3D Lung Organoid Culture Protocol

Lung epithelial cells (Ter119⁻/Cd31⁻/Cd45⁻/Epcam⁺) isolated from 7-10-week-old Cracd WT mice or Cracd KO were cultured with lung stromal cells in a 3D organoid air-liquid interface, as described previously^{24 (link),45 (link)}. In brief, freshly sorted lung epithelial cells were resuspended in 3D organoid media (Dulbecco’s modified Eagle’s medium [DMEM]/F12 [Gibco, USA]), 10% FBS [Thermo Fisher Scientific], 1′ penicillin-streptomycin-glutamine [Thermo Fisher Scientific], and 1′ insulin-transferrin-selenium [Thermo Fisher Scientific.]) and mixed with LuECs at a ratio of 1:1. Cells containing 3D media were mixed with growth factor-reduced Matrigel (BD Biosciences) at a ratio of 1:1. The 100 ml of mixtures containing lung epithelial cells (5 X 10³) and LuECs (5 X 10⁴) were placed in the transwell insert (0.4-mm pore, Corning, Lowell, MA). After incