Human nasal epithelial cell organoids as a platform for subsequent passage stability, ALI culture differentiation and influenza virus infection modeling

Human nasal epithelial cell organoids as a platform for subsequent passage stability, ALI culture differentiation and influenza virus infection modeling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Human nasal epithelial cell organoids as a platform for subsequent passage stability, ALI culture differentiation and influenza virus infection modeling Hyun Jik Kim, Siyeon Jin, Sujin Kim, Minseop Kim, Hyunkyung Cha, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9241349/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The cultures of human nasal epithelial cells (NECs) are good surrogate models for studying immune responses. Three-dimensional (3D) organoids have emerged as an alternative for nasal epithelium, serving as robust platforms for modeling respiratory viral infections. We aimed to generate human NEC organoids and to determine their cellular characteristics as an in vitro model for air-liquid interface (ALI) culture and influenza A virus (IAV) infection. We successfully developed human NEC organoids with using a Matrigel-based 3D culture system that recapitulates the structural characteristics and cellular composition of the human nasal epithelium. Histological results, real-time PCR, and single-cell RNA sequencing (scRNA-seq) revealed that the organoids consisted of seven different cell types and displayed a difference in the composition between the cells. The organoids displayed a much higher rate of basal progenitor cells whereas ciliated cells were less dominant. Unlike ALI cultures in which passage (P) 2 was the limit, the organoids exhibited successful and subsequent passages up to P5. Our data determined that NEC organoids are an adequate in vitro model for IAV infection and showed a sharp induction of mRNA levels for interferons (IFNs) and IFN-stimulated genes following IAV infection. These results demonstrate that human NEC organoids serve as a robust in vitro model, successfully recapitulating the biological characteristics of nasal epithelium, and could be an innovative tool for exploring distinct IFN-related innate immune responses following influenza infection. Biological sciences/Cell biology/Cell growth Health sciences/Medical research/Experimental models of disease Organoid Nasal epithelium Influenza virus Innate immune responses Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction ໿ The mucosal surfaces of the human respiratory tract are in direct contact with the external environment, making them susceptible to invasion of respiratory viruses. 1,2 Studies on the clearance reaction of the host’s airway mucosa increasingly take into consideration the contribution of immune responses and specific protection against infection from external pathogens to integrate environmental signals. 3–5 Inhaled viruses encounter the host immune system in the respiratory mucosa, and mounting evidence indicates that the nasal epithelium is the primary site where respiratory viruses like SARS-CoV-2 and influenza begin to replicate. 6–8 Nasal epithelial cells (NECs) not only provide a physical barrier to potentially harmful insults, but they also play a crucial role in the first line of immunological defense. 9,10 Cultured human NECs are good research tools for studying respiratory epithelial physiology and have been instrumental in increasing our knowledge of antiviral immune responses in the upper airway. To date, many kinds of epithelial cell culture methods, such as submerged, suspension, floating, and air-liquid interface (ALI), have been introduced and widely utilized for primary human NEC culture. 9,11,12 ALI cultures of NECs have been successfully obtained from both nasal biopsies and nasal brushings and it is of great interest whether NECs are an ideal surrogate. 3,4,9 However, primary NEC cultures take a long time to establish fully differentiated epithelium, and the cell condition deteriorates with each passage, limiting their use in repetitive experiments. Furthermore, primary NEC cultures via the ALI system proliferate and differentiate in vitro for a finite number of passages before gradually losing their original characteristics. 13–15 The scarcity of sample obtained from a patient’s nasal tissue is a significant limitation to in vitro studies using these culture techniques. Three-dimensional (3D) organoids grown from stem cells or patient biopsies can self-assemble over long periods (months) of culture while maintaining genetic and phenotypic stability, providing an almost unlimited supply of primary cells. 16 Airway organoids have recently emerged as a promising experimental tool to model the human respiratory tract and have been successfully used in experimental research in respiratory biology. 16–18 Recent research has demonstrated the utility of human airway organoids for respiratory diseases, with applications ranging from basic scientific research to pharmaceutical development and clinical diagnosis. 19,20 We assumed that NEC organoids, instead of primary NEC cultures with ALI, would have many advantages, such as shortening cell culture time and increasing cell stability over multiple passages, which would be useful for diverse in vitro experiments. We established NEC organoids directly from human nasal mucosa (NM) using Matrigel. These organoids were stably expanded up to passage 5 without any cell purification procedure. We then generated an ALI NEC culture that morphologically and functionally phenocopy the native nasal epithelium through the organoids. NEC organoids have become a suitable research tool for studying influenza virus infection and interferon (IFN)-related innate immune responses. We estimate that human NEC organoids could be a good in vitro research method for applying ALI NEC culture and respiratory virus infection with stable expansion of passages and repeated experiments. Material & Methods Sample collection and processing This study was performed according to the Helsinki Declaration and was approved by the Institutional Review Board of Seoul National University College of Medicine, Seoul, Korea (IRB No. 2204-097-1316). Written informed consent was obtained from all participants before sample collection. The subjects were all referred to the Department of Otorhinolaryngology at Seoul National University Hospital (Seoul, Korea) between November and December 2024, primarily for nasal surgery. All subjects were free of clinical signs of rhinosinusitis and upper airway infection and had no history of other allergic diseases. Nasal cavity of each subject was accurately observed using an intranasal endoscope under general anesthesia due to nasal surgery and sampling of NM was performed around the middle turbinate by an otorhinolaryngologist (HJ Kim). The NM tissue was collected individually from 5 subjects and each NM sample was placed in an individual tube containing transfer media [Dulbecco’s Modified Eagle’s Medium (DMEM)/Nutrient Mixture F12 medium + 1% Penicillin/Streptomycin]. After collection, the NM samples were incubated in 0.1% protease, prepared in transfer medium, at 4°C overnight. Then, the detached epithelial cell layer was collected and transferred to a new tube, followed by centrifugation at 1,200 rpm, 4°C for 5 min. The supernatant was aspirated, and the specimen was resuspended in washing buffer (transfer media + 10% Fetal bovine serum). After pipetting or inverting several times, tubes were centrifuged at 1,200 rpm, 4°C for 5 min. After aspirating the supernatant, the specimen was resuspended in 1X RBC lysis solution. After pipetting several times, tubes were centrifuged at 1,200 rpm, 4°C for 5 min. After supernatant was discarded, the pellet was resuspended in culture medium and centrifuged again. Establishment of NEC organoid Nasal cell pellets were resuspended in cold Growth factor reduced Matrigel (CORNING, Corning, NY, USA), and 150 ㎕ drops of Matrigel-cell suspension were allowed to solidify on pre-warmed 12-well cell culture plates at 37℃ for 20 min. After solidification, 1 mL of airway organoid medium 16 was added to each well and organoid cells were incubated in 37℃/5% CO2. Medium was changed every 4 days and organoids were passaged every 2–3 weeks. After Matrigel was lysed in cell recovery solution, organoids were dissociated by resuspension in 5 mL TrypLE Express (Thermo Fisher Scientific, Waltham, MA, USA), incubation for 5 min in a water bath, and pipetting. Following the addition of 5 mL airway organoid medium and centrifugation at 300 x g, dissociated cells were resuspended in cold Matrigel and reseeded. Air-liquid interface cell culture Normal human nasal epithelial (NHNE) cells were cultured as described previously. 9 Briefly, passage-3 NECs (1 x 105 cells/culture) were seeded in 0.5 ml of culture medium on Transwell clear culture inserts (12-mm, with a 0.4-㎛ pore size; CORNING, Corning, NY, USA). Cells were cultured in a 1:1 mixture of basal epithelial growth medium and DMEM containing previously described supplements. 21 Cultures were grown while submerged for the first 9 days. The culture medium was changed on Day 1, and every other day thereafter. An air–liquid interface (ALI) was created on Day 9 by removing the apical medium and feeding the cultures from the basal compartment only. The culture medium was changed daily after the initiation of the ALI. We added antibiotics such as 1% penicillin and streptomycin into all media for subculture and culture stages. All experiments described here used cultured nasal epithelial cells at 14 days after the creation of the ALI. Real-Time PCR Total RNA was isolated from nasal epithelial cells at 14 days after confluence, using TRIzol (QIAGEN, Venlo, Netherland). The cDNA was synthesized from 1 µg of RNA with random hexamer primers, using Moloney Murine Leukemia Virus reverse transcriptase (enzynomics, Daejeon, Republic of Korea). Commercial reagents (TaqMan Universal PCR Master Mix; Thermo Fisher Scientific) were selected and conditions were set according to the manufacturer’s protocol. The total reaction volume of 12 µL contained 2 µL of cDNA (reverse transcription mixture), oligonucleotide primers at a final concentration of 800 nM, and the TaqMan hybridization probe at 200 nM. The real-time PCR probe was labeled at the 5’ end with carboxylfluorescein, and at the 3’ end with the quencher carboxytetramethylrhodamine. Primers for human KRT5 (Hs00361185_m1), TP63 (Hs00978340_m1), MUC5AC (Hs01365616_m1), FOXJ1 (Hs00230964_m1) and influenza A virus polymerase acidic protein (PA) were purchased from Thermo Fisher Scientific. Real-time PCR was performed using the QuantStudio 3 Sequence Detection System. The thermocycler parameters included 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. Target mRNA levels were quantified using target-specific primer and probe sets for KRT5, TP63, MUC5AC, FOXJ1 and glyceraldehyde 3–phosphate dehydrogenase (GAPDH). All PCR assays were analyzed using the ΔΔCt method and are presented as relative expression values. All probes were designed to span an intron, and did not react with genomic DNA. All reactions were performed in triplicate, and the results were normalized against GAPDH as an endogenous control. Electron microscopy Electron microscopic analysis was performed, as previously described, with modifications. 