3D Co-Culture of Lung Epithelial and Stromal Cells

Freshly sorted secretory cells were resuspended in culture medium (3D basic medium (DMEM/F12, Gibco) supplemented with 10% FBS (Gibco) and ITS (Insulin‐Transferrin‐Selenium, Corning)). These cells were mixed with cultured lung stromal cells negatively isolated by microbeads of CD326/EpCAM, CD45, and CD31 via MACS (Miltenyi Biotech), followed by resuspension in growth factor‐reduced Matrigel (BD Biosciences) at a ratio of 1:5. A 100 μl mixture was placed in a 24‐well Transwell insert with a 0.4 μm pore (Corning) (Lee et al, 2014 (link)). Approximately 5 × 10³ epithelial cells were seeded in each insert. 500 μl of culture medium was placed in the lower chamber, and the medium was changed every other day. For amino acid media change experiment, 500 µl of 3D basic media, 10% dialyzed FBS in BME media (Thermofisher, 21010046), or 10% dialyzed FBS in BME media with EAA (1 mM of L‐Histidine, L‐Isoleucine, L‐Leucine, L‐Methionine, L‐Threonine, L‐Valine, L‐Glutamine, L‐Arginine, L‐Cysteine) was replaced every day.