Bepicm

HBECs (Sciencell, California, USA) were cultured in bronchial epithelial cell medium (BEpiCM, Sciencell, California, USA), trypsinized and viable cells counted using trypan blue staining (Life Technologies, Paisley, UK). Cells were seeded to be confluent at 24 h onto either Falcon 0.4 μm pore size polyethylene terephthalate (PET; Becton Dickinson Labware, Claix, France) as a positive control or POSS-PCU sheets clipped with Cellcrown (Sigma–Aldrich, Gillingham, UK) to create a POSS-PCU transwell insert. 500 μL of Pneumacult-ALI (STEMCELL Technologies, Cambridge, UK) was added into the lower chamber of each well while cells were seeded in a 200 μL volume in the upper compartment. After three days of submerged culture, scaffolds were air-lifted by adding 1 mL of Pneumacult-ALI to the basal chamber and removing media from the upper chamber to generate an air-liquid interface.

Cell culture: HTrF were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% Fetal Bovine Serum (FBS, Gibco), and HTEpiC were cultured in Bronchial Epithelial Cell Medium (BEpiCM, ScienCell) containing 1% Bronchial Epithelial Cell Growth Stimulus (BEpiCGS, ScienCell). The cells were cultured in an incubator at 37 °C with 95% humidity and 5% CO₂. The culture dish used for HTEpiC was coated with Poly-d-Lysine hydrobromide (PDL, Salarbio) at a concentration of 0.1 mg/mL for 2 h in advance and washed with PBS thrice before cells were seeded. The culture medium was changed daily.
Drug Preparation: (1) Paclitaxel, mitomycin C, and rapamycin powder were weighed and dissolved in Dimethyl Sulfoxide (DMSO, Solarbio, Beijing, China) to an initial concentration of 10⁻³ mol/L. The drugs were then diluted to 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, and 10⁻¹¹ mol/L in complete medium. (2) Rapamycin solutions of different concentrations (1 × 10⁻⁵, 2 × 10⁻⁵, 4 × 10⁻⁵, 6 × 10⁻⁵, 8 × 10⁻⁵, and 10 × 10⁻⁵ mol/L) were prepared.

Human bronchial epithelial cells (HBEpiCs/HBECs, ScienCell and Lonza/Fischer Scientific) were cultured in BEpiCM (ScienCell) at 37 °C in an atmosphere containing 5% CO₂ until 80–90% confluence. Next, cells were washed once with PBS, and treated with trypsin (ACF Enzymatic Dissociation Solution, Stemcell Technologies, Vancouver, Canada) followed by incubation at 37 °C in 5% CO₂ for 5 min. Once the cells were dissociated, an equal volume of trypsin solution was added, followed by centrifugation at 230 × g for 5 min before aspirating the medium and re-suspending the cell pellet in 1 mL fresh medium. For experiments, 24-well plates were coated with poly-L-Lysine (ScienCell) for at least 1 h at 37 °C. Wells were washed once with Dulbecco’s PBS before seeding the cells (30 000 cells/well). Upon reaching 80% confluence, cells were stimulated with 5% (v/v) CSE for 24 h at 37 °C. At the end of the incubation period, plates were centrifuged at 230 × g for 5 min before collecting cells and supernatants for further experiments.

Sinus mucosal samples obtained during surgery were incubated with 0.5% dispase (GenDEPOT, Katy, TX, USA) for 24 h. Thereafter, epithelial cells were isolated from mucosa by mechanical detachment using a cell scraper and cultured in culture plates containing Bronchial epithelial cell medium (BEpiCM; ScienCell, Carlsbad, CA, USA). After the culture surface was fully occupied by epithelial cells, cells harvested with trypsin were cultured under an air–liquid interface (ALI) in the SPL Insert system (SPL, Gyeonggi-do, Republic of Korea). ALI culture was used for all experiments.

16HBE cells (Jennio Biotechnology, CHN), a transformed human bronchial epithelial cell line were cultured with 10% fetal bovine serum (FBS) (10099141; Gibco, USA) as previously outlined^{36 (link)}. Normal primary human bronchial epithelial cells (HBEpiCs) (3210; ScienCell, USA) were maintained in Bronchial Epithelial Cell Medium (BEpiCM) (3211; ScienCell, USA) with 1% bronchial epithelial cell growth supplement. Cells were cultured in humidified air with 5% CO₂ at 37 °C. Prior to O₃ or H₂O₂ (H6520; Sigma-Aldrich, CHN) exposure, cells were equilibrated by medium containing 1% FBS or 0.1% supplement medium.

BEAS-2B (ATCC
CRL-9609) and A549 (ATCCCCL-185) cell lines were purchased from ATCC.
For culturing BEAS-2B cells, ATCC recommended the bronchial epithelial
cell growth medium (BEGM), complete with supplements and growth factors
(BEpiCM, ScienCell, U.S.A.).
Depending on the experiment, A549
cells were cultured in Dulbecco’s modified eagle’s medium
(DMEM, Institute of Immunology and Experimental Technology, Wrocław,
Poland) or phenol-red free DMEM in 5% or 10% heat-inactivated fetal
bovine serum (FBS), l-glutamine (L/G, 2 mM) and penicillin-streptomycin
(P/S, 100 mg mL⁻¹). All chemicals were purchased from Sigma-Aldrich (U.S.A.). Trypsin-EDTA
(0.25% solution with phenol red, Sigma-Aldrich, U.S.A.) was used to
routinely detach the adherent cells for passaging. Both cell lines
were cultured in tissue culture (TC) treated T75 flasks (Cell Star,
Greiner Bio-One, Austria) and maintained at 37 °C in a 5% CO₂ humidified incubator during the course of the experiment.
Depending on the type of the experiment, cells were seeded onto 96-well
advanced TC treated μclear microplates (Greiner Bio-One, Austria).
For all experiments, BEAS-2B cells underwent 1–16 passages,
while A549 cells underwent 3–20 passages.