9 Specimens were fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hours at 4°C, washed, and then incubated with 1% osmium tetroxide in 0.1 M PB for 2 hours at 25°C. For scanning electron microscope (SEM), specimens were dehydrated in graded baths to 100% ethanol, critical-point-dried under liquid carbon dioxide, gold sputter-coated, and visualized on a JSM-7401F microscope (JEOL Ltd., Tokyo, Japan). For transmission electron microscope (TEM), osmium-stained samples were further fixed in 7% uranyl acetate, thin-sectioned (70 nm) in Polybed 812 (Polysciences, Warrington, PA), post-stained in uranyl acetate and lead citrate, and visualized on a JEM-1400. Histologic analysis and immunohistochemistry For NEC organoids, nasal organoids were collected by scraping Matrigel domes from 12-well plates and centrifuged at 200 x g, 4℃ for 5 min. The pellet was resuspended in cold AO medium and incubated on a shaker at 4℃ for 10 minutes, followed by centrifugation and removal of the supernatant. This wash step was repeated to ensure complete removal of the Matrigel. Organoids were then fixed in 10% formalin at room temperature, centrifuged and resuspended in PBS before being mixed with 2% agarose. The organoid-agarose mixture was gently dispensed onto parafilm to form a dome, which, after solidification, was placed into a cassette and processed following standard tissue-sample procedures. For NHNE cells, cells were fixed by adding 10% formalin to apical and basolateral compartments at room temperature. The excised Transwell membrane was embedded in a cassette. De-waxed sections were stained with either hematoxylin and eosin (H&E), or a periodic acid-Schiff (PAS) kit, according to the manufacturer’s instructions (Bioptica, Milan, Italy). Immunostaining was performed on formalin-fixed paraffin sections (4 µm) of NEC organoid and cultured NHNE cells. Formalin-fixed paraffin sections (4 µm) were de-waxed in xylene (Sigma Chemicals, St Louis, MO, USA), rehydrated in successive ethanol baths, and subjected to antigen retrieval by microwave in 0.01 mol/L sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% methanolic hydrogen peroxide for 10 minutes at room temperature. Nonspecific binding was blocked by incubation with 10% normal serum from VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. Primary antibodies (1:200 dilution) were applied at 4°C for 24 hours. Following washing in TBS, slides were incubated with peroxidase-conjugated goat anti-mouse/rabbit antibodies (1:200 dilution, Vector Laboratories) for 30 minutes at room temperature. Signal was amplified using the indirect immunoperoxidase technique using the DAKO Envision kit (Dako, Kingsgrove, Australia). Staining was visualized using an Olympus UTV0.63XC microscope with the DP Controller software (Hamburg, Germany). The experiment was performed using NHNE cells and inferior turbinate specimens from four healthy volunteers. Immunofluorescence Staining and Imaging To evaluate protein expression profiles, NEC organoids were harvested from Matrigel domes and washed with ice-cold PBS to remove residual Matrigel. The organoids were fixed with 4% paraformaldehyde (PFA; Green Bee Bio, PC2031-100-00) for 30 minutes at room temperature (RT). For cryoprotection, fixed organoids were dehydrated in a 30% sucrose solution and subsequently embedded in OCT compound (Scigen, 4586) to create cryo-blocks. The blocks were sectioned at a thickness of 10 µm using a cryostat. The sections were permeabilized with 0.1% Triton X-100 and blocked with 3% bovine serum albumin (BSA) in PBS to minimize non-specific binding. The sections were then incubated overnight at 4°C with primary antibodies against KRT8/18 (Progen, GP11), KRT5 (Invitrogen, MA5-12596), p63 (Abcam, ab124762), acetylated ɑ-tubulin (Cell Signaling, 5335), and MUC5AC (Invitrogen, MA5-12178), all at a 1:200 dilution. Following three washes with PBS, the sections were incubated for 2 hours at RT with appropriate fluorescence-conjugated secondary antibodies (1:400), along with phalloidin (1:400; Invitrogen, R415) for F-actin visualization and DAPI (1:1000; Invitrogen, D1306) for nuclear counterstaining. Immunofluorescence images were captured using a confocal laser scanning microscope (LSM 700; Carl Zeiss, Jena, Germany). Virus infection Influenza A virus strain A/Wilson–Smith/1933 H1N1 (IAV A/WS/33; American Type Culture Collection, Manassas, VA, USA) was used in this study to model acute viral infection. Virus stocks were grown in Madin–Darby canine kidney (MDCK) cells in viral growth medium according to a standard procedure. 22 Briefly, after 48 hours of incubation at 37°C, the supernatants were harvested and spun by centrifugation at 1,000 x g for 5 minutes to remove cellular debris. The clarified supernatant was transferred to a centrifugal filter tube and centrifuged at 5,000 x g for 30 minutes. The virus retained on the filter was resuspended in an appropriate volume of virus growth medium and stored at − 80°C. Dissociated organoid cells were inoculated with WSN/33 (H1N1) at a multiplicity of infection (MOI) of 1 and incubated at 37℃ for 2 hours. Following incubation, the cells were centrifuged at 1,200 rpm for 5 minutes, after which the virus-containing supernatant was carefully removed and the cell pellet was washed once with PBS. The cells were subsequently centrifuged again at 1,200 rpm for 5 minutes and the supernatant was discarded. The resulting cell pellet was resuspended in cold Matrigel and reseeded. Viral titer determination Viral titers were determined using a plaque assay. Virus samples were serially diluted with PBS containing 1% FBS. Confluent monolayers of MDCK cells in 6-well plates were washed twice with PBS, then infected in duplicate with 250 µL/well dilution of each virus. The plates were incubated at room temperature for 2 hours to facilitate virus adsorption. Following adsorption, a 1.5% agarose overlay in complete MEM supplemented with TPCK trypsin (2 µg/mL) and penicillin/streptomycin was applied. The plates were then incubated at 37°C, and cells were fixed with 10% formalin at 2 days post-infection (dpi). Western blot analysis The IAV NP protein levels were assessed using western blot analysis. IAV NP antibody was purchased from BIOSYNTH (Staad, Switzerland, Cat #10-1097). The NHNE cells were lysed with 5X lysis buffer (125 mM Tris, 10% SDS, 10% β-mercaptoethanol, 0.25% bromophenol blue, and 50% glycerol). Cell lysate (15 µg of protein) was electrophoresed in 10% SDS gels and transferred to polyvinylidene difluoride membranes in Tris-buffered saline (TBS) (50 mM Tris-Cl, pH of 7.5, 150 mM of NaCl) for one hour at 4℃. The membrane was incubated overnight with primary antibody (1:1000) in 2% BSA. After washing with Tween-TBS, the blot was incubated for one hour at room temperature with secondary anti-mouse antibody (1:5,000, Santa Cruz Biotechnology, Dallas, TX, USA) in TTBS and was visualized using an ECL system (Amersham, Little Chalfont, England). Single cell RNA sequencing (scRNA-seq) Library construction was performed using 10× chromium single-cell 3’ version 3.1 reagent kits (10× Genomics, Pleasanton, CA, USA). Samples were sequenced using the NovaSeq 6000 platform (Illumina, San Diego, CA, USA), and preliminary sequencing results were converted to FASTQ files using the Cell Ranger pipeline (10× Genomics). We followed the 10× Genomics standard sequence protocol by trimming the barcode and unique molecular identifier end to 26 bp and the mRNA end to 98 bp, respectively. Then, the FASTQ files were aligned to the human reference genome (GRCh38). Subsequently, we applied Cell Ranger for preliminary data analysis and generated a file that contained a barcode table, a gene table, and a gene-expression matrix. Downstream analyses were performed in R (version 4.5.2) using Seurat. Cells with 10% were removed. Data were normalized using Seurat’s LogNormalize method (scale factor = 10,000) followed by log-transformation and identification of highly variable genes (2000 features) using Seurat’s vst method. Data were scaled and regressed for percent mitochondrial reads. Principal component analysis was performed on the variable genes. Lineage relationships were inferred using Slingshot with root cluster (cycling basal cell) specified as the starting state. The data are available under accession number GSE324883 and can be accessed using the reviewer token: edkdwmasvxwhbsv. Statistical analyses For in vitro study, at least three independent experiments were performed with cultured cells from each donor, and the results are presented as mean ± standard deviation (SD) values of triplicate cultures. Differences between treatment groups were evaluated by repeated measure two-way analysis of variance (ANOVA) and two sample t-test was used to analyze scRNA-seq data. GraphPad Prism (version 10; GraphPad Software, La Jolla, CA, USA) were used for these statistical analyses and differences were considered significant at p-value < 0.05. Results Establishment of Matrigel-based human NEC organoids The dissociated epithelial cells from human NM tissue were embedded in Matrigel and submerged in organoid culture medium to establish primary NEC organoids (0.5 x 105 cells/each Matrigel, Fig. 1 a). Microscopically, organoid formation was observed at passage (P) 0 and organoids were successfully expanded to P1,P3, and P5 with highly differentiated NECs, forming hollow spheres (Fig. 1 b). Then, we compared the gene expression of representative epithelial cell markers, including basal, secretory, and ciliated cells, in NEC organoid, human NM, and fully differentiated NHNE cells. We measured the mRNA levels of KRT5, TP63, MUC5AC, and FOXJ1 in P1 NEC organoid, human NM tissue, and P1 NHNE cells to quantify each component of epithelial cells. The results revealed that mRNA levels of both KRT5 and TP63 were highly elevated in these in vitro and ex vivo samples (Fig. 1 c). Although mRNA levels were relatively lower, MUC5AC and FOXJ1 gene expressions were also detected in three samples. Interestingly, the expression patterns of genes in each epithelial cell were similar among NEC organoids, cultured NHNE cells, and nasal mucosa. To further assess the morphologic characteristics of NEC organoids, we then carried out histologic examination with optical microscopy and immunofluorescence staining using P1 organoids obtained from two donors. Both H&E and PAS staining results showed that NEC organoids in donor 1 and donor 2 displayed analogous histology at the microscale to the corresponding nasal epithelium, mirroring its epithelial organization. The organoids grew in the expansion medium and initiated differentiation with a 3-day incubation with a basal medium to enlarge the central lumen (Fig. 1 d). Optical microscopic findings showed that Matrigel-embedded NECs shaped cystic organoids on day 3 and enlarged gradually with overlaid organoid expansion (Fig. 1 e). To gain further insight into the cellular composition and ensure the phenotypic consistency of the organoids, we employed immunofluorescence imaging. Keratin8, Keratin5, and P63 were used to identify basal cells, alpha-tubulin was used to identify ciliated cells, and MUC5AC was used to identify goblet cells (Fig. 1 f). This imaging revealed that NEC organoids exhibited comparable distribution of these cell types, indicating the differentiation process in the organoids. Collectively, we successfully developed 3D-differentiated NEC organoids with a Matrigel-based culture system that recapitulates the structural characteristics and cellular composition of human nasal epithelium. Cellular characterization of human NEC organoids To ascertain the cellular composition of the organoids, we performed single-cell RNA sequencing (scRNA-seq) using P0-NEC organoids. This generated 6,358 high-quality epithelial cells for downstream analysis. UMAP feature plots showed broad expression of EPCAM, an epithelial marker, across clusters. PTPRC (immune), PECAM1 (endothelial), and COL1A1 (fibroblast) signals were negligible. These findings support high epithelial purity of the P0 organoid scRNA-seq dataset. UMAP embedding of the scRNA-seq data revealed seven major clusters corresponding to canonical airway epithelial populations: basal, suprabasal, cycling basal, non-mucin secretory, mucin secretory, ionocyte, and ciliated cells (Fig. 2 a). Cell identities were assigned based on canonical marker genes (Fig. 2 b, 2 c) reported in previous studies. 23–27 Basal cells represented the largest fraction (3,352 cells; 52.7%), followed by non-mucin secretory cells (1,249 cells; 19.6%), cycling basal cells (628 cells; 9.9%), mucin secretory cells (529 cells; 8.3%), suprabasal cells (437 cells; 6.9%), ionocytes (88 cells; 1.4%), and ciliated cells (75 cells; 1.2%). Basal and suprabasal lineages were easily distinguished by cytokeratin and transcription factor expression (Fig. 2 b, 2 c). Basal and cycling basal clusters showed strong expression of KRT5, KRT15, and TP63, consistent with an undifferentiated progenitor phenotype, whereas cycling basal cells were further defined by robust expressions of proliferation-associated markers, including MKI67 and TOP2A. The suprabasal cluster shared basal keratin expression but was distinguished by high levels of KRT4 with additional expression of KRT13 and loss of TP63 expression. Expression analyses showed that suprabasal cells exhibited low but detectable levels of selected secretory markers such as BPIFB1, BPIFA1, MUC5AC, and MUC5B, suggesting that this population represents an intermediate differentiation state transitioning from basal cells toward mature secretory fates. Secretory lineages formed two major clusters that were annotated as non-mucin secretory and mucin secretory cells. Non-mucin secretory cells predominantly expressed SCGB1A1, BPIFB1, and BPIFA1, with minimal or absent expression of gel-forming mucin genes such as MUC5AC and MUC5B, consistent with a serous/antimicrobial secretory phenotype. In contrast, mucin secretory cells were characterized by high expression of MUC5AC and MUC5B together with secretory markers including SCGB1A1, BPIFB1, and BPIFA1, consistent with a mucin-producing goblet-like identity (Fig. 2 b, 2 c). Along the principal trajectory, cells were ordered from cycling basal and basal populations to suprabasal cells, followed by mucin secretory and non-mucin secretory populations, and ultimately ciliated cells, consistent with a basal-to-secretory/ciliated differentiation hierarchy within the organoid (Fig. 2 d). Higher resolution subclustering of the mucin secretory compartment resolved two transcriptionally distinct subpopulations corresponding to club and goblet cells (Fig. 3 A). Club cells showed high and selective expressions of SCGB1A1, BPIFB1, BPIFA1, and MSMB, consistent with a non-gel-forming, serous/antimicrobial secretory phenotype that may be poised to undergo goblet metaplasia (Fig. 3 b, 3 c). In contrast, goblet cells exhibited strong expression of the gel-forming mucins MUC5AC and MUC5B, together with additional goblet-associated markers such as TFF3 and SPDEF (Fig. 3 b, 3 c). Further subclustering of the ciliated compartment also resolved two discrete populations corresponding to multiciliated and deuterosomal cells (Fig. 3 d). Multiciliated cells, representing terminally differentiated ciliated epithelium, expressed high levels of core ciliogenesis and axoneme-associated genes, including FOXJ1, TPPP3, PIFO, LRRC23, IFT57, CDC20B, and CCNO (Fig. 3 e- 3 g). Deuterosomal cells, identified as precursors to multiciliated cells, exhibited strong expression of DEUP1 (Fig. 3 e- 3 g). Taken together, the human NEC organoids captured major airway epithelial lineages. Basal cells were the predominant population, with abundant secretory cells (club and goblet) and evidence of ciliated differentiation, as indicated by deuterosomal and multiciliated cell states. Consecutive passages of NEC organoids and differentiation to NHNE cells Our previous study on ALI culture of NHNE cells showed that nasal epithelia cells of P2 or higher showed abnormal differentiation and proliferation at the subculture step, making them unsuitable for in vitro experiments. 9 To determine whether NEC organoids could overcome these limitations, we established organoids for three different donors and consecutively passaged them every 14 days up to P5 over 80 days. We measured the mRNA levels of KRT5, TP63, MUC5AC, and FOXJ1 in P1, P3, and P5 NEC organoids to examine the expression of compartment-specific genes in nasal epithelium after consecutive passages. Interestingly, the real-time PCR results showed that the expression patterns of the four genes KRT5, TP63, MUC5AC, and FOXJ1 were similar in P1, P3, and P5 organoids (Fig. 4 a). In the optical microscopy findings, Matrigel-embedded NEC organoids from P1 to 5 showed a stable cystic shape, and the growth rate did not decrease until P5, indicating maintenance of proliferation activity (Fig. 4 b). In addition, organoids displayed analogous histologic findings in H&E and PAS staining up to P5 (Fig. 4 c). We then propagated NEC-derived P3 and P5 organoid cells in the culture medium for ALI NHNE cultures, which were grown for 14 days in medium containing retinoic acid to induce mucociliary differentiation (Fig. 5 a). Light microscopy findings showed optimal cell growth and proliferation in the organoids (P3 and P5) (Fig. 5 b). Then, P3 and P5 organoid cells were seeded at a mean density of 1 x 10 5 cells/well on Transwell clear culture inserts, and ALI culture was created after 14 days confluence. Confluent NHNE cells were small, polygonal, and tightly joined; they had the typical cobblestone morphology of epithelial cells. SEM of apical cells in ALI culture at 14 days after confluence showed polygonal cells tightly attached to each other, with well-differentiated cilia and mucus secretion. TEM also showed ciliated and secretory cells that were well differentiated, with intact tight junctions (Fig. 5 b). Histological findings, including H&E and PAS, showed that ALI cultures at 14 days after confluence were four to five layers in thickness. The cells in the ALI cultures were well-differentiated, stratified, columnar epithelial cells and included secretory and ciliated cells (Fig. 5 c). In addition, the real-time PCR results showed that the expression patterns of the four genes KRT5, TP63, MUC5AC, and FOXJ1 were similar in ALI NHNE cells from P3 and P5 NEC organoids (Fig. 5 c). The current findings demonstrate that NEC organoids might be a useful in vitro tool for fully differentiated NHNE cell culture with stable differentiation and proliferation, even in consecutive passages. Infection of NEC organoids with influenza A virus and innate immune signatures To further elucidate the innate immune response to an external pathogen, P3 NEC organoids were subjected to infection with influenza A virus (IAV) (Fig. 6 a). Organoids were infected with WSN/33 (H1N1) at a multiplicity of infection (MOI) of 1 to assess susceptibility to IAV. Cell lysates and supernatant were harvested 1, 2, and 3 days post-infection (dpi). Real-time PCR results revealed that IAV polymerase acidic protein (PA) mRNAs in the organoid cell lysate were significantly higher until 3 dpi following IAV infection with the highest titer observed at 1 dpi (mRNA level: 7.9x107) (Fig. 6 b). Subsequently, levels of IAV nucleoprotein (NP) were measured through western blot analysis, and IAV NP expression was considerably increased in IAV-infected organoids from 1 dpi (Fig. 6 c) in concert with induction of viral titer (Fig. 6 d). Finally, we evaluated the distinct interferon (IFN)-related immune and inflammatory responses elicited by temporal effects following IAV infection. NEC organoids from 5 healthy subjects were inoculated with WSN/33 (H1N1) at MOI 1 and the mRNA levels of IFN-α, IFN-β, IFN-λs, and IFN-γ were measured at 0, 1, 2, and 3 dpi by real-time PCR. The result revealed that the mRNA levels of IFN-α, IFN-β, IFN-λ 1 , IFN-λ 2/3 , and IFN-λ 4 were significantly elevated at 1 dpi compared with mRNA levels of 0 dpi and gradually decreased until 3 dpi. By contrast, mRNA levels of IFN-γ were not induced after IAV infection in organoids (Fig. 6 e). As a next step, the transcription levels of representative IFN-stimulated genes related to antiviral innate immune responses in nasal mucosa were measured after IAV infection. The mRNA levels of CXCL10, OAS1, IFIT1, and Mx1 were highly elevated in IAV-infected organoids at 1 dpi and then gradually decreased until 3 dpi (Fig. 6 f). To evaluate the induction of the acute inflammatory responses and the activation of inflammatory cytokines, we quantified the expression levels of TNF-α, IL-1β, and IL-8 using real-time PCR. The results also showed that the highest transcription of inflammatory cytokines was observed at 1 dpi following IAV infection (Fig. 6 g). The same experimental results were obtained when P5 NEC organoids were infected with IAV (MOI 1.0) (Supplementary Fig. 1a-1f). These results demonstrate that NEC organoids are suitable as a model for influenza virus infection and are particularly useful for observing IFN-related innate immune responses activated in nasal epithelium. Discussion ໿In this study, we successfully established human NEC organoids using human nasal mucosa extracted from healthy volunteers. This in vitro system is stable for subsequent passages and long-term expansion of NECs, even with a limited amount of human nasal mucosa. In particular, we proved that NEC organoids are not only useful for culturing fully differentiated NHNE cells but also helpful in unraveling the epithelium-derived innate immune responses following influenza virus infection. While ALI culture systems have long been regarded as the gold standard for recapitulating the characteristics of human nasal epithelium, NEC organoids offer distinct advantages in terms of cellular stability across multiple passages and versatility for both organoid-based studies and ALI culture applications. The mucosal epithelium lining the upper airway from the nose to the nasopharynx is the entry portal and primary target of respiratory pathogens including influenza virus, thus nasal epithelial cells express the entry factors of respiratory virus among multiple interrogated human tissue. 28,29 The susceptibility of nasal epithelium to respiratory virus, robust viral replication in these cells and subsequent viral shedding constitute the biological basis underlying viral pathogenesis and its transmissibility. 4,12,23 Indeed, the nasal epithelium not only serves as a mechanical barrier to protect the host against environmental factors, microorganisms, allergens, and toxins, but also participates in both innate and adaptive immune responses. 30,31 Therefore, a better understanding of the antiviral defense mechanisms involved in suppression of respiratory viruses requires analysis of nasal epithelium obtained from human samples. 32 The majority of in vitro studies for primary NECs have been conducted using ALI culture, and ALI NEC culture has been used to study influenza virus and SARS-CoV-2 virus infection in the upper airway. 33 Based on our previous studies, the fully differentiated appearance of the nasal epithelium was observed 14 days after confluence in ALI culture and such cultures not only mimic the morphologic features of the stratified columnar epithelium, but also exhibit secretory and ciliated cell differentiation. 9,22 However, ALI cultures are unable to expand long term due to limited in vitro proliferation and cellular differentiation, which substantially restricts their application for routine airway biologic research. 9,34 The NEC organoids we established using human nasal mucosa extracted from the middle turbinate enable highly stable expansion and remarkable passages of human NECs. While primary ALI NEC cultures typically lose their differentiation capacity and original characteristics beyond P2-3, our NEC organoids maintained stable cellular composition, morphology, and gene expression patterns up to P7. This observation is consistent with recent studies demonstrating that organoid cultures can maintain genetic and phenotypic stability over extended culture periods. 35–39 The ability to passage organoids indefinitely while maintaining in vivo characteristics represents a major advancement for respiratory virus research, addressing one of the most critical limitations of primary epithelial cell cultures—the scarcity of human tissue samples and the finite lifespan of cultured cells. It is important to acknowledge that NEC organoids fully recapitulate the in vivo cellular composition of human nasal epithelium. Our scRNA-seq analysis revealed that NEC organoids displayed a higher proportion of basal progenitor cells (expressing KRT5 and TP63), while ALI cultures showed greater ciliated cell differentiation (FOXJ1-positive). This finding aligns with established understanding that ALI culture conditions, which expose epithelial cells to air on the apical surface, promote robust mucociliary differentiation and better mimic the in vivo airway environment. Interestingly, the critical advantage of NEC organoids lies in their dual functionality: the organoids not only serve as a stable, expandable cell source but can also be successfully differentiated into ALI cultures. Our results demonstrated that P3 and P5 NEC organoid-derived cells could be differentiated into NHNE cells with proper mucociliary differentiation, comparable to those derived from freshly isolated primary cells. This approach essentially overcomes the "passage limitation" that has long hindered repetitive experiments with ALI NEC culture. By establishing a bank of cryopreserved NEC at early passages, researchers can generate multiple batches of ALI cultures with consistent characteristics, eliminating the need for repeated tissue biopsies from donors. This is particularly valuable for longitudinal studies and high-throughput screening applications. It has also demonstrated the feasibility of converting ALI-differentiated nasal epithelia back into organoids for functional assays, suggesting a bidirectional relationship between these culture systems that can be leveraged for various experimental designs. The applicability of NEC organoids is well presented in our in vitro study of IAV infection with subsequent antiviral immune responses. The current findings demonstrated that the cells in NEC organoids are susceptible to influenza virus, supporting productive viral replication as evidenced by increasing viral mRNA levels and protein expression. Importantly, the infected NEC organoid mounted robust innate immune responses characterized by rapid induction of type I and III IFNs (IFN-α, IFN-β, IFN-λ 1 , IFN-λ 2/3 , IFN-λ 4 ), along with IFN-stimulated genes (CXCL10, OAS1, IFIT1, Mx1) and pro-inflammatory cytokines (TNF-α, IL-1β, IL-8). The temporal pattern of these responses closely mirrors the innate immune kinetics observed in human airway epithelium and in ALI epithelial cultures infected with diverse influenza strains. 39–41 Previously, the ALI transwell (bronchial epithelial cell) and airway organoid models from lung tissue, in general, provide comparable outcomes regarding virus infection kinetics and host response to infection, with subtle differences in the cell type composition and tissue differentiation status. 31 The fact that our NEC organoid model recapitulates these cell-intrinsic antiviral mechanisms supports its validity for studying host-pathogen interactions in the nasal epithelium. Nevertheless, our study has several limitations that should be acknowledged. The altered cellular composition in NEC organoids compared to native nasal epithelium, particularly the predominance of basal cells over differentiated ciliated cells, may affect certain aspects of viral pathogenesis and immune responses that depend on specific cell types. Additionally, while our NEC organoids capture epithelial cell responses, they lack the stromal, immune, and vascular components present in vivo, which contribute to the complete picture of mucosal immunity. Future iterations of the model could incorporate co-cultures with immune cells or utilize more complex tissue engineering approaches to better represent the multicellular nasal microenvironment. In summary, our study demonstrates that NEC organoids represent a valuable and versatile platform as an alternative experimental model for ALI culture, influenza virus infections, and innate immune responses in nasal epithelium. While ALI culture systems remain superior for achieving complete mucociliary differentiation that fully recapitulates native nasal epithelium, NEC organoids offer critical advantages including long-term passage stability, expandability, and applicability to ALI culture differentiation. The dual functionality of NEC organoids might act as an expandable cell source and an innovative tool that complements existing methodologies for virus and immunology research in the upper airway. Future studies should focus on integrating immune and stromal components into the NEC organoid to better recapitulate the in vivo nasal microenvironment. Declarations Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. Consent for publication Not applicable Author contribution Conceptualization: S.J., S.K., M.K. Methodology: S.L., M.L., H.C. Investigation: H.C., S.C., H.J.K. Supervision: S.C., H.J.K. Writing: S.J., M.K., H.J.K. Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education (2022R1A2C2011867) awarded to H.J.K and by the Ministry of Science and ICT (RS 2023-00222762), Korea awarded to H.J.K. as well as (RS-2022-NR067280) awarded to S.C and H.J.K References Gallo, O., Locatello, LG., Mazzoni, A., Novelli, L. & Annunziato, F. The central role of the nasal microenvironment in the transmission, modulation, and clinical progression of SARS-CoV-2 infection. Mucosal Immunol . 14 , 305-316 (2021). Cha, H. et al . Innate immune signatures in the nasopharynx after SARS-CoV-2 infection and links with the clinical outcome of COVID-19 in Omicron-dominant period. Cell Mol Life Sci . 81 , 364 (2024). Kim, HJ. et al. Nasal commensal Staphylococcus epidermidis enhances interferon-λ-dependent immunity against influenza virus. Microbiome . 7 , 80 (2019). Jo, A. et al . Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium. NPJ Biofilms Microbiomes . 8 , 26 (2022). Kim, G. et al . A Moonlighting Protein Secreted by a Nasal Microbiome Fortifies the Innate Host Defense Against Bacterial and Viral Infections. Immune Netw . https://doi.org/10.4110/in.2023.23.e31. (2023) Sojati, J., Parks, OB., Eddens, T., Lan, J., Johnson, M. & Williams, JV. Limited nasal IFN production contributes to delayed respiratory virus clearance and suboptimal vaccine responses. JCI Insight . https://doi.org/10.1172/jci.insight.182836. (2025) Han, S. et al. Intranasal nanophotosensitizer enables safe and broad-spectrum photodynamic inactivation of respiratory viruses. J Photochem Photobiol B . 272 ,113261 (2025). Ji, JY. et al. 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Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures. Development . 146 , dev177428 (2019). Ahn, JH. et al . Nasal ciliated cells are primary targets for SARS-CoV-2 replication in the early stage of COVID-19. J Clin Invest . https://doi.org/10.1172/JCI148517. (2021) Plasschaert, LW. et al . A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature . 560 , 377-381 (2018). Stölting, H. et al . Distinct airway epithelial immune responses after infection with SARS-CoV-2 compared to H1N1. Mucosal Immunol . 15 , 952-963 (2022). Otter, CJ., Renner, DM., Fausto, A., Tan, LH., Cohen, NA. & Weiss, SR. Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance. Proc Natl Acad Sci U S A . https://doi.org/10.1073/pnas.2402540121. (2024) Jeon, YJ., Gil, CH., Won, J., Jo, A. & Kim, HJ. Symbiotic microbiome Staphylococcus aureus from human nasal mucus modulates IL-33-mediated type 2 immune responses in allergic nasal mucosa. BMC Microbiol . 20 , 301 (2020). Su, A. et al . Infection Studies with Airway Organoids from Carollia perspicillata Indicate That the Respiratory Epithelium Is Not a Barrier for Interspecies Transmission of Influenza Viruses. Microbiol Spectr . https://doi.org/10.1128/spectrum.03098-22. (2023) Ziegler, CGK. et al. SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues. Cell . 181 , 1016-1035.e19 (2020). Woodall, MNJ. et al. Age-specific nasal epithelial responses to SARS-CoV-2 infection. Nat Microbiol . 9 , 1293-1311 (2024). Wu, CT. et al . SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming. Cell . 186 , 112-130.e20 (2023). Ekanger, CT. et al. Comparison of air-liquid interface transwell and airway organoid models for human respiratory virus infection studies. Front Immunol . 16 ,1532144 (2025). Shao, W., Xu, H., Zeng, K., Ye, M., Pei, R. & Wang, K. Advances in liver organoids: replicating hepatic complexity for toxicity assessment and disease modeling. Stem Cell Res Ther . 16 , 27 (2025). Thorel, L. et al . Patient-derived tumor organoids: a new avenue for preclinical research and precision medicine in oncology. Exp Mol Med . 56 , 1531-1551 (2024). Georgakopoulos, N. et al. Long-term expansion, genomic stability and in vivo safety of adult human pancreas organoids. BMC Dev Biol . 20 , 4 (2020). Van de Wetering, M. et al . Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell . 161 , 933-945 (2015). Major, J. et al. Type I and III interferons disrupt lung epithelial repair during recovery from viral infection. Science . 369 , 712-717 (2020). Broggi, A. et al . Type III interferons disrupt the lung epithelial barrier upon viral recognition. Science . 369 , 706-712 (2020). Thyrsted, J. et al . Influenza A induces lactate formation to inhibit type I IFN in primary human airway epithelium. iScience . 24 , 103300 (2021). Additional Declarations There is no conflict of interest Supplementary Files Supplementaryfigure1.docx Supplementary fig.1 Supplemental information The online version contains supplementary figures available. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9241349","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":619650312,"identity":"2b9fee0d-415b-4df0-b820-11104cec2254","order_by":0,"name":"Hyun Jik Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACAxAhASLYG6BCPERr4TlAihYwkEggUou5RI7ZA4uKe/kGN58/e8zDYCfPwHP2AV4tljNyzA0kzhRbbridY27Mw5Bs2MDbboBXi8GNHDMJybYEA4PbOWzSPAzMCQz8bAT8AtbyD6jl5vFnQC31xGppAGq5wWAG1HI4gYG3jYCWM8/KJCSOJRhInskxk5xjcNywjecYAS3Hk7dJS9QkGPAdP/5M4k1FtTw/Txp+LQwCCQzMoKhUOAA2gYGBgE+AgP8AA+MHIC3fQFDpKBgFo2AUjFQAANj8OyHrUqTIAAAAAElFTkSuQmCC","orcid":"","institution":"Seoul national university college of medicine","correspondingAuthor":true,"prefix":"","firstName":"Hyun","middleName":"Jik","lastName":"Kim","suffix":""},{"id":619650313,"identity":"1192d904-5363-40c6-8c19-7ab2d0e414f3","order_by":1,"name":"Siyeon Jin","email":"","orcid":"","institution":"Seoul national university college of medicine","correspondingAuthor":false,"prefix":"","firstName":"Siyeon","middleName":"","lastName":"Jin","suffix":""},{"id":619650314,"identity":"6453abc9-5709-42e4-89a7-efd7d4a23d18","order_by":2,"name":"Sujin Kim","email":"","orcid":"","institution":"Seoul national university college of medicine","correspondingAuthor":false,"prefix":"","firstName":"Sujin","middleName":"","lastName":"Kim","suffix":""},{"id":619650315,"identity":"7d4daf11-e6ca-4f9e-8be3-a84fc016e44a","order_by":3,"name":"Minseop Kim","email":"","orcid":"https://orcid.org/0000-0002-4794-272X","institution":"KU-KIST Graduate School of Converging Science and Technology, Korea University","correspondingAuthor":false,"prefix":"","firstName":"Minseop","middleName":"","lastName":"Kim","suffix":""},{"id":619650316,"identity":"0b19be84-61b3-4b52-bb0c-13f5dfd0a64e","order_by":4,"name":"Hyunkyung Cha","email":"","orcid":"","institution":"Soonchunhyang University","correspondingAuthor":false,"prefix":"","firstName":"Hyunkyung","middleName":"","lastName":"Cha","suffix":""},{"id":619650317,"identity":"2f209454-08bc-4d3c-aae8-1d36f2e126a2","order_by":5,"name":"Seok Chung","email":"","orcid":"https://orcid.org/0000-0002-1735-8338","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Seok","middleName":"","lastName":"Chung","suffix":""}],"badges":[],"createdAt":"2026-03-27 07:15:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9241349/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9241349/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107104628,"identity":"4f26130b-5559-4b92-92fc-c1968d2f4886","added_by":"auto","created_at":"2026-04-16 20:25:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6184861,"visible":true,"origin":"","legend":"\u003cp\u003eEstablishment of human nasal mucosa-derived 3D NEC organoids and histological characterization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Schematic overview of the workflow. Nasal mucosa was obtained from the middle turbinate of healthy donors undergoing septoturbinoplasty (n = 5). Epithelial cells were isolated by mechanical disruption and enzymatic dissociation, embedded in Matrigel (0.5 × 10\u003csup\u003e5\u003c/sup\u003e cells per Matrigel droplet), and cultured under submerged organoid conditions to generate primary NEC organoids (P1, P3, P5) (original magnification x40). \u003cstrong\u003eb\u003c/strong\u003e Representative bright-field images. It shows organoid formation and serial expansion across passages (P0, P1, P3, and P5), with the development of hollow, cystic spheroids. \u003cstrong\u003ec\u003c/strong\u003e The mRNA levels of \u003cem\u003eKRT5\u003c/em\u003e, \u003cem\u003eTP63\u003c/em\u003e, \u003cem\u003eMUC5AC\u003c/em\u003e, and \u003cem\u003eFOXJ1\u003c/em\u003e in NEC organoid (left), in human nasal mucosa (middle), and in fully differentiated NHNE cells (right). Values are expressed as mean \u003cu\u003e+\u003c/u\u003e standard deviation (SD). \u003cstrong\u003ed\u003c/strong\u003e Representative images of H\u0026amp;E and PAS staining on 14 days after seeding. H\u0026amp;E staining (left panel) showed the structure of the human NEC organoid including ciliated and secretory cells. Well-stained secretory cells were observed in the NEC organoid through PAS staining (right panel) (scale bar: 50 uM). \u003cstrong\u003ee\u003c/strong\u003e Representative light microscopy images of the NEC organoid 14 days after seeding (original magnification x40 (left), x100 (middle), and x400 (right)). \u003cstrong\u003ef\u003c/strong\u003e Confocal microscopy images illustrate the cellular architecture and protein expression profiles within nasal epithelial organoids. F-actin (Phalloidin) delineates the structural framework. KRT8 labels the luminal epithelial cells, while KRT5 and p63 identify the basal cell population, with p63 specifically marking the nuclei. Acetylated a-tubulin and MUC5AC denote ciliated and goblet cell populations, respectively. (Scale bar = 100µm)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/ecb66ecabad1cc171c92059d.png"},{"id":107481778,"identity":"7fdf2579-e2fa-4105-8113-1a321c5bef87","added_by":"auto","created_at":"2026-04-22 02:19:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2817210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell transcriptomic mapping of airway epithelial lineages and differentiation trajectory in a single organoid. a\u003c/strong\u003e UMAP visualization and seven major populations: Basal, Suprabasal, Cycling basal, Non-mucin secretory, Mucin secretory, Ionocyte, and Ciliated cells. \u003cstrong\u003eb\u003c/strong\u003e UMAP feature plots showing expression of representative marker genes for each lineage, including basal markers (KRT5, KRT15, TP63), cycling/proliferation markers (MKI67, TOP2A, BIRC5, CDC20B), suprabasal markers (KRT4, KRT13), secretory markers (SCGB1A1, BPIFB1, BPIFA1, MSMB, MUC1, MUC5AC, MUC5B), ciliated and deuterosomal markers (FOXJ1, DNAH5, PIFO, TPPP3, SNTN, DEUP1, CDC20B, CCNO), and ionocyte markers (FOXI1, CFTR). \u003cstrong\u003ec\u003c/strong\u003e Dot plot showing the proportion of cells expressing each marker (dot size) and the scaled average expression (color intensity) within each cluster. \u003cstrong\u003ed\u003c/strong\u003e Ridge plots of Slingshot pseudotime distributions for each cluster, demonstrating a basal-to-secretory/ciliated hierarchy, with Cycling basal and Basal cells enriched at early pseudotime, followed by Suprabasal and secretory (Mucin and Non-mucin secretory) populations, and Ciliated cells predominant at late pseudotime.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/3b6bbe5fc7bf6d4ae838adff.png"},{"id":107104631,"identity":"74634b43-cf5f-4f5c-80f6-b104d16bc405","added_by":"auto","created_at":"2026-04-16 20:25:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2628519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubclustering of the mucin secretory compartment (identifies Goblet and Club cell) and ciliated compartment (identifies Multiciliated and Deuterosomal epithelial cells). a\u003c/strong\u003e UMAP embedding of the secretory cell subset showing two transcriptionally distinct clusters annotated as Goblet (red) and Club (turquoise) cells. \u003cstrong\u003eb\u003c/strong\u003e UMAP feature plots of representative secretory and goblet/club-associated markers, including SCGB1A1, BPIFB1, BPIFA1, MUC1, SPDEF, MUC5AC, MUC5B, TFF3, MSMB, and VMO1, illustrating their spatial expression across the two clusters. \u003cstrong\u003ec\u003c/strong\u003e Violin plots comparing expression levels of the same marker genes between Goblet and Club cells, highlighting enrichment of SCGB1A1, BPIFB1, BPIFA1, MSMB, and VMO1 in Club cells and of SPDEF, MUC5AC, MUC5B, and TFF3 in Goblet cells. \u003cstrong\u003ed\u003c/strong\u003e UMAP embedding of the ciliated cell subset showing two transcriptionally distinct clusters annotated as Deuterosomal (red) and Multiciliated (turquoise) cells. \u003cstrong\u003ee\u003c/strong\u003e UMAP feature plots of key ciliogenesis and deuterosome-associated genes, including FOXJ1, TPPP3, PIFO, LRRC23, IFT57, DEUP1, CDC20B, and CCNO, illustrating their spatial expression across the two clusters. \u003cstrong\u003ef\u003c/strong\u003e Violin plots comparing expression levels of the same markers between Deuterosomal and Multiciliated cells. \u003cstrong\u003eg\u003c/strong\u003e Dot plot summarizing, for each cluster, the proportion of cells expressing each marker (dot size) and the scaled average expression (color intensity).