This study was performed according to the ethical standards of the declaration of Helsinki and following approval of the ethical committees of the university of Witten/Herdecke (No. 209/2020). Donors gave informed consent for the research use of their nasal polyp tissue. Pseudonymisation was carried out, and patient data were treated confidentially.

Nasal polyps obtained from patients with CRSwNP and undergoing endoscopic endonasal sinus surgery are the source of the tissue material. We performed a preoperative clinical endoscopic examination and a computed tomography scan of sinus. The patients reported typical symptoms of chronic rhinosinusitis, such as rhinorrhea, cephalgia, and obstructed nasal breathing. Patients with a unilateral finding were excluded due to possible neoplasia and papilloma.

We include tissues from 6 patients for this study (Table 1). The evaluation of the isolation, proliferation, and flow cytometric analysis was carried out with Polyp 1, Polyp 2, and Polyp 3. ELISA was performed with epithelial cells from Polyp 4, Polyp 5, and Polyp 6.

All isolation and culture procedures were performed in a sterile condition with sterile instruments. Tissues were rinsed with 7.5% povidone-iodine solution (Braunol®, B.Braun, Germany) and rinsed twice with 0.9% sodium chloride immediately after harvest from sinus surgery. The tissue was stored in Dulbecco's Modified Eagle Medium (Gibco, cat #: 1,437,676, Carlsbad, CA, USA) with 10% fetal bovine serum (v/v; Bio&SELL, cat #: FBS.S 0615 HI, Feucht, Germany) and 1% penicillin/streptomycin (Bio&SELL, cat #: BS.A 2213, Feucht, Germany) at 4°C. The following treatment was carried out within 6 h after surgical removal. The tissue was washed once with Dulbecco's phosphate-buffered saline without calcium and magnesium (PBS; Gibco, cat #: 14,190,169, Carlsbad, CA, USA). Polyps were cut into small pieces on the petri dish with surgical scissors (approx. 1–1.5 mm diameter) (Fig. 6A). The small pieces were placed in the 12-well culture plate (2 pieces/well) and placed under the safety cabinet without lid for 5–10 min to dry and stick to the bottom of the culture dish. Subsequently, 350 μl of Bronchial Epithelial Growth Medium (BEGM; Lonza, cat #: CC-3170, Basel, Switzerland) was added carefully without mobilizing the tissue pieces from the dish (Fig. 6B); the wells should be completely covered with medium, but the medium volume must be as low as possible to avoid floating of the tissue pieces in the cell culture vessel (approx. 350 µl medium in our conditions, as mentioned above). The medium was renewed every 2 days without mobilizing the tissue pieces. The incubation lasted for 8 days, and the outgrowth of the epithelial cells from polyp tissues was evaluated microscopically every 2 days when the medium was changed. The cells were treated with 300 µl trypsin 0.25%/EDTA 0.02% for 2–3 min after rinsing with PBS twice, and the trypsin was neutralized with the same volume of soybean trypsin inhibitor (1 mg/ml in PBS; Sigma, cat #: SLCF8902, Steinheim, Germany). The cells were subsequently washed and centrifuged twice with PBS (300 × g) and then seeded on a 6-well plate (5,000 cells per well with 1.5 ml BEGM). The cells were grown in a humidified incubator with 5% CO2 at 37°C. The medium was renewed every 2 days. Cells were trypsinized at 70%–80% confluence and were re-passaged. The cell count was determined with the haemocytometer after mixing cell suspension with 0.1% trypan blue 1:1 (v/v) to identify vital cells.

Tissue is on the petri dish placed and 1 ml PBS is added to keep the tissue from becoming dry A. The tissue is cut into small pieces of approx. 1–1.5 mm with surgical scissors. Small pieces were placed on the 12-well culture plate (2 pieces/well) and stand under the safety cabinet without lid for 5–10 min to dry and stick on the surface of the culture dish. Approx. 350 μl of BEGM was then given carefully without mobilizing tissue pieces from the dish (B)

Flow cytometry with anti-cytokeratin-PE (Miltenyi Biotec, clone: REA831, Bergisch Gladbach, Germany) and anti-p63 (Ventana Medical, clone: 4A4, AZ, USA) was performed to identify epithelial cells and evaluate differentiation. Simultaneously, the expression of Ki-67 (anti-Ki-67-FITC, Miltenyi Biotec, clone: REA183, Bergisch Gladbach, Germany) was determined to evaluate proliferation. Cells from the 2nd or 3rd passage were used for the analysis. Cells were cultured in a 75 cm2 cell culture flask with 10 ml BEGM, renewing the medium every 2 days. In 300 µl incubation buffer (0.5% BSA in PBS), 5 × 105—1 × 106 cells were collected. Supernatant was removed after centrifuging at 300 × g for 10 min at 4°C, and the cells were washed 3 times in total in the same way, by adding 300 µl incubation buffer followed by centrifugation. (The same washing process was carried out after each step and will not be mentioned from here on.) Cells were fixed in 500 µl of 4% formaldehyde for 15 min at room temperature. After removing the formaldehyde, permeabilization was carried out with 300 µl precooled permeabilization buffer (0.5% Octoxynol-9, 0.68 µM ethylenediaminetetraacetic acid, and 1% bovine serum albumin in distilled aqua) for 10 min on ice. The permeabilization buffer was removed, and cells were incubated first with 300 µl unconjugated anti-p63 (dilution 1:200 in incubation buffer) overnight at 4°C and then with secondary anti-mouse AlexaFluor633-conjugated antibody (dilution 1:200 in incubation buffer; Molecular Probes, cat #: A-21052, Eugene, OR, USA) for 1 h in a darkened chamber at room temperature. Cells were washed and incubated with fluorescent-dye conjugated anti-cytokeratin-PE and anti-Ki-67-FITC (each dilution 1:200 in incubation buffer) overnight at 4°C in a darkened chamber. Cells were washed and resuspended in 100 µl incubation buffer to perform a flow cytometric analysis. Compensation was set up before analysis, and positive and negative populations were predefined accordingly.

Epithelial cell function was assessed by the production of typical cytokines of type 2 inflammation, representatively IL-33 and periostin. Concentrations of IL33 and periostin in culture supernatants were measured by ELISA according to the manufacturer’s instructions (Invitrogen, cat #: BMS2048TEN, Carlsbad, CA, USA; R&D Systems, cat #: DY3548B, Minneapolis, MN, USA) and as described previously [21]. To obtain the supernatants, 5,000 cells were seeded in a 24-well plate with 1 ml BEGM, and medium was renewed every 2 days until 70%–80% confluence. Then 1 ml of BEGM was added, and supernatants were collected after 24, 48, and 72 h. Cells from 2nd or 3rd passage were used for the analysis.