Animal component free cell dissociation kit

We obtained primary HBE cells from Lonza (CC–2540S or CC–2540 for healthy donors, 00196979 for the CF donor). They were cultured in T–25 flasks using PneumaCult–Ex Plus medium (Stemcell Technologies) for no more than 3 passages. When they reached confluence, the cells were detached from the flask using the Animal Component–Free Cell Dissociation Kit (Stemcell Technologies) and centrifuged before being resuspended in PneumaCult–Ex Plus to a density of approximately 20,000 cells/μl.

Primary human bronchial epithelial (HBE) cells (LONZA) were seeded as passage 1 (P1) into 100 mm dishes on PneumaCult-Ex Plus media (StemCell) and incubated at 37°C, 5% CO₂. Once cells reached 70–80% confluency, they were dissociated using Animal Component-Free Cell Dissociation Kit (StemCell) and seeded on 12 mm Transwells (Corning) coated with 0.3 mg/mL Collagen type IV from human placenta (Sigma-Aldrich). Upon reaching confluency, the apical medium was removed to obtain an air liquid interface (ALI), and the basal medium replaced with PneumaCult-ALI medium (StemCell). Medium was changed every second day and apical surfaces washed with PBS (Ca²⁺, Mg²⁺) twice per week. Cultures were used for experiments after reaching full differentiation (∼ 3-4 wk on air) as assessed by visual confirmation of beating cilia and mucus (28 (link)). BEAS-2B cells, a SV-40-transformed human bronchial epithelial cell line (ATCC CRL-9609), were maintained in DMEM:F12, 5% FBS medium while VERO (ATCC, CRL-1586) were grown on EMEM, 10% FBS. All media contained penicillin (100 U/ml) and streptomycin (100 μg/ml).

Human bone marrow and adipose-derived MSCs were purchased from American Type Culture Collection (Catalog PCS-500-012, PCS-500-011, ATCC, MD, USA). They were cultured in MesenCult-ACF Plus Medium (catalog number 05448, STEMCELL Technologies, Vancouver, Canada) supplemented with 1% penicillin–streptomycin. They were cultured at 37 ℃ with 5% CO₂. The cells were passaged at a seeding density of 4 × 10³ viable cells/cm² when reaching 80% confluence using Animal Component-Free Cell Dissociation Kit (catalog number 05426, STEMCELL Technologies, Vancouver, Canada).

The immunophenotype of ECFCs was determined by flow cytometry on 3 biological replicates of ECFCs using fluorescence-conjugated antibodies. ECFCs were sorted into CD44^hi/lo populations as previously described (12 (link)). Briefly, cells were detached (Animal Component-Free Cell Dissociation Kit, STEMCELL Technologies, 05426) and PBS washed. ECFCs were incubated for 20 minutes on ice with 20 μL of APC-conjugated primary murine monoclonal antibodies against human CD44 antibody (clone G44-57, catalog 559942, BD Pharmingen) in 0.4 mL stain buffer (PBS [Dulbecco’s, no Ca²⁺, no Mg²⁺, Thermo Fisher Scientific, 14190250], 5% FBS, with 0.5 mM EDTA), washed 3 times, and analyzed by FACSAria flow cytometer (BD) with FlowJo (TreeStar) software. Duplicate and dead cells were excluded from the sort using forward and side scatter to analyze their 2-dimensional profile. Fluorescence voltages were set using negative controls, and the same strategy for setting parameters and gating were applied to all samples. The same staining protocol was applied for all FACS analyses of ECFCs using antibodies and concentrations listed in Supplemental Table 2, and the same gating strategy was used for all cell sorting experiments.

Primary human nasal epithelial cells (HNECs) were isolated by brushing from the nasal cavity of a male donor after informed consent. The isolated cells were cultured and maintained in PneumaCult™-Ex Plus Basal Medium supplemented with 1x PneumaCult™-Ex Plus Supplement, 1x hydrocortisone (200x stock solution 96 µg/ml) and 1x penicillin-streptomycin (all from StemCell Technologies). For cell expansion, the culture flasks and plates were coated with Collagen Solution according to manufacturer’s instructions, and Animal Component-Free Cell Dissociation Kit was used for dissociation and passaging (all from StemCell Technologies).
For the cell viability assay, HNECs were cultured on an opaque-walled 96-well plate (10 000 cells/well) for 24 h, and then treated with a 2-fold concentration series of TriSb92 diluted in complete culture medium (starting from 1 mg/ml) for 24 h at 37 °C. NaN₃ (5%) served as a cytotoxic drug control. Cell viability was measured using CellTiter-Glo 2.0 Assay (Promega; Cat# E2710) according to manufacturer’s instructions. Average and standard deviations of a representative assay performed in triplicates were calculated.