refer to... Figure 2Figures a and b are shown in the middle. Figure 2 Figure a shows the morphology of iPS cell aggregation transforming into endoderm cells during the pre-induction phase (exemplarily the first day of organoid induction). Figure 2 Figure b shows the morphology of iPS cells transforming into endoderm cells in the later stage (exemplarily day 4 of organoid induction). Figure 2 This study demonstrates how highly viable iPS cells aggregate over time to form cell spheres and gradually increase in size. Ultimately, these endoderm cells become spherical, with a diameter of approximately 10 μm to 50 μm; therefore, they can also be referred to as endoderm cell spheres. The interior of the endoderm cell spheres is dense, with good cell contact and clear edges. Gentle blowing on these endoderm cell spheres does not cause them to completely disintegrate.

[0058] When completely aspirating the supernatant from the wells, a smaller pipette tip can be used, including but not limited to 20 μL white tips and 200 μL yellow tips. It is important to note that incomplete removal of the supernatant at this stage can have a critical impact on subsequent organoid induction, such as affecting the concentration of added reagents, and the possibility that certain components remaining in the supernatant may affect the proliferation status of iPS cells, thus interfering with the iPS cell differentiation process.

[0059] In some embodiments, the amount of reagent B added to the cell sphere in each well is 50 μL to 200 μL.

[0060] Step S14: Perform organoid immunofluorescence detection on endoderm cells and determine whether the endoderm cells are SOX17 positive.

[0061] Please refer to this again. Figure 3 After obtaining the endoderm cells, the method may further include: detecting the endoderm cell spheres using organoid immunofluorescence technology and determining whether the endoderm cells are SOX17 positive. The detection showed that the endoderm cell spheres were SOX17 positive. SOX17 refers to a gene in the High Mobility Group (HMG) gene family associated with the Sex-determining Region Y gene (SRY). SOX17 expression is a marker of endoderm cells. Specifically, red fluorescence represents the marker factor SOX17; blue fluorescence represents DAPI, used to visualize the cell nucleus; and green fluorescence represents phalloidine, used to visualize the cytoskeleton.

[0062] In some embodiments, before the humanized iPS cells are directed to differentiate into endoderm cells, the humanized iPS cells can be expanded to obtain a large number of iPS cells.

[0063] The specific amplification method includes: Step 1, preparing cell plates. The specific process may include: (1) diluting Matrigel with DMEM / F-12 medium at 4°C. The volume ratio of DMEM / F-12 medium to Matrigel is 100:1; (2) at 4°C, aspirating the diluted Matrigel solution into a 6-well cell plate (referred to as cell plate), 2 ml per well; (3) incubating the Matrigel solution at 37°C for 40 min, discarding the supernatant, and obtaining Matrigel-coated cell plates.

[0064] Step 2, iPS cell expansion, the specific process may include: (1) resuscitating humanized iPS cells, the number of cells being 1~2×10 6 300× g Centrifuge for 5 min. Discard the supernatant and resuspend the lower cell pellet in 1 mL of iPS-specific medium containing 10 μM Y-27632. Transfer the iPS cell suspension to a Matrigel-coated cell plate and incubate at 37°C and 5% CO2 for 24 h. (2) Discard the supernatant and incubate in 1 mL of iPS-specific medium at 37°C and 5% CO2 for 24 h. Repeat this step once a day. (3) After 3-5 days of culture, when the iPS cells are approximately 80% confluenced, the iPS cells can be passaged. Pre-passaging treatment: After culturing cells for 3-5 days, discard the supernatant, add 1 mL of D-PBS to wash away cell debris, and discard the supernatant again; add 200 μL of non-animal-derived trypsin, incubate at 37°C, 5% CO2 for 1-3 minutes; add 1.6 mL of D-PBS, gently pipette the cells to detach them from the cell plate, collect the cell suspension, and then pass it through a flow meter at 300× g Centrifuge for 5 minutes. Discard the supernatant and resuspend the iPS cells in iPS-specific culture medium containing 10 μM Y-27632 to obtain expanded iPS cells. These cells can be passaged at a 1:6 ratio or used for further processing.

[0065] Step S2: Endoderm cells are encapsulated with Matrigel and cultured in DMEM / F-12 medium A, which contains Dorsomorphin and SB431542, to obtain foregut organoids.

[0066] Specifically, the following steps are included:

[0067] Step S21: The endoderm cells obtained in step S1 are suspended in Matrigel and incubated at 37°C and 5% CO2 for 45 min.

[0068] Step S22: After Matrigel solidifies, Matrigel microspheres with a 3D structure are formed. The Matrigel microspheres are then transferred to a cell culture plate.

[0069] Step S23: Remove excess liquid, add DMEM / F-12 medium A containing Dorsomorphin (Dor) and SB431542 (SB), and incubate at 37°C and 5% CO2 for 24 hours.

[0070] Step S24: Repeat the steps of transferring the Matrigel microspheres to the cell culture plate and incubating them in the DMEM / F-12 medium A multiple times (step S23) to obtain the foregut organoids.

[0071] Dor and SB are two small molecule inhibitors commonly used in stem cell differentiation and organoid culture. They work by regulating specific signaling pathways to promote or inhibit certain differentiation pathways, thereby inducing the formation of specific cell types. In this step, Dor and SB are added as inducers to DMEM / F-12 medium to form DMEM / F-12 medium A.

[0072] Dor primarily exerts its effects by inhibiting the BMP (bone morphogenetic protein) signaling pathway. The BMP signaling pathway plays a crucial regulatory role in embryonic development, particularly in various types of differentiation, including endoderm formation and organogenesis. Dor promotes the differentiation of endoderm cells into foregut organoids by inhibiting BMP receptors, thereby reducing BMP signal transduction and decreasing other unwanted differentiation pathways, such as ectoderm or mesoderm differentiation. SB is an inhibitor of the TGF-β (transforming growth factor β) signaling pathway, specifically its major receptor ALK5. By inhibiting the TGF-β / ALK5 pathway, SB reduces its inhibitory effect on cell differentiation, thus promoting further endoderm formation and differentiation, and facilitating foregut organoid development. In this culture phase, the combined use of Dor and SB effectively promotes foregut organoid generation because they respectively regulate two key signaling pathways (BMP and TGF-β), inducing endoderm differentiation, promoting foregut development, and reducing cell differentiation into other non-target directions.

[0073] In some embodiments, endoderm cells encapsulated in Matrigel exhibit a three-dimensional (3D) structure. Firstly, Matrigel provides a supporting matrix for the endoderm cells, aiding in their growth, differentiation, and organization in three-dimensional space. Compared to traditional two-dimensional culture, three-dimensional culture better simulates the in vivo physiological environment, making cell growth, differentiation, and interactions more closely resemble physiological states. Furthermore, this application allows for the use of specialized Matrigel with low growth factor content to construct organoids, ensuring controllable reagent types and concentrations throughout the organoid induction process and effectively reducing the inappropriate induction of iPS cells by certain factors within Matrigel.

[0074] In some embodiments, the concentration of Dor in DMEM / F-12 medium A can be 1.5 μM to 4 μM, and the concentration of SB can be 5 μM to 20 μM. By adjusting the concentrations of Dor and SB, the BMP and TGF-β signaling pathways can be more precisely controlled, improving the differentiation of endoderm cells into foregut organoids. This not only improves the formation efficiency of foregut organoids but also enhances their structural stability, functionality, and healthy growth, reducing unnecessary cell death or improper differentiation. The Dor concentration can further be 2 μM to 4 μM, and exemplary values ​​can be any value within the range of any two of the values ​​composed of 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, or more. The SB concentration can further be 8 μM to 15 μM, and exemplary values ​​can be any value within the range of any two of the values ​​composed of 5 μM, 8 μM, 10 μM, 13 μM, 15 μM, 20 μM, or more.

[0075] In some embodiments, the repetition in step S24 can be 2 to 3 times.

[0076] Step S3: The foregut organoids are cultured in DMEM / F-12 medium B, which contains FGF10 protein, CHIR-99021, retinoic acid and BMP4 protein, to obtain airway epithelial progenitor cell organoids.

[0077] Specifically, excess fluid outside the foregut organoids was aspirated, and DMEM / F-12 medium B containing FGF10 protein (HH), CHIR-99021 (CH), retinoic acid (RA), and BMP4 protein (B) was added to the foregut organoids. The mixture was incubated at 37°C and 5% CO2 for 48 hours. This process was repeated 4 to 5 times to obtain the airway epithelial progenitor cell organoids.

[0078] HH, CH, RA, and BMP4 proteins were added as inducers to DMEM / F-12 medium, which formed medium B. HH, a member of the fibroblast growth factor family, has been reported to play a key role in early embryonic development and is a key protein involved in the morphogenesis of multiple organs, including the lungs and skin. It promotes the differentiation of iPS cells along the airway epithelial progenitor to nasopharyngeal epithelial cell pathway by participating in the Hedgehog and MAPK signaling pathways. CH is a selective GSK3β (glycogen synthase kinase 3β) inhibitor that activates the Wnt / β-catenin signaling pathway by inhibiting GSK3β. This signaling pathway promotes the differentiation of airway epithelial progenitor cells, supports the organization of airway epithelium, and regulates pattern formation and histological structure of airway epithelial cells during airway development. RA is the active form of vitamin A, which regulates gene expression by activating nuclear receptors, affecting cell differentiation, proliferation, and pattern formation. RA can also induce the differentiation of airway epithelial cells. BMP4 protein participates in regulating cell differentiation by modulating the Smad signaling pathway, promoting the formation, development, and differentiation of airway epithelial progenitor cells. In this culture phase, foregut organoids induced by HH, CH, RA, and BMP4 proteins promote the transformation of foregut organoids into airway epithelial progenitor cell organoids through the synergistic action of multiple signaling pathways.

[0079] In some embodiments, in DMEM / F-12 medium B, the concentration of HH can be 5 ng / mL to 20 ng / mL, the concentration of CH can be 1.5 μM to 4 μM, the concentration of RA can be 1 μM to 10 μM, and the concentration of BMP4 protein can be 5 ng / mL to 20 ng / mL. Concentrations of each inducer within these ranges are beneficial for further enhancing synergistic effects, precisely regulating the differentiation, proliferation, and pattern formation of airway epithelial progenitor cells, and promoting the successful induction of airway epithelial progenitor cell organoids.

[0080] The HH content can be further 10 ng / mL - 15 ng / mL, for example, it can be any value within the range of any two values ​​of 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL or above.

[0081] The CH content can be further 2μM~3μM, and for example, it can be any value within the range of any two values ​​of 1.5μM, 2μM, 2.5μM, 3μM or above.

[0082] The content of RA can be further ranged from 3 μM to 10 μM. For example, it can be any value within the range of any two values ​​of 1 μM, 2 μM, 3 μM, 5 μM, 8 μM, 10 μM or above.

[0083] The content of BMP4 protein can be further 8 ng / mL to 15 ng / mL, for example, it can be any value within the range of any two of the following values: 5 ng / mL, 8 ng / mL, 10 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL or above.

[0084] Step S4: The airway epithelial progenitor cell organoids are cultured in DMEM / F-12 medium C, which contains HH, RA and BMP4 proteins, to obtain nasopharyngeal epithelial organoids.

[0085] Specifically, excess fluid outside the airway epithelial progenitor cell organoids was aspirated, and DMEM / F-12 medium C containing FGF10 protein, RA and BMP4 protein was added to the airway epithelial progenitor cell organoids. The organoids were incubated at 37°C and 5% CO2 for 48 hours. This process was repeated 5 to 8 times to obtain the nasopharyngeal epithelial organoids.

[0086] FGF10, RA, and BMP4 proteins were added as inducers to DMEM / F-12 medium, forming medium C. FGF10 protein, a member of the fibroblast growth factor family, has been reported to play a crucial role in early embryonic development and is a key protein involved in the morphogenesis of multiple organs, including the lungs and skin. It differentiates along the airway epithelial progenitor cell to nasopharyngeal epithelial cell pathway by participating in signaling pathways such as Hedgehog and MAPK. RA promotes the transformation of airway epithelial progenitor cell organoids into nasopharyngeal epithelial organoids at this stage and promotes nasopharyngeal epithelial organoid development. BMP4 protein, by continuing to activate the Smad signaling pathway at this stage, regulates the differentiation of airway epithelial progenitor cells, promotes the hierarchical and polarized development of epithelial structures, thereby promoting the transformation of airway epithelial progenitor cells into nasopharyngeal epithelial cell types. This helps to promote the successful differentiation of airway epithelial progenitor cells into nasopharyngeal epithelial organoids through precise regulation of cell proliferation, differentiation, and patterning. The addition of three inducers, HH, RA, and BMP4, during this culture phase helps to promote the successful differentiation of airway epithelial progenitor cells into nasopharyngeal epithelial organoids by precisely regulating cell proliferation, differentiation, and patterning.

[0087] In some embodiments, in DMEM / F-12 medium C, the concentration of FGF10 protein can be 5 ng / mL to 20 ng / mL, the concentration of RA can be 1 μM to 10 μM, and the concentration of BMP4 protein can be 5 ng / mL to 20 ng / mL. Concentrations of each inducer within these ranges are beneficial for further synergistic effects and promoting the induction of nasopharyngeal epithelial organoids.

[0088] The concentration of FGF10 protein can further be 8 ng / mL to 15 ng / mL. For example, it can be any value within the range of 5 ng / mL, 8 ng / mL, 10 ng / mL, 13 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, or any two of the above values.

[0089] The content of RA can be further ranged from 2 μM to 8 μM. For example, it can be any value within the range of any two values ​​of 1 μM, 3 μM, 5 μM, 8 μM, 10 μM or above.

[0090] The content of BMP4 protein can be further ranged from 8 ng / mL to 15 ng / mL. For example, it can be any value within the range of any two of the above values: 5 ng / mL, 8 ng / mL, 10 ng / mL, 13 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL.

[0091] In some embodiments, serum-free KFSM-type culture media can be used for maintenance culture of nasopharyngeal epithelial organoids, providing a stable, serum-free culture environment to promote their long-term growth and maturation. Following step S4, the procedure further includes:

[0092] Step S5: Perform fluorescence detection on the mature nasopharyngeal epithelial organoids and determine whether the nasopharyngeal epithelial organoids are SMO positive.

[0093] Please see Figure 4 The mature nasopharyngeal epithelial organoids (Mature) and the immature nasopharyngeal organoids (Control) were observed using fluorescence microscopy. Figure 4 Figure a shows the fluorescence detection results of immature nasopharyngeal epithelial organoids. Figure 4Figure b in the diagram shows the fluorescence detection results of mature nasopharyngeal epithelial organoids. Compared to the control group, the nasopharyngeal epithelial organoids highly expressed the ciliary marker protein SMO, exhibiting SMO positivity. The protein encoded by SMO is a G protein-coupled receptor that interacts with the receptor of hedgehog protein. Previous studies have considered SMO expression to be a marker of epithelial cilia. High SMO expression indicates that this nasopharyngeal epithelial organoid can effectively form and maintain functional cilia, which is of great significance for mimicking the physiological function of the nasopharyngeal epithelium and for research on related diseases (such as nasopharyngeal carcinoma). Among them, Merge is an imaging image of superimposed fluorescence signal channels at 405nm, 488nm, 594nm, and 647nm; DAPI is a nucleic acid dye that can display the cell nucleus, and this DAPI has excitation light near a 405nm laser; FoxA1 is a biomarker of nasopharyngeal epithelium; β-Tubulin can be used for the fluorescence detection of microtubules in cells or tissues; the protein encoded by SMO is a G protein-coupled receptor that can interact with the receptor of hedgehog protein and is considered a marker of epithelial cilia.

[0094] Please refer to Figure 5 In step S5, the mature nasopharyngeal epithelial organoid can also be observed under a bright-field microscope. The presence of ciliated structures at the edges indicates that the nasopharyngeal epithelial organoid has well-formed functional cilia. Figure 5 Figure a shows a relatively long ciliary. Figure 5 Image b shows a relatively dense area of ​​cilia.

[0095] The method for preparing iPS-driven nasopharyngeal epithelial organoids provided in this application first involves the directed differentiation of humanized iPS cells into endoderm cells. After encapsulation with Matrigel, the endoderm cells undergo multi-stage continuous differentiation using DMEM / F-12 medium containing different combinations of inducing agents, ultimately forming nasopharyngeal epithelial organoids. This method allows for the large-scale, reproducible, and uniform production of nasopharyngeal epithelial organoids, while effectively reducing the preparation cost and shortening the preparation cycle.

[0096] This application also provides a preparation system for performing the aforementioned iPS-driven nasopharyngeal epithelial organoid preparation method. The preparation system includes a kit comprising DMEM / F-12 culture medium A, DMEM / F-12 culture medium B, and DMEM / F-12 culture medium C.

[0097] DMEM / F-12 medium A contains 1.5 μM–4 μM Dorsomorphin and 5 μM–20 μM SB431542. For example, taking DMEM / F-12 medium A containing 2 μM Dorsomorphin and 10 μM SB431542 as an example, its preparation method is as follows: 1 mg of Dorsomorphin is added to 1.2526 mL of DMSO solvent and incubated in a 50°C water bath until the solute is completely dissolved, obtaining a 2 mM stock solution; 5 mg of SB431542 is added to 1.3008 mL of DMSO solvent and incubated until the solute is completely dissolved, obtaining a 10 mM stock solution. Both stock solutions are added to an appropriate amount of DMF / F12 medium at a volume ratio of 1:1000 to prepare medium A.

[0098] DMEM / F-12 medium B contains 5 ng / mL to 20 ng / mL of FGF10 protein, 1.5 μM to 4 μM of CHIR-99021, 1 μM to 10 μM of retinoic acid (RA), and 5 ng / mL to 20 ng / mL of BMP4 protein. For example, taking DMEM / F-12 medium B containing 10 ng / mL FGF10 protein, 3 μM CHIR-99021, 5 μM RA, and 10 ng / mL BMP4 protein as an example, the preparation method is as follows: 10 μg of FGF10 protein is dissolved in 1 mL of PBS to prepare a 10 μg / mL stock solution; 1 mg of CHIR-99021 is dissolved in 0.7163 mL of DMSO to prepare a 3 mM stock solution; 5 mg of RA is dissolved in 3.3289 mL of DMSO until the solute is completely dissolved to prepare a 5 mM stock solution; 10 μg of BMP4 protein is dissolved in 1 mL of PBS to prepare a 10 μg / mL stock solution. All four stock solutions are added to an appropriate amount of DMF / F12 medium at a volume ratio of 1:1000 to prepare medium B.

[0099] DMEM / F-12 medium C contains 5 ng / mL to 20 ng / mL FGF10 protein, 1 μM to 10 μM RA, and 5 ng / mL to 20 ng / mL BMP4 protein. For example, taking DMEM / F-12 medium C containing 10 ng / mL FGF10 protein, 5 μM RA, and 10 ng / mL BMP4 protein as an example, its preparation method is as follows: 10 μg of FGF10 protein is dissolved in 1 mL of PBS to prepare a 10 μg / mL stock solution; 5 mg of RA is dissolved in 3.3289 mL of DMSO solvent until the solute is completely dissolved to obtain a 5 mM stock solution; 10 μg of BMP4 protein is dissolved in 1 mL of PBS to prepare a 10 μg / mL stock solution. All three stock solutions are added to an appropriate amount of DMF / F12 medium at a volume ratio of 1:1000 to prepare medium C.

[0100] This application also provides a nasopharyngeal epithelial organoid, which is prepared using the aforementioned iPS-driven nasopharyngeal epithelial organoid preparation method.

[0101] Compared to existing technologies, this nasopharyngeal epithelial organoid can be obtained in large quantities and in a standardized manner. Furthermore, since it is produced using humanized iPS cells, it can be efficiently infected by highly pathogenic NPC factors such as EBV, making it an ideal experimental biological model for EBV infection and providing an important experimental tool for related research. This is of great significance for long-term culture of nasopharyngeal epithelial organoids, research on related disease models, and drug screening.

[0102] Furthermore, this application also provides a method for constructing an EBV virus-infected nasopharyngeal epithelial organoid model, the method comprising:

[0103] Infecting nasopharyngeal epithelial organoids with EBV virus, wherein the organoids are the aforementioned nasopharyngeal epithelial organoids; and

[0104] Immunofluorescence detection was performed on the nasopharyngeal epithelial organoids that were not infected with EBV and those that were infected. The FoxA1 factor expression level in the infected nasopharyngeal epithelial organoids was lower than that in the uninfected nasopharyngeal epithelial organoids, indicating that the organoid structure was damaged.

[0105] Please see Figure 7 and Figure 8 Fluorescence detection was performed on nasopharyngeal organoids that were neither infected with EBV nor infected with EBV. The observation results for nasopharyngeal organoids that were not infected with EBV are as follows: Figure 7 The results of observation of nasopharyngeal organoids infected with EBV are as follows: Figure 8 .in, Figure 7 Figure a and Figure 8Figure a shows the Merge image, which is an image of the superimposed fluorescence signal channels at 405nm, 488nm, 594nm, and 647nm. Figure 7 The b-image and Figure 8 Image b shows the DAPI signal. DAPI is a nucleic acid dye that can visualize the cell nucleus and is excited by a 405nm laser. Figure 7 The C diagram and Figure 8 Figure c shows FoxA1, a biomarker for nasopharyngeal epithelium; Figure 7 d-graph and Figure 8 Figure d shows SMO, which encodes a G protein-coupled receptor that interacts with receptors in hedgehog proteins and is considered a marker of epithelial cilia. This result indicates that the organoid structure is disrupted, with internal cavities and morphological dissociation, suggesting that EBV has invaded and begun to damage the organoids.

[0106] This technology not only provides an ideal model for studying EBV infection mechanisms, virus-host interactions, and immune evasion, but also promotes research on nasopharyngeal carcinoma and EBV-related diseases. This model offers great potential for efficient screening of anti-EBV drugs, vaccine development, and research into the molecular mechanisms by which viruses induce tumors. It also boasts advantages such as high throughput and ethical feasibility, which can drive the development of early diagnosis and treatment options for related diseases.

[0107] The following specific examples further illustrate the aforementioned iPS-driven nasopharyngeal epithelial organoids, their preparation methods, and applications.

[0108] Example 1: iPS cell expansion

[0109] 1. Cell plate preparation:

[0110] (1) Dilute Matrigel with DMEM / F-12 medium at 4℃, with a volume ratio of DMEM / F-12 medium to Matrigel of 100:1;

[0111] (2) At 4°C, the diluted Matrigel solution was pipetted into a 6-well cell plate (referred to as a cell plate), 2 ml per well;

[0112] (3) Incubate Matrigel solution at 37°C for 40 min, discard the supernatant, and obtain Matrigel-coated cell plates.

[0113] 2. iPS cell expansion

[0114] (1) Resuscitate humanized iPS cells, with a cell count of 1~2×10⁻⁶. 6 300× gCentrifuge for 5 min. Discard the supernatant and resuspend the lower cell pellet in 1 mL of iPS-specific medium containing 10 μM Y-27632. Transfer the iPS cell suspension to a Matrigel-coated cell plate and incubate at 37°C with 5% CO2 for 24 h.

[0115] (2) Discard the supernatant and culture it in 1 mL of iPS-specific medium at 37°C and 5% CO2. Repeat this step once a day.

[0116] (3) After culturing for 3-5 days, when the iPS cells reach approximately 80% confluence, the iPS cells can be passaged. Pre-passage treatment: After culturing for 3-5 days, discard the supernatant, add 1 mL of D-PBS to wash away cell debris, and discard the supernatant again; add 200 μL of non-animal-derived trypsin, incubate at 37℃, 5% CO2 for 1-3 minutes; add 1.6 mL of D-PBS, gently pipette the cells to detach them from the cell plate, collect the cell suspension, and pass the suspension at 300× g Centrifuge for 5 minutes. Discard the supernatant and resuspend the iPS cells in iPS-specific culture medium containing 10 μM Y-27632 to obtain expanded iPS cells. These cells can be passaged at a 1:6 ratio or used for further processing.

[0117] Example 2: Obtaining Endoderm Cells (Day 0-Day 6)

[0118] (1) Using Corning Incorporated 96-well Clear Round Bottom Ultra-Low Attachment Microplate (catalog number 7007), the cell wells in the 96-well plate were rinsed with D-PBS.

[0119] (2) The iPS cells expanded in Example 2 were resuspended in iPS-specific culture medium containing 10 μM Y-27632 to obtain an iPS cell suspension with an iPS cell concentration of 0.8~1.6×10⁻⁶ cells / mL. 6 / ml.

[0120] (3) Add 50 μL of iPS cell suspension to some wells of the above-mentioned round-bottom low-absorption 96-well plate. Add the same volume of D-PBS to the adjacent wells containing iPS cell suspension to balance the vapor pressure within the 96-well plate. Throughout the entire endoderm cell acquisition process, the volume of D-PBS should be kept consistent with the volume of the cell suspension. Incubate at 37°C and 5% CO2 for 24 h. Record this day as organoid induction Day 0.

[0121] (4) After incubation for about 24 hours, the cells at the bottom of the cell plate are spherical with a diameter of about 10 μm, and a large number of cells are diffused around the surface of the sphere.

[0122] (5) Using smaller pipette tips, including but not limited to 20 μL white tips and 200 μL yellow tips, completely aspirate the supernatant from the wells. It should be noted that failure to completely remove the supernatant at this stage will have a critical impact on subsequent organoid induction. During this process, it is acceptable to aspirate and discard loosely bound cells, but if the cell spheres are broken up, the cells in that well must be completely discarded.

[0123] (6) Carefully add 50 μL of reagent A from the endoderm KIT kit to each culture well, avoiding breaking up or dissociating the cell spheres during the process. Incubate at 37°C and 5% CO2 for 24 h. Record this day as organoid induction Day 1;

[0124] (7) Carefully use a small pipette tip to completely aspirate the supernatant. The evaluation criteria for whether cell spheroids can continue to be induced can be found in step (5) of this embodiment.

[0125] (8) Add 50 μL of reagent B from the endoderm KIT kit. Record this day as organoid induction Day 2.

[0126] (9) Repeat steps (7) and (8) in this embodiment until Day 6.