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/a23213a755f09d911c1fe894.png"},{"id":107481092,"identity":"87e6bcd1-793d-4bfa-b617-1f7a806ae2f6","added_by":"auto","created_at":"2026-04-22 02:15:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6266039,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubsequent passages of NEC organoids.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e The mRNA levels of \u003cem\u003eKRT5\u003c/em\u003e, \u003cem\u003eTP63\u003c/em\u003e, \u003cem\u003eMUC5AC\u003c/em\u003e, and \u003cem\u003eFOXJ1\u003c/em\u003ein NEC organoids of passage 1 (P1), passage 3 (P3), and passage 5 (P5). Values are expressed as mean \u003cu\u003e+\u003c/u\u003e standard deviation (SD). \u003cstrong\u003eb\u003c/strong\u003e Representative light microscopy images (left) and growth rate (right) of P1, P3, and P5 NEC organoid (Upper panel: scale bar, 100 uM and lower panel: scale bar, 50uM) \u003cstrong\u003ec\u003c/strong\u003eRepresentative images of H\u0026amp;E and PAS staining of P1, P3, and P5 NEC organoid (scale bar: 50 uM).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/28bb88030d8637279a4dc875.png"},{"id":107104634,"identity":"874ddc5c-519f-40c9-8ad3-287ebd89702e","added_by":"auto","created_at":"2026-04-16 20:25:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3093061,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentiation of NHNE cells in ALI culture from P3 and P5 human NEC organoids.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003eA schematic figure outlines the ALI culture of NHNE cells from P3 and P5 NEC organoids. \u003cstrong\u003eb\u003c/strong\u003e Differentiation of NHNE cells in ALI culture from human NEC organoids. Representative light microscopy images of an NEC organoid. Scanning electron micrograph (SEM) and transmission electron micrograph (TEM) findings of fully differentiated NHNE cells cultured from human NEC organoids. SEM findings of cultured NHNE cells showing well-differentiated ciliated and secretory cells (left, original magnification x500). TEM findings of a ciliated cell at the apical surface of the epithelium and a columnar secretory cell containing electron-lucent secretory granules (C, original magnification x5000). \u003cstrong\u003ec\u003c/strong\u003e Primary nasal epithelial cells growing on cross-section (5 um) of NHNE cells, at 14 days after confluence, stained with H\u0026amp;E from P3 (left panel) and P5 (right panel) (scale bar: 50 uM). \u003cstrong\u003ed \u003c/strong\u003eThe mRNA levels of \u003cem\u003eKRT5\u003c/em\u003e, \u003cem\u003eTP63\u003c/em\u003e, \u003cem\u003eMUC5AC\u003c/em\u003e, and \u003cem\u003eFOXJ1\u003c/em\u003e in NHNE cells cultured from P3 (left panel) and P5 (right panel) NEC organoids. Values are expressed as mean \u003cu\u003e+\u003c/u\u003estandard deviation (SD).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/07bb54c50f143c183cf8f66e.png"},{"id":107104633,"identity":"009f0e11-76e2-4897-ba6b-0d98c4bcb247","added_by":"auto","created_at":"2026-04-16 20:25:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1628979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInnate immune responses in NEC organoids following IAV infection.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e The schematic figure of IAV infection (MOI 1.0) to NEC organoid (1 x10\u003csup\u003e6\u003c/sup\u003e cells / Matrigel). P3 NEC organoids were inoculated with WSN/33 (H1N1) for 0 (no infection), 1, 2, and 3 days at an MOI of 1. \u003cstrong\u003eb\u003c/strong\u003e Real-time PCR showed that the IAV mRNA level was higher in NEC organoids from 1 day after infection, and the biggest difference in mRNA level was observed at 1 dpi. \u003cstrong\u003ec\u003c/strong\u003e Western blot analysis revealed that the level of IAV nucleoprotein (NP) was increased from 1 dpi in NEC organoid after IAV infection. \u003cstrong\u003ed\u003c/strong\u003e A plaque assay also showed that the viral titer in the supernatant of IAV-infected NEC organoid was significantly higher from 1 dpi. Real-time PCR showed that\u003cstrong\u003e e\u003c/strong\u003e the IFN mRNA levels, \u003cstrong\u003ef\u003c/strong\u003e representative IFN-stimulated genes, and \u003cstrong\u003eg\u003c/strong\u003e inflammatory cytokines were increased in NEC organoids from 1 day after infection excepting IFN-Υ, and the biggest difference in mRNA level was observed at 1 dpi. Results are presented here as the mean ± standard deviation (SD) from 3 independent experiments (*\u003csup\u003e \u003c/sup\u003ep\u0026lt;0.05 comparing the levels with no infection). (PI: post of infection)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/812997147a98a3314cc1dd2b.png"},{"id":108183193,"identity":"4c7fc662-4d4d-4ab0-b48d-1d2cdd63d512","added_by":"auto","created_at":"2026-04-30 08:59:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20939056,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/a6bd2923-53f2-477c-a23b-32246aad1432.pdf"},{"id":107481161,"identity":"74520155-dddc-48d7-9146-6d51633da11a","added_by":"auto","created_at":"2026-04-22 02:16:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":242807,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary fig.1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary figures available.\u003c/p\u003e","description":"","filename":"Supplementaryfigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9241349/v1/c93441dbceaa95a3bc084a33.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Human nasal epithelial cell organoids as a platform for subsequent passage stability, ALI culture differentiation and influenza virus infection modeling","fulltext":[{"header":"Introduction","content":"\u003cp\u003e໿ The mucosal surfaces of the human respiratory tract are in direct contact with the external environment, making them susceptible to invasion of respiratory viruses.\u003csup\u003e1,2\u003c/sup\u003e Studies on the clearance reaction of the host\u0026rsquo;s airway mucosa increasingly take into consideration the contribution of immune responses and specific protection against infection from external pathogens to integrate environmental signals.\u003csup\u003e3\u0026ndash;5\u003c/sup\u003e Inhaled viruses encounter the host immune system in the respiratory mucosa, and mounting evidence indicates that the nasal epithelium is the primary site where respiratory viruses like SARS-CoV-2 and influenza begin to replicate.\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e Nasal epithelial cells (NECs) not only provide a physical barrier to potentially harmful insults, but they also play a crucial role in the first line of immunological defense.\u003csup\u003e9,10\u003c/sup\u003e Cultured human NECs are good research tools for studying respiratory epithelial physiology and have been instrumental in increasing our knowledge of antiviral immune responses in the upper airway. To date, many kinds of epithelial cell culture methods, such as submerged, suspension, floating, and air-liquid interface (ALI), have been introduced and widely utilized for primary human NEC culture.\u003csup\u003e9,11,12\u003c/sup\u003e ALI cultures of NECs have been successfully obtained from both nasal biopsies and nasal brushings and it is of great interest whether NECs are an ideal surrogate.\u003csup\u003e3,4,9\u003c/sup\u003e However, primary NEC cultures take a long time to establish fully differentiated epithelium, and the cell condition deteriorates with each passage, limiting their use in repetitive experiments. Furthermore, primary NEC cultures via the ALI system proliferate and differentiate in vitro for a finite number of passages before gradually losing their original characteristics.\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e The scarcity of sample obtained from a patient\u0026rsquo;s nasal tissue is a significant limitation to in vitro studies using these culture techniques.\u003c/p\u003e \u003cp\u003eThree-dimensional (3D) organoids grown from stem cells or patient biopsies can self-assemble over long periods (months) of culture while maintaining genetic and phenotypic stability, providing an almost unlimited supply of primary cells.\u003csup\u003e16\u003c/sup\u003e Airway organoids have recently emerged as a promising experimental tool to model the human respiratory tract and have been successfully used in experimental research in respiratory biology.\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e Recent research has demonstrated the utility of human airway organoids for respiratory diseases, with applications ranging from basic scientific research to pharmaceutical development and clinical diagnosis.\u003csup\u003e19,20\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWe assumed that NEC organoids, instead of primary NEC cultures with ALI, would have many advantages, such as shortening cell culture time and increasing cell stability over multiple passages, which would be useful for diverse in vitro experiments. We established NEC organoids directly from human nasal mucosa (NM) using Matrigel. These organoids were stably expanded up to passage 5 without any cell purification procedure. We then generated an ALI NEC culture that morphologically and functionally phenocopy the native nasal epithelium through the organoids. NEC organoids have become a suitable research tool for studying influenza virus infection and interferon (IFN)-related innate immune responses. We estimate that human NEC organoids could be a good in vitro research method for applying ALI NEC culture and respiratory virus infection with stable expansion of passages and repeated experiments.\u003c/p\u003e"},{"header":"Material \u0026 Methods","content":"\n\u003ch3\u003eSample collection and processing\u003c/h3\u003e\n\u003cp\u003e This study was performed according to the Helsinki Declaration and was approved by the Institutional Review Board of Seoul National University College of Medicine, Seoul, Korea (IRB No. 2204-097-1316). Written informed consent was obtained from all participants before sample collection. The subjects were all referred to the Department of Otorhinolaryngology at Seoul National University Hospital (Seoul, Korea) between November and December 2024, primarily for nasal surgery. All subjects were free of clinical signs of rhinosinusitis and upper airway infection and had no history of other allergic diseases. Nasal cavity of each subject was accurately observed using an intranasal endoscope under general anesthesia due to nasal surgery and sampling of NM was performed around the middle turbinate by an otorhinolaryngologist (HJ Kim). The NM tissue was collected individually from 5 subjects and each NM sample was placed in an individual tube containing transfer media [Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM)/Nutrient Mixture F12 medium\u0026thinsp;+\u0026thinsp;1% Penicillin/Streptomycin]. After collection, the NM samples were incubated in 0.1% protease, prepared in transfer medium, at 4\u0026deg;C overnight. Then, the detached epithelial cell layer was collected and transferred to a new tube, followed by centrifugation at 1,200 rpm, 4\u0026deg;C for 5 min. The supernatant was aspirated, and the specimen was resuspended in washing buffer (transfer media\u0026thinsp;+\u0026thinsp;10% Fetal bovine serum). After pipetting or inverting several times, tubes were centrifuged at 1,200 rpm, 4\u0026deg;C for 5 min. After aspirating the supernatant, the specimen was resuspended in 1X RBC lysis solution. After pipetting several times, tubes were centrifuged at 1,200 rpm, 4\u0026deg;C for 5 min. After supernatant was discarded, the pellet was resuspended in culture medium and centrifuged again.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of NEC organoid\u003c/h2\u003e \u003cp\u003eNasal cell pellets were resuspended in cold Growth factor reduced Matrigel (CORNING, Corning, NY, USA), and 150 ㎕ drops of Matrigel-cell suspension were allowed to solidify on pre-warmed 12-well cell culture plates at 37℃ for 20 min. After solidification, 1 mL of airway organoid medium\u003csup\u003e16\u003c/sup\u003e was added to each well and organoid cells were incubated in 37℃/5% CO2. Medium was changed every 4 days and organoids were passaged every 2\u0026ndash;3 weeks. After Matrigel was lysed in cell recovery solution, organoids were dissociated by resuspension in 5 mL TrypLE Express (Thermo Fisher Scientific, Waltham, MA, USA), incubation for 5 min in a water bath, and pipetting. Following the addition of 5 mL airway organoid medium and centrifugation at 300 x g, dissociated cells were resuspended in cold Matrigel and reseeded.