Airway epithelial cell growth medium supplement pack

We used HPC patient-derived xenograft cell lines (HPCM1^{7 (link),8 (link)} and HPCM2^{7 (link),8 (link)} cells), which were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), 100 unit/mL penicillin, and 100 μg/mL streptomycin. MCC148c cells^{13 (link)}, established by patient-derived xenografts of cancer tissue from a lung squamous cell carcinoma patient^{5 (link)}, were maintained in DMEM supplemented with 10% FBS, 0.4 mg/mL hydrocortisone, 2.5 mM Y-27632 (Focus Biomolecules, Plymouth, PA, USA), and penicillin/streptomycin. DMEM supplemented with 10% FBS and penicillin/streptomycin was used to maintain 293 T cells (RIKEN BioResource Center, Kyoto, Japan). Het-1A cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in airway epithelial cell basal medium from the airway epithelial cell growth medium supplement pack (PromoCell, Heidelberg, Germany) supplemented with 4% FBS and penicillin/streptomycin. IMR-32 cells were purchased from ATCC and maintained in DMEM supplemented with 10% FBS and 0.1 mM non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA). HSC-3 cells were purchased from RIKEN (Saitama, Japan) and maintained in Eagle’s minimal essential medium supplemented with 10% FBS and penicillin/streptomycin.

Each brush, with attached cells, was placed in sterile phosphate buffered saline (PBS) mixed with transport medium (DMEM, 0.1% Penicillin/streptomycin (1:1 V/V)). Paediatric nasal cells were removed from the brushes and expanded in airway epithelial cell basal medium supplemented with an airway epithelial cell growth medium supplement pack (Promocell, Heidelberg, Germany) using established methods.[9 (link),15 ] Upon reaching 70–80% confluency, the cells were seeded at passage 3 onto collagen-coated 6 mm diameter Transwells (Corning, Tewksbury, Massachusetts) at a density of 1x10⁵ cells/Transwell. After reaching confluency, air-liquid interface (ALI) conditions were established and maintained until complete differentiation occurred. Following complete differentiation, which was defined by extensive cilia coverage and mucus production under light microscopy, transepithelial electrical resistances (TEER) were measured, as described previously[9 (link)]

For calcium imaging, we used the human tracheal epithelial cell line (HTEpC, C12644, PromoCell, Heidelberg, Germany). Cells were cultivated in an Airway Epithelial Cell Growth Medium Kit (C-21160) containing the Airway Epithelial Cell Growth Medium Supplement Pack (C-39160, PromoCell, Heidelberg, Germany). The airway epithelial cells were kept in a humidified chamber at 37 °C with air containing 5% CO₂. For the Ca²⁺ imaging, cells were seeded onto laminin-coated coverslips. Dye loading with 2.5 µM FURA-2 AM (Biotium, Fremont, CA, USA) in darkness was performed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer for 30 min at 37 °C. For the contents of the HEPES buffer, see the section on drugs and buffer solutions. After the loading period, the cells were rinsed in fresh HEPES buffer and were transferred to the recording chamber of an upright fluorescence microscope equipped with 20 × immersion lenses (BX50 WI, Olympus, Hamburg, Germany), which contains 2 ml HEPES. The excitation light was provided by a 50 W xenon lamp. The microscope was equipped with a dichromatic excitation longpass mirror (400 nm).
The ratiometric dye, FURA-2 AM, was excited at 340 nm and 380 nm (± 8 nm) when equipped with bandpass excitation filters. The emitted fluorescence was directed through a dichromatic shortpass filter of 560 nm to a bandpass filter of 510 nm.