[0127] (10) At this point, iPS-driven endoderm cell spheres are obtained. These endoderm cell spheres are spherical, with a diameter of approximately 10–50 μm. The interior of the endoderm cell spheres is dense, with good cell contact and clear edges. Gentle blowing on the endoderm cell spheres does not cause them to completely dissociate. At this point, organoid immunofluorescence technology can be used to detect that the endoderm cell spheres are SOX17 positive.

[0128] Example 3: Preparation of iPS-driven nasopharyngeal epithelial organoids (Day 7-Day 30+)

[0129] (1) Remove the sealing film from the clean bench and cut it into 2×2cm pieces. Spray with alcohol and sterilize under UV light for at least 40 minutes. Note that since antibiotics cannot be used throughout the organoid induction process, this step is the most likely to cause contamination and must be thoroughly sterilized.

[0130] (2) Using a pipette tip, such as a 1 mL blue tip, carefully aspirate the endoderm cell spheres prepared in Example 3 and place the liquid droplet of the endoderm cell spheres onto the sealing film. Each 2-3 endoderm cell spheres can be placed in one liquid droplet.

[0131] (3) Remove the liquid from the droplet and add 30 μL to 50 μL of Matrigel. Suspend the endoderm cell spheres in the hemispherical droplet formed by Matrigel. Incubate at 37°C and 5% CO2 for 45 min.

[0132] (4) After Matrigel gel solidifies, Matrigel beads are formed. Use a pipette tip to draw up DMEM / F-12 medium and gently blow off the Matrigel beads. Transfer the Matrigel beads to a 24-well cell culture plate.

[0133] (5) Remove excess liquid from the 24-well cell culture plate and add 1 mL of DMEM / F-12 medium A containing Dor and SB. Incubate at 37°C and 5% CO2 for 24 h.

[0134] (6) Repeat step (5) of this embodiment 2 to 3 times to obtain foregut organoids.

[0135] (7) Remove excess liquid from the 24-well cell culture plate and add 1.5 mL of DMEM / F-12 medium B containing HH, CH, RA and BMP4 proteins. Incubate at 37°C and 5% CO2 for 48 h.

[0136] (8) Repeat step (7) of this embodiment 4 to 5 times to obtain airway epithelial progenitor cell organoids.

[0137] (9) Remove excess liquid from the 24-well cell culture plate and add 1.5 mL of DMEM / F-12 medium containing HH, RA and BMP4 proteins. Incubate at 37°C and 5% CO2 for 48 h.

[0138] (10) Repeat step (9) of this embodiment 5 to 8 times to obtain iPS-driven nasopharyngeal epithelial organoids.

[0139] (11) The nasopharyngeal epithelial organoid can be maintained using serum-free KFSM-type culture medium.

[0140] Example 4: EBV infection model of organoids

[0141] (1) Using a Transwell cell culture plate with a pore size of 0.4 μm, B95 cells with 80% cell confluence and capable of autonomously producing EBV particles were seeded in the cell culture plate.

[0142] (2) Remove the culture medium. Please refer to [the instructions]. Figure 6As shown, the organoid, specifically the nasopharyngeal epithelial organoid prepared in Example 4, was placed in a Transwell. An appropriate amount of DMEM / F-12 medium containing PM, MA, and VA was added to the cell culture plate and Transwell. PM is disodium pamidronate, which induces the accumulation of isopentenyl pyrophosphate within cells, effectively triggering the cell death signaling pathway and leading to the lysis of EBV-transformed lymphoblastoid B cell lines. MA is mevastatin, a competitive HMG-CoA reductase inhibitor. VA is valproic acid, possessing potent histone deacetylase inhibitory activity, which can be used to activate the expression of lysing viral genes in EBV-positive tumors. This reagent combination is commonly used for the lysis of B95 cells and the activation of EBV virus. The liquid level was just above the organoid, ensuring that material exchange between the cell culture plate and Transwell occurred only through a semi-permeable membrane. The cells were cultured at 37°C and 5% CO2 for 96 hours.

[0143] (3) Remove the organoids and perform fluorescence detection on the nasopharyngeal organoids that were not infected with EBV and those that were infected with EBV. Please refer to the results again. Figure 7 and Figure 8 The results showed differences in the expression and morphological changes of nasopharyngeal organoid-related factors before and after EBV infection.

[0144] The above description of the embodiments is only for the purpose of helping to understand the method and core idea of ​​this application; in addition, those skilled in the art can make various other corresponding changes and modifications based on the technical concept of this application, and all such changes and modifications should fall within the protection scope of the claims of this application.

