Pneumacult ali 10x supplement

All cells were grown at 37 °C in a humidified atmosphere with 5% (v/v) CO₂. Culture conditions for CFBE and HT₂₉ cells have been described earlier [25 (link)]. In brief, airway epithelial cells were grown in DMEM/Ham’s F-12 with L-Glutamine medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine 200mM and 1% (v/v) HEPES 1M (all from Capricorn Scientific, Ebsdorfergrund, Germany). CFBE parental cells were grown in MEM with Earle’s Salts with L-Glutamine medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% FBS. The airway epithelial cell line H441 was grown in RPMI and DMEM media. The immortalized human airway basal cell line BCi-NS1 (kindly provided by Prof. Ron Crystal, Weill Cornell Medical College, New York City, NY, USA) was maintained in Bronchial Epithelial Growth Media (Lonza). Cells were differentiated by growing on permeable supports (Snapwell, Corning, NY, USA) in an air-liquid interface (ALI) for up to 30 days in PnemaCult-ALI medium supplemented with PneumaCult-ALI 10X Supplement, PneumaCult-ALI Maintenance Supplement, hydrocortisone and heparin (all from StemCell Technologies, Vancouver, BC, Canada), and 1% penicilin-steptomycin (10 000 U/mL; Gibco, ThermoFisherScientific, Waltham, MA, USA).

Human primary small airway epithelial cells (SAEC, PCS-301-010™) were purchased from ATCC^® (Manassas, VA, USA) and cultured using the air–liquid interface (ALI) cell culture system. The media and reagents for the ALI system, including PneumaCult™-Ex Plus Medium (#05002), PneumaCult™-ALI Basal Medium (#05002), PneumaCult™-ALI 10X Supplement (#05003), and PneumaCult™-ALI Maintenance Supplement 100X (#05006) were purchased from STEMCELL Technologies (Vancouver, BC, Canada). The CelTox Sampler was purchased from MedTec Biolab Inc. (Hillsborough, NC, USA) for the in vitro exposure assessment. For the toxicological assays, CellTiter 96 Aqueous One Solution (Promega, Madison, WI, USA) and GSH-Glo^TM Glutathione reagent (Promega, Madison, WI, USA) were used to measure cellular viability and total glutathione, respectively, in SAEC.

For an ALI culture, HBE cells were seeded on Transwell^® inserts (#3460, Corning Incorporated, Corning, NY). HBE cells were seeded at a density of 1 × 10⁵ cells/1.1 cm² and maintained in submerged with BEGM medium. After the cells reached 100% confluence, the cultures were airlifted by removing BEGM medium and feed from basal chamber only with PneumaCult^TM-ALI medium which is composed of PneumaCult^TM-ALI basal medium (05002, StemCell Technology, Vancouver, BC) supplemented with PneumaCult™-ALI 10X Supplement (05003, StemCell Technology, Vancouver, BC) and PneumaCult™-ALI Maintenance Supplement (100X) (05006). The cells are well-differentiation after 28 days post airlift.