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAir-liquid interface cell culture\u003c/h3\u003e\n\u003cp\u003eNormal human nasal epithelial (NHNE) cells were cultured as described previously.\u003csup\u003e9\u003c/sup\u003e Briefly, passage-3 NECs (1 x 105 cells/culture) were seeded in 0.5 ml of culture medium on Transwell clear culture inserts (12-mm, with a 0.4-㎛ pore size; CORNING, Corning, NY, USA). Cells were cultured in a 1:1 mixture of basal epithelial growth medium and DMEM containing previously described supplements.\u003csup\u003e21\u003c/sup\u003e Cultures were grown while submerged for the first 9 days. The culture medium was changed on Day 1, and every other day thereafter. An air\u0026ndash;liquid interface (ALI) was created on Day 9 by removing the apical medium and feeding the cultures from the basal compartment only. The culture medium was changed daily after the initiation of the ALI. We added antibiotics such as 1% penicillin and streptomycin into all media for subculture and culture stages. All experiments described here used cultured nasal epithelial cells at 14 days after the creation of the ALI.\u003c/p\u003e\n\u003ch3\u003eReal-Time PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from nasal epithelial cells at 14 days after confluence, using TRIzol (QIAGEN, Venlo, Netherland). The cDNA was synthesized from 1 \u0026micro;g of RNA with random hexamer primers, using Moloney Murine Leukemia Virus reverse transcriptase (enzynomics, Daejeon, Republic of Korea). Commercial reagents (TaqMan Universal PCR Master Mix; Thermo Fisher Scientific) were selected and conditions were set according to the manufacturer\u0026rsquo;s protocol. The total reaction volume of 12 \u0026micro;L contained 2 \u0026micro;L of cDNA (reverse transcription mixture), oligonucleotide primers at a final concentration of 800 nM, and the TaqMan hybridization probe at 200 nM. The real-time PCR probe was labeled at the 5\u0026rsquo; end with carboxylfluorescein, and at the 3\u0026rsquo; end with the quencher carboxytetramethylrhodamine. Primers for human KRT5 (Hs00361185_m1), TP63 (Hs00978340_m1), MUC5AC (Hs01365616_m1), FOXJ1 (Hs00230964_m1) and influenza A virus polymerase acidic protein (PA) were purchased from Thermo Fisher Scientific. Real-time PCR was performed using the QuantStudio 3 Sequence Detection System. The thermocycler parameters included 95\u0026deg;C for 20 seconds, followed by 40 cycles of 95\u0026deg;C for 1 second and 60\u0026deg;C for 20 seconds. Target mRNA levels were quantified using target-specific primer and probe sets for KRT5, TP63, MUC5AC, FOXJ1 and glyceraldehyde 3\u0026ndash;phosphate dehydrogenase (GAPDH). All PCR assays were analyzed using the ΔΔCt method and are presented as relative expression values. All probes were designed to span an intron, and did not react with genomic DNA. All reactions were performed in triplicate, and the results were normalized against GAPDH as an endogenous control.\u003c/p\u003e\n\u003ch3\u003eElectron microscopy\u003c/h3\u003e\n\u003cp\u003eElectron microscopic analysis was performed, as previously described, with modifications.\u003csup\u003e9\u003c/sup\u003e Specimens were fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 hours at 4\u0026deg;C, washed, and then incubated with 1% osmium tetroxide in 0.1 M PB for 2 hours at 25\u0026deg;C. For scanning electron microscope (SEM), specimens were dehydrated in graded baths to 100% ethanol, critical-point-dried under liquid carbon dioxide, gold sputter-coated, and visualized on a JSM-7401F microscope (JEOL Ltd., Tokyo, Japan). For transmission electron microscope (TEM), osmium-stained samples were further fixed in 7% uranyl acetate, thin-sectioned (70 nm) in Polybed 812 (Polysciences, Warrington, PA), post-stained in uranyl acetate and lead citrate, and visualized on a JEM-1400.\u003c/p\u003e\n\u003ch3\u003eHistologic analysis and immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFor NEC organoids, nasal organoids were collected by scraping Matrigel domes from 12-well plates and centrifuged at 200 x g, 4℃ for 5 min. The pellet was resuspended in cold AO medium and incubated on a shaker at 4℃ for 10 minutes, followed by centrifugation and removal of the supernatant. This wash step was repeated to ensure complete removal of the Matrigel. Organoids were then fixed in 10% formalin at room temperature, centrifuged and resuspended in PBS before being mixed with 2% agarose. The organoid-agarose mixture was gently dispensed onto parafilm to form a dome, which, after solidification, was placed into a cassette and processed following standard tissue-sample procedures. For NHNE cells, cells were fixed by adding 10% formalin to apical and basolateral compartments at room temperature. The excised Transwell membrane was embedded in a cassette.\u003c/p\u003e \u003cp\u003eDe-waxed sections were stained with either hematoxylin and eosin (H\u0026amp;E), or a periodic acid-Schiff (PAS) kit, according to the manufacturer\u0026rsquo;s instructions (Bioptica, Milan, Italy). Immunostaining was performed on formalin-fixed paraffin sections (4 \u0026micro;m) of NEC organoid and cultured NHNE cells. Formalin-fixed paraffin sections (4 \u0026micro;m) were de-waxed in xylene (Sigma Chemicals, St Louis, MO, USA), rehydrated in successive ethanol baths, and subjected to antigen retrieval by microwave in 0.01 mol/L sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% methanolic hydrogen peroxide for 10 minutes at room temperature. Nonspecific binding was blocked by incubation with 10% normal serum from VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. Primary antibodies (1:200 dilution) were applied at 4\u0026deg;C for 24 hours. Following washing in TBS, slides were incubated with peroxidase-conjugated goat anti-mouse/rabbit antibodies (1:200 dilution, Vector Laboratories) for 30 minutes at room temperature. Signal was amplified using the indirect immunoperoxidase technique using the DAKO Envision kit (Dako, Kingsgrove, Australia). Staining was visualized using an Olympus UTV0.63XC microscope with the DP Controller software (Hamburg, Germany). The experiment was performed using NHNE cells and inferior turbinate specimens from four healthy volunteers.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence Staining and Imaging\u003c/h2\u003e \u003cp\u003eTo evaluate protein expression profiles, NEC organoids were harvested from Matrigel domes and washed with ice-cold PBS to remove residual Matrigel. The organoids were fixed with 4% paraformaldehyde (PFA; Green Bee Bio, PC2031-100-00) for 30 minutes at room temperature (RT). For cryoprotection, fixed organoids were dehydrated in a 30% sucrose solution and subsequently embedded in OCT compound (Scigen, 4586) to create cryo-blocks. The blocks were sectioned at a thickness of 10 \u0026micro;m using a cryostat. The sections were permeabilized with 0.1% Triton X-100 and blocked with 3% bovine serum albumin (BSA) in PBS to minimize non-specific binding. The sections were then incubated overnight at 4\u0026deg;C with primary antibodies against KRT8/18 (Progen, GP11), KRT5 (Invitrogen, MA5-12596), p63 (Abcam, ab124762), acetylated ɑ-tubulin (Cell Signaling, 5335), and MUC5AC (Invitrogen, MA5-12178), all at a 1:200 dilution. Following three washes with PBS, the sections were incubated for 2 hours at RT with appropriate fluorescence-conjugated secondary antibodies (1:400), along with phalloidin (1:400; Invitrogen, R415) for F-actin visualization and DAPI (1:1000; Invitrogen, D1306) for nuclear counterstaining. Immunofluorescence images were captured using a confocal laser scanning microscope (LSM 700; Carl Zeiss, Jena, Germany).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eVirus infection\u003c/h3\u003e\n\u003cp\u003eInfluenza A virus strain A/Wilson\u0026ndash;Smith/1933 H1N1 (IAV A/WS/33; American Type Culture Collection, Manassas, VA, USA) was used in this study to model acute viral infection. Virus stocks were grown in Madin\u0026ndash;Darby canine kidney (MDCK) cells in viral growth medium according to a standard procedure.\u003csup\u003e22\u003c/sup\u003e Briefly, after 48 hours of incubation at 37\u0026deg;C, the supernatants were harvested and spun by centrifugation at 1,000 x g for 5 minutes to remove cellular debris. The clarified supernatant was transferred to a centrifugal filter tube and centrifuged at 5,000 x g for 30 minutes. The virus retained on the filter was resuspended in an appropriate volume of virus growth medium and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Dissociated organoid cells were inoculated with WSN/33 (H1N1) at a multiplicity of infection (MOI) of 1 and incubated at 37℃ for 2 hours. Following incubation, the cells were centrifuged at 1,200 rpm for 5 minutes, after which the virus-containing supernatant was carefully removed and the cell pellet was washed once with PBS. The cells were subsequently centrifuged again at 1,200 rpm for 5 minutes and the supernatant was discarded. The resulting cell pellet was resuspended in cold Matrigel and reseeded.\u003c/p\u003e\n\u003ch3\u003eViral titer determination\u003c/h3\u003e\n\u003cp\u003eViral titers were determined using a plaque assay. Virus samples were serially diluted with PBS containing 1% FBS. Confluent monolayers of MDCK cells in 6-well plates were washed twice with PBS, then infected in duplicate with 250 \u0026micro;L/well dilution of each virus. The plates were incubated at room temperature for 2 hours to facilitate virus adsorption. Following adsorption, a 1.5% agarose overlay in complete MEM supplemented with TPCK trypsin (2 \u0026micro;g/mL) and penicillin/streptomycin was applied. The plates were then incubated at 37\u0026deg;C, and cells were fixed with 10% formalin at 2 days post-infection (dpi).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eThe IAV NP protein levels were assessed using western blot analysis. IAV NP antibody was purchased from BIOSYNTH (Staad, Switzerland, Cat #10-1097). The NHNE cells were lysed with 5X lysis buffer (125 mM Tris, 10% SDS, 10% β-mercaptoethanol, 0.25% bromophenol blue, and 50% glycerol). Cell lysate (15 \u0026micro;g of protein) was electrophoresed in 10% SDS gels and transferred to polyvinylidene difluoride membranes in Tris-buffered saline (TBS) (50 mM Tris-Cl, pH of 7.5, 150 mM of NaCl) for one hour at 4℃. The membrane was incubated overnight with primary antibody (1:1000) in 2% BSA. After washing with Tween-TBS, the blot was incubated for one hour at room temperature with secondary anti-mouse antibody (1:5,000, Santa Cruz Biotechnology, Dallas, TX, USA) in TTBS and was visualized using an ECL system (Amersham, Little Chalfont, England).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSingle cell RNA sequencing (scRNA-seq)\u003c/h2\u003e \u003cp\u003eLibrary construction was performed using 10\u0026times; chromium single-cell 3\u0026rsquo; version 3.1 reagent kits (10\u0026times; Genomics, Pleasanton, CA, USA). Samples were sequenced using the NovaSeq 6000 platform (Illumina, San Diego, CA, USA), and preliminary sequencing results were converted to FASTQ files using the Cell Ranger pipeline (10\u0026times; Genomics). We followed the 10\u0026times; Genomics standard sequence protocol by trimming the barcode and unique molecular identifier end to 26 bp and the mRNA end to 98 bp, respectively. Then, the FASTQ files were aligned to the human reference genome (GRCh38). Subsequently, we applied Cell Ranger for preliminary data analysis and generated a file that contained a barcode table, a gene table, and a gene-expression matrix. Downstream analyses were performed in R (version 4.5.2) using Seurat. Cells with \u0026lt;\u0026thinsp;200 detected genes or mitochondrial reads\u0026thinsp;\u0026gt;\u0026thinsp;10% were removed. Data were normalized using Seurat\u0026rsquo;s LogNormalize method (scale factor\u0026thinsp;=\u0026thinsp;10,000) followed by log-transformation and identification of highly variable genes (2000 features) using Seurat\u0026rsquo;s vst method. Data were scaled and regressed for percent mitochondrial reads. Principal component analysis was performed on the variable genes. Lineage relationships were inferred using Slingshot with root cluster (cycling basal cell) specified as the starting state. The data are available under accession number GSE324883 and can be accessed using the reviewer token: edkdwmasvxwhbsv.