Claims

1. A method for preparing iPS-driven nasopharyngeal epithelial organoids, characterized in that, include: Humanized iPS cells were directed to differentiate into endoderm cells; The endoderm cells were encapsulated with Matrigel and cultured in DMEM / F-12 medium A, which contained 1.5 μM to 4 μM of Dorsomorphin and 5 μM to 20 μM of SB431542, to obtain foregut organoids. The foregut organoids were cultured in DMEM / F-12 medium B, which contained 5 ng / mL to 20 ng / mL of FGF10 protein, 1.5 μM to 4 μM of CHIR-99021, 1 μM to 10 μM of retinoic acid and 5 ng / mL to 20 ng / mL of BMP4 protein to obtain airway epithelial progenitor cell organoids. as well as The airway epithelial progenitor cell organoids were cultured in DMEM / F-12 medium C, which contained 5 ng / mL to 20 ng / mL of FGF10 protein, 1 μM to 10 μM of retinoic acid and 5 ng / mL to 20 ng / mL of BMP4 protein to obtain the nasopharyngeal epithelial organoids.

2. The method for preparing iPS-driven nasopharyngeal epithelial organoids according to claim 1, characterized in that, The step of encapsulating the endoderm cells with Matrigel and culturing them in DMEM / F-12 medium A includes: The endoderm cells were suspended in Matrigel and incubated at 37°C and 5% CO2 for 30 to 60 minutes. After the Matrigel solidifies, forming Matrigel microspheres, these Matrigel microspheres are transferred to a cell culture plate. Remove excess liquid, add DMEM / F-12 medium A containing Dorsomorphin and SB431542, and incubate at 37°C and 5% CO2 for 48 hours; and The foregut organoids were obtained by repeating the steps of transferring the Matrigel microspheres to cell culture plates and incubating them in the DMEM / F-12 medium A multiple times.

3. The method for preparing iPS-driven nasopharyngeal epithelial organoids according to claim 1, characterized in that, The step of culturing the foregut organoids in DMEM / F-12 medium B includes: Remove excess fluid from the foregut organoids and add DMEM / F-12 medium B to the foregut organoids. Incubate at 37°C and 5% CO2 for 48 hours. Repeat this process multiple times to obtain the airway epithelial progenitor cell organoids.

4. The method for preparing iPS-driven nasopharyngeal epithelial organoids according to claim 1, characterized in that, The step of culturing the airway epithelial progenitor cell organoids in DMEM / F-12 medium C includes: Remove excess fluid from the airway epithelial progenitor cell organoids, add DMEM / F-12 medium C containing FGF10 protein, retinoic acid and BMP4 protein to the airway epithelial progenitor cell organoids, and incubate at 37°C and 5% CO2 for 48 h; repeat multiple times to obtain the nasopharyngeal epithelial organoids.

5. A preparation system for performing the method for preparing iPS-driven nasopharyngeal epithelial organoids as described in claim 1, characterized in that, The kit includes DMEM / F-12 medium A, DMEM / F-12 medium B, and DMEM / F-12 medium C. The DMEM / F-12 medium A contains 1.5 μM to 4 μM Dorsomorphin and 5 μM to 20 μM SB431542; the DMEM / F-12 medium B contains 5 ng / mL to 20 ng / mL FGF10 protein, 1.5 μM to 4 μM CHIR-99021, 1 μM to 10 μM retinoic acid and 5 ng / mL to 20 ng / mL BMP4 protein; and the DMEM / F-12 medium C contains 5 ng / mL to 20 ng / mL FGF10 protein, 1 μM to 10 μM retinoic acid and 5 ng / mL to 20 ng / mL BMP4 protein.

6. A nasopharyngeal epithelial organoid, characterized in that, Obtained by the method for preparing iPS-driven nasopharyngeal epithelial organoids according to any one of claims 1 to 4.

7. A method for constructing an EBV virus-infected nasopharyngeal epithelial organoid model, characterized in that, include: Infecting nasopharyngeal epithelial organoids with EBV virus, wherein the nasopharyngeal epithelial organoids are nasopharyngeal epithelial organoids prepared by the method for preparing iPS-driven nasopharyngeal epithelial organoids as described in any one of claims 1 to 4; and Fluorescence detection was performed on the nasopharyngeal epithelial organoids that were not infected with EBV and those that were infected with EBV. The FoxA1 factor expression level in the EBV-infected nasopharyngeal epithelial organoids was lower than that in the uninfected EBV-infected nasopharyngeal epithelial organoids.

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