Alveolar Type II Cell Culture

Human distal lung samples were obtained from a 13-month-old male donor (pediatric lung cells) and a 32-year-old male donor with no smoking history (adult lung cells). Both donors died of traumatic injuries, and their lungs were deemed unsuitable for transplantation. Distal human lung samples were treated with Liberase (MilliporeSigma) for 60 minutes at 37°C and disaggregated by pipetting and filtered through 40 μm cell strainers (Falcon) twice. Cells were stained for EpCAM (BioLegend), CD31 (BioLegend), CD45 (BioLegend), NGFR (BioLegend), and HTII-280 (Terrace Biotech). EpCAM⁺CD45^–CD31^–NGFR^–HTII-280⁺ cells (AT2s) were sorted on a BD Fusion FACS instrument.
After sorting, 5000 AT2s and 25000 MRC-5 fibroblasts (ATCC) in Growth Factor Reduced Matrigel (Corning) were seeded per 6.5 mm diameter Transwell cell culture inserts (Corning) with CK+DCI medium (14 (link), 21 (link)) in the basolateral compartment.