Isolation and Culture of Primary Airway Mesenchymal Cells

The primary AM or OKC cells were isolated as previously described [23 (link)]. The suspended primary AM cell were seeded in the 0.1% gelatin-coated (PRIMARY CELL SOLUTION, PCS-999-027) culture dishes (1×10⁶) in defined Keratinocyte Basal Medium-2 (KBM-2, LONZA, CC-3103) supplemented with KGM^Ⓡ-2 SingleQuots^Ⓡ (LONZA, CC-4152) at 37 °C in a humidified atmosphere with 5% carbon dioxide (CO₂). After 48 h, the non-adherent cells were removed, and fresh KGM^Ⓡ-2 media were replenished every 3 days. Early passages of primary cells were cryopreserved, and less than six passages were used for further experiments. For calcium imaging experiment or histological analysis, AM cells were seeded in the KGM^Ⓡ-2 media with DMSO (0.1% v/v), Bay-k8644 (10 nM) or VPM (10 µM) and cultured for 12 h.