Type 4 collagen coated 12 well transwell inserts

Primary HBE cells were isolated from patients who underwent surgical lung resection for pulmonary diseases in Nanjing Children’s Hospital, as described previously [11 (link)]. HBE cells were plated onto type I and III collagen-coated six-well tissue culture plates and cultured in BEGM media (Lonza, Germany) supplemented with the required additives (Lonza). When the cells reached 80%–90% confluence, traditional monolayer two-dimensional (2D) HBE cells were dissociated using trypsin, and 3 × 10⁵ cells were seeded on type IV collagen-coated 12-well transwell inserts (Costar, ME, USA). The medium was renewed for both the apical and basolateral surfaces every other day. The medium was then changed to air–liquid interface (ALI) medium (BEGM + DMEM + additives) until the HBE cells reached full confluence. After 5 days, the HBE cells were exposed to air, and only the basolateral compartment was cultured in ALI medium. The ALI culture was continued for 4–6 weeks, during which time the cells differentiated into 3D pseudostratified HBE cells. Prior to the experiments, all cultures were maintained at 37 °C in a 5% CO₂ incubator.

Primary HAE cells were isolated from patients who underwent surgical lung resection for pulmonary diseases in Nanjing Children’s Hospital, as described previously [19 ]. HAE cells were plated onto type I and III collagen-coated six-well tissue culture plates and cultured in BEGM media (Lonza, Germany) supplemented with the required additives (Lonza). When the cells reached 80–90% confluence, traditional monolayer two-dimensional (2D) HAE cells were dissociated with trypsin, and 3 × 10⁵ cells were seeded on type IV collagen-coated 12-well transwell inserts (Costar, ME, USA). Medium was renewed for both the apical and basolateral surfaces every other day. The medium was then changed to air–liquid interface (ALI) medium (BEGM+DMEM+additives) until the HAE cells reached full confluence. After 5 days, the HAE cells were exposed to air, and only the basolateral compartment was cultured with ALI medium. The ALI culture was continued for 4–6 weeks, during which time the cells differentiated into 3D pseudostratified HAE cells. Prior to the experiments, all cultures were maintained at 37 °C in a 5% CO₂ incubator.