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eFor in vitro study, at least three independent experiments were performed with cultured cells from each donor, and the results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) values of triplicate cultures. Differences between treatment groups were evaluated by repeated measure two-way analysis of variance (ANOVA) and two sample t-test was used to analyze scRNA-seq data. GraphPad Prism (version 10; GraphPad Software, La Jolla, CA, USA) were used for these statistical analyses and differences were considered significant at p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of Matrigel-based human NEC organoids\u003c/h2\u003e \u003cp\u003eThe dissociated epithelial cells from human NM tissue were embedded in Matrigel and submerged in organoid culture medium to establish primary NEC organoids (0.5 x 105 cells/each Matrigel, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Microscopically, organoid formation was observed at passage (P) 0 and organoids were successfully expanded to P1,P3, and P5 with highly differentiated NECs, forming hollow spheres (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Then, we compared the gene expression of representative epithelial cell markers, including basal, secretory, and ciliated cells, in NEC organoid, human NM, and fully differentiated NHNE cells. We measured the mRNA levels of KRT5, TP63, MUC5AC, and FOXJ1 in P1 NEC organoid, human NM tissue, and P1 NHNE cells to quantify each component of epithelial cells. The results revealed that mRNA levels of both KRT5 and TP63 were highly elevated in these in vitro and ex vivo samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Although mRNA levels were relatively lower, MUC5AC and FOXJ1 gene expressions were also detected in three samples. Interestingly, the expression patterns of genes in each epithelial cell were similar among NEC organoids, cultured NHNE cells, and nasal mucosa.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess the morphologic characteristics of NEC organoids, we then carried out histologic examination with optical microscopy and immunofluorescence staining using P1 organoids obtained from two donors. Both H\u0026amp;E and PAS staining results showed that NEC organoids in donor 1 and donor 2 displayed analogous histology at the microscale to the corresponding nasal epithelium, mirroring its epithelial organization. The organoids grew in the expansion medium and initiated differentiation with a 3-day incubation with a basal medium to enlarge the central lumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Optical microscopic findings showed that Matrigel-embedded NECs shaped cystic organoids on day 3 and enlarged gradually with overlaid organoid expansion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). To gain further insight into the cellular composition and ensure the phenotypic consistency of the organoids, we employed immunofluorescence imaging. Keratin8, Keratin5, and P63 were used to identify basal cells, alpha-tubulin was used to identify ciliated cells, and MUC5AC was used to identify goblet cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). This imaging revealed that NEC organoids exhibited comparable distribution of these cell types, indicating the differentiation process in the organoids.\u003c/p\u003e \u003cp\u003eCollectively, we successfully developed 3D-differentiated NEC organoids with a Matrigel-based culture system that recapitulates the structural characteristics and cellular composition of human nasal epithelium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCellular characterization of human NEC organoids\u003c/h2\u003e \u003cp\u003eTo ascertain the cellular composition of the organoids, we performed single-cell RNA sequencing (scRNA-seq) using P0-NEC organoids. This generated 6,358 high-quality epithelial cells for downstream analysis. UMAP feature plots showed broad expression of EPCAM, an epithelial marker, across clusters. PTPRC (immune), PECAM1 (endothelial), and COL1A1 (fibroblast) signals were negligible. These findings support high epithelial purity of the P0 organoid scRNA-seq dataset. UMAP embedding of the scRNA-seq data revealed seven major clusters corresponding to canonical airway epithelial populations: basal, suprabasal, cycling basal, non-mucin secretory, mucin secretory, ionocyte, and ciliated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Cell identities were assigned based on canonical marker genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) reported in previous studies.\u003csup\u003e23\u0026ndash;27\u003c/sup\u003e Basal cells represented the largest fraction (3,352 cells; 52.7%), followed by non-mucin secretory cells (1,249 cells; 19.6%), cycling basal cells (628 cells; 9.9%), mucin secretory cells (529 cells; 8.3%), suprabasal cells (437 cells; 6.9%), ionocytes (88 cells; 1.4%), and ciliated cells (75 cells; 1.2%).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBasal and suprabasal lineages were easily distinguished by cytokeratin and transcription factor expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Basal and cycling basal clusters showed strong expression of KRT5, KRT15, and TP63, consistent with an undifferentiated progenitor phenotype, whereas cycling basal cells were further defined by robust expressions of proliferation-associated markers, including MKI67 and TOP2A. The suprabasal cluster shared basal keratin expression but was distinguished by high levels of KRT4 with additional expression of KRT13 and loss of TP63 expression. Expression analyses showed that suprabasal cells exhibited low but detectable levels of selected secretory markers such as BPIFB1, BPIFA1, MUC5AC, and MUC5B, suggesting that this population represents an intermediate differentiation state transitioning from basal cells toward mature secretory fates. Secretory lineages formed two major clusters that were annotated as non-mucin secretory and mucin secretory cells. Non-mucin secretory cells predominantly expressed SCGB1A1, BPIFB1, and BPIFA1, with minimal or absent expression of gel-forming mucin genes such as MUC5AC and MUC5B, consistent with a serous/antimicrobial secretory phenotype. In contrast, mucin secretory cells were characterized by high expression of MUC5AC and MUC5B together with secretory markers including SCGB1A1, BPIFB1, and BPIFA1, consistent with a mucin-producing goblet-like identity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Along the principal trajectory, cells were ordered from cycling basal and basal populations to suprabasal cells, followed by mucin secretory and non-mucin secretory populations, and ultimately ciliated cells, consistent with a basal-to-secretory/ciliated differentiation hierarchy within the organoid (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eHigher resolution subclustering of the mucin secretory compartment resolved two transcriptionally distinct subpopulations corresponding to club and goblet cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Club cells showed high and selective expressions of SCGB1A1, BPIFB1, BPIFA1, and MSMB, consistent with a non-gel-forming, serous/antimicrobial secretory phenotype that may be poised to undergo goblet metaplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In contrast, goblet cells exhibited strong expression of the gel-forming mucins MUC5AC and MUC5B, together with additional goblet-associated markers such as TFF3 and SPDEF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Further subclustering of the ciliated compartment also resolved two discrete populations corresponding to multiciliated and deuterosomal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Multiciliated cells, representing terminally differentiated ciliated epithelium, expressed high levels of core ciliogenesis and axoneme-associated genes, including FOXJ1, TPPP3, PIFO, LRRC23, IFT57, CDC20B, and CCNO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Deuterosomal cells, identified as precursors to multiciliated cells, exhibited strong expression of DEUP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, the human NEC organoids captured major airway epithelial lineages. Basal cells were the predominant population, with abundant secretory cells (club and goblet) and evidence of ciliated differentiation, as indicated by deuterosomal and multiciliated cell states.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConsecutive passages of NEC organoids and differentiation to NHNE cells\u003c/h2\u003e \u003cp\u003eOur previous study on ALI culture of NHNE cells showed that nasal epithelia cells of P2 or higher showed abnormal differentiation and proliferation at the subculture step, making them unsuitable for in vitro experiments.\u003csup\u003e9\u003c/sup\u003e To determine whether NEC organoids could overcome these limitations, we established organoids for three different donors and consecutively passaged them every 14 days up to P5 over 80 days. We measured the mRNA levels of KRT5, TP63, MUC5AC, and FOXJ1 in P1, P3, and P5 NEC organoids to examine the expression of compartment-specific genes in nasal epithelium after consecutive passages. Interestingly, the real-time PCR results showed that the expression patterns of the four genes KRT5, TP63, MUC5AC, and FOXJ1 were similar in P1, P3, and P5 organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the optical microscopy findings, Matrigel-embedded NEC organoids from P1 to 5 showed a stable cystic shape, and the growth rate did not decrease until P5, indicating maintenance of proliferation activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, organoids displayed analogous histologic findings in H\u0026amp;E and PAS staining up to P5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then propagated NEC-derived P3 and P5 organoid cells in the culture medium for ALI NHNE cultures, which were grown for 14 days in medium containing retinoic acid to induce mucociliary differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Light microscopy findings showed optimal cell growth and proliferation in the organoids (P3 and P5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Then, P3 and P5 organoid cells were seeded at a mean density of 1 x 10\u003csup\u003e5\u003c/sup\u003e cells/well on Transwell clear culture inserts, and ALI culture was created after 14 days confluence. Confluent NHNE cells were small, polygonal, and tightly joined; they had the typical cobblestone morphology of epithelial cells. SEM of apical cells in ALI culture at 14 days after confluence showed polygonal cells tightly attached to each other, with well-differentiated cilia and mucus secretion. TEM also showed ciliated and secretory cells that were well differentiated, with intact tight junctions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Histological findings, including H\u0026amp;E and PAS, showed that ALI cultures at 14 days after confluence were four to five layers in thickness. The cells in the ALI cultures were well-differentiated, stratified, columnar epithelial cells and included secretory and ciliated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In addition, the real-time PCR results showed that the expression patterns of the four genes KRT5, TP63, MUC5AC, and FOXJ1 were similar in ALI NHNE cells from P3 and P5 NEC organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe current findings demonstrate that NEC organoids might be a useful in vitro tool for fully differentiated NHNE cell culture with stable differentiation and proliferation, even in consecutive passages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eInfection of NEC organoids with influenza A virus and innate immune signatures\u003c/h2\u003e \u003cp\u003eTo further elucidate the innate immune response to an external pathogen, P3 NEC organoids were subjected to infection with influenza A virus (IAV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Organoids were infected with WSN/33 (H1N1) at a multiplicity of infection (MOI) of 1 to assess susceptibility to IAV. Cell lysates and supernatant were harvested 1, 2, and 3 days post-infection (dpi). Real-time PCR results revealed that IAV polymerase acidic protein (PA) mRNAs in the organoid cell lysate were significantly higher until 3 dpi following IAV infection with the highest titer observed at 1 dpi (mRNA level: 7.9x107) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Subsequently, levels of IAV nucleoprotein (NP) were measured through western blot analysis, and IAV NP expression was considerably increased in IAV-infected organoids from 1 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) in concert with induction of viral titer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, we evaluated the distinct interferon (IFN)-related immune and inflammatory responses elicited by temporal effects following IAV infection. NEC organoids from 5 healthy subjects were inoculated with WSN/33 (H1N1) at MOI 1 and the mRNA levels of IFN-α, IFN-β, IFN-λs, and IFN-γ were measured at 0, 1, 2, and 3 dpi by real-time PCR. The result revealed that the mRNA levels of IFN-α, IFN-β, IFN-λ\u003csub\u003e1\u003c/sub\u003e, IFN-λ\u003csub\u003e2/3\u003c/sub\u003e, and IFN-λ\u003csub\u003e4\u003c/sub\u003e were significantly elevated at 1 dpi compared with mRNA levels of 0 dpi and gradually decreased until 3 dpi. By contrast, mRNA levels of IFN-γ were not induced after IAV infection in organoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). As a next step, the transcription levels of representative IFN-stimulated genes related to antiviral innate immune responses in nasal mucosa were measured after IAV infection. The mRNA levels of CXCL10, OAS1, IFIT1, and Mx1 were highly elevated in IAV-infected organoids at 1 dpi and then gradually decreased until 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). To evaluate the induction of the acute inflammatory responses and the activation of inflammatory cytokines, we quantified the expression levels of TNF-α, IL-1β, and IL-8 using real-time PCR. The results also showed that the highest transcription of inflammatory cytokines was observed at 1 dpi following IAV infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eThe same experimental results were obtained when P5 NEC organoids were infected with IAV (MOI 1.0) (Supplementary Fig.\u0026nbsp;1a-1f). These results demonstrate that NEC organoids are suitable as a model for influenza virus infection and are particularly useful for observing IFN-related innate immune responses activated in nasal epithelium.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e໿In this study, we successfully established human NEC organoids using human nasal mucosa extracted from healthy volunteers. This in vitro system is stable for subsequent passages and long-term expansion of NECs, even with a limited amount of human nasal mucosa. In particular, we proved that NEC organoids are not only useful for culturing fully differentiated NHNE cells but also helpful in unraveling the epithelium-derived innate immune responses following influenza virus infection. While ALI culture systems have long been regarded as the gold standard for recapitulating the characteristics of human nasal epithelium, NEC organoids offer distinct advantages in terms of cellular stability across multiple passages and versatility for both organoid-based studies and ALI culture applications.\u003c/p\u003e \u003cp\u003eThe mucosal epithelium lining the upper airway from the nose to the nasopharynx is the entry portal and primary target of respiratory pathogens including influenza virus, thus nasal epithelial cells express the entry factors of respiratory virus among multiple interrogated human tissue.\u003csup\u003e28,29\u003c/sup\u003e The susceptibility of nasal epithelium to respiratory virus, robust viral replication in these cells and subsequent viral shedding constitute the biological basis underlying viral pathogenesis and its transmissibility.\u003csup\u003e4,12,23\u003c/sup\u003e Indeed, the nasal epithelium not only serves as a mechanical barrier to protect the host against environmental factors, microorganisms, allergens, and toxins, but also participates in both innate and adaptive immune responses.\u003csup\u003e30,31\u003c/sup\u003e Therefore, a better understanding of the antiviral defense mechanisms involved in suppression of respiratory viruses requires analysis of nasal epithelium obtained from human samples.\u003csup\u003e32\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe majority of in vitro studies for primary NECs have been conducted using ALI culture, and ALI NEC culture has been used to study influenza virus and SARS-CoV-2 virus infection in the upper airway.\u003csup\u003e33\u003c/sup\u003e Based on our previous studies, the fully differentiated appearance of the nasal epithelium was observed 14 days after confluence in ALI culture and such cultures not only mimic the morphologic features of the stratified columnar epithelium, but also exhibit secretory and ciliated cell differentiation.\u003csup\u003e9,22\u003c/sup\u003e However, ALI cultures are unable to expand long term due to limited in vitro proliferation and cellular differentiation, which substantially restricts their application for routine airway biologic research.\u003csup\u003e9,34\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe NEC organoids we established using human nasal mucosa extracted from the middle turbinate enable highly stable expansion and remarkable passages of human NECs. While primary ALI NEC cultures typically lose their differentiation capacity and original characteristics beyond P2-3, our NEC organoids maintained stable cellular composition, morphology, and gene expression patterns up to P7. This observation is consistent with recent studies demonstrating that organoid cultures can maintain genetic and phenotypic stability over extended culture periods.\u003csup\u003e35\u0026ndash;39\u003c/sup\u003e The ability to passage organoids indefinitely while maintaining in vivo characteristics represents a major advancement for respiratory virus research, addressing one of the most critical limitations of primary epithelial cell cultures\u0026mdash;the scarcity of human tissue samples and the finite lifespan of cultured cells. It is important to acknowledge that NEC organoids fully recapitulate the \u003cem\u003ein vivo\u003c/em\u003e cellular composition of human nasal epithelium. Our scRNA-seq analysis revealed that NEC organoids displayed a higher proportion of basal progenitor cells (expressing KRT5 and TP63), while ALI cultures showed greater ciliated cell differentiation (FOXJ1-positive). This finding aligns with established understanding that ALI culture conditions, which expose epithelial cells to air on the apical surface, promote robust mucociliary differentiation and better mimic the \u003cem\u003ein vivo\u003c/em\u003e airway environment.\u003c/p\u003e \u003cp\u003eInterestingly, the critical advantage of NEC organoids lies in their dual functionality: the organoids not only serve as a stable, expandable cell source but can also be successfully differentiated into ALI cultures. Our results demonstrated that P3 and P5 NEC organoid-derived cells could be differentiated into NHNE cells with proper mucociliary differentiation, comparable to those derived from freshly isolated primary cells. This approach essentially overcomes the \"passage limitation\" that has long hindered repetitive experiments with ALI NEC culture. By establishing a bank of cryopreserved NEC at early passages, researchers can generate multiple batches of ALI cultures with consistent characteristics, eliminating the need for repeated tissue biopsies from donors. This is particularly valuable for longitudinal studies and high-throughput screening applications. It has also demonstrated the feasibility of converting ALI-differentiated nasal epithelia back into organoids for functional assays, suggesting a bidirectional relationship between these culture systems that can be leveraged for various experimental designs.\u003c/p\u003e \u003cp\u003eThe applicability of NEC organoids is well presented in our in vitro study of IAV infection with subsequent antiviral immune responses. The current findings demonstrated that the cells in NEC organoids are susceptible to influenza virus, supporting productive viral replication as evidenced by increasing viral mRNA levels and protein expression. Importantly, the infected NEC organoid mounted robust innate immune responses characterized by rapid induction of type I and III IFNs (IFN-α, IFN-β, IFN-λ\u003csub\u003e1\u003c/sub\u003e, IFN-λ\u003csub\u003e2/3\u003c/sub\u003e, IFN-λ\u003csub\u003e4\u003c/sub\u003e), along with IFN-stimulated genes (CXCL10, OAS1, IFIT1, Mx1) and pro-inflammatory cytokines (TNF-α, IL-1β, IL-8). The temporal pattern of these responses closely mirrors the innate immune kinetics observed in human airway epithelium and in ALI epithelial cultures infected with diverse influenza strains.\u003csup\u003e39\u0026ndash;41\u003c/sup\u003e Previously, the ALI transwell (bronchial epithelial cell) and airway organoid models from lung tissue, in general, provide comparable outcomes regarding virus infection kinetics and host response to infection, with subtle differences in the cell type composition and tissue differentiation status.\u003csup\u003e31\u003c/sup\u003e The fact that our NEC organoid model recapitulates these cell-intrinsic antiviral mechanisms supports its validity for studying host-pathogen interactions in the nasal epithelium.\u003c/p\u003e \u003cp\u003eNevertheless, our study has several limitations that should be acknowledged. The altered cellular composition in NEC organoids compared to native nasal epithelium, particularly the predominance of basal cells over differentiated ciliated cells, may affect certain aspects of viral pathogenesis and immune responses that depend on specific cell types. Additionally, while our NEC organoids capture epithelial cell responses, they lack the stromal, immune, and vascular components present in vivo, which contribute to the complete picture of mucosal immunity. Future iterations of the model could incorporate co-cultures with immune cells or utilize more complex tissue engineering approaches to better represent the multicellular nasal microenvironment.\u003c/p\u003e \u003cp\u003eIn summary, our study demonstrates that NEC organoids represent a valuable and versatile platform as an alternative experimental model for ALI culture, influenza virus infections, and innate immune responses in nasal epithelium. While ALI culture systems remain superior for achieving complete mucociliary differentiation that fully recapitulates native nasal epithelium, NEC organoids offer critical advantages including long-term passage stability, expandability, and applicability to ALI culture differentiation. The dual functionality of NEC organoids might act as an expandable cell source and an innovative tool that complements existing methodologies for virus and immunology research in the upper airway. Future studies should focus on integrating immune and stromal components into the NEC organoid to better recapitulate the in vivo nasal microenvironment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAuthor contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization: S.J., S.K., M.K. Methodology: S.L., M.L., H.C. Investigation: H.C., S.C., H.J.K. Supervision: S.C., H.J.K. Writing: S.J., M.K., H.J.K.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education (2022R1A2C2011867) awarded to H.J.K and by the Ministry of Science and ICT (RS 2023-00222762), Korea awarded to H.J.K. as well as (RS-2022-NR067280) awarded to S.C and H.J.K\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGallo, O., Locatello, LG., Mazzoni, A., Novelli, L. \u0026amp; Annunziato, F. The central role of the nasal microenvironment in the transmission, modulation, and clinical progression of SARS-CoV-2 infection. \u003cem\u003eMucosal Immunol\u003c/em\u003e. \u003cstrong\u003e14\u003c/strong\u003e, 305-316 (2021).\u003c/li\u003e\n\u003cli\u003eCha, H. \u003cem\u003eet al\u003c/em\u003e. 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Influenza A induces lactate formation to inhibit type I IFN in primary human airway epithelium. \u003cem\u003eiScience\u003c/em\u003e. \u003cstrong\u003e24\u003c/strong\u003e, 103300 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Organoid, Nasal epithelium, Influenza virus, Innate immune responses","lastPublishedDoi":"10.21203/rs.3.rs-9241349/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9241349/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe cultures of human nasal epithelial cells (NECs) are good surrogate models for studying immune responses. Three-dimensional (3D) organoids have emerged as an alternative for nasal epithelium, serving as robust platforms for modeling respiratory viral infections. We aimed to generate human NEC organoids and to determine their cellular characteristics as an in vitro model for air-liquid interface (ALI) culture and influenza A virus (IAV) infection.\u003c/p\u003e \u003cp\u003eWe successfully developed human NEC organoids with using a Matrigel-based 3D culture system that recapitulates the structural characteristics and cellular composition of the human nasal epithelium. Histological results, real-time PCR, and single-cell RNA sequencing (scRNA-seq) revealed that the organoids consisted of seven different cell types and displayed a difference in the composition between the cells. The organoids displayed a much higher rate of basal progenitor cells whereas ciliated cells were less dominant. Unlike ALI cultures in which passage (P) 2 was the limit, the organoids exhibited successful and subsequent passages up to P5.\u003c/p\u003e \u003cp\u003eOur data determined that NEC organoids are an adequate in vitro model for IAV infection and showed a sharp induction of mRNA levels for interferons (IFNs) and IFN-stimulated genes following IAV infection. These results demonstrate that human NEC organoids serve as a robust in vitro model, successfully recapitulating the biological characteristics of nasal epithelium, and could be an innovative tool for exploring distinct IFN-related innate immune responses following influenza infection.\u003c/p\u003e","manuscriptTitle":"Human nasal epithelial cell organoids as a platform for subsequent passage stability, ALI culture differentiation and influenza virus infection modeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 20:25:10","doi":"10.21203/rs.3.rs-9241349/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"57f73402-4be2-43c5-9b0c-e3665647c4a0","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject after peer review","date":"2026-04-30T04:28:15+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65940571,"name":"Biological sciences/Cell biology/Cell growth"},{"id":65940572,"name":"Health sciences/Medical research/Experimental models of disease"}],"tags":[],"updatedAt":"2026-04-30T04:30:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 20:25:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9241349","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9241349","identity":"rs-9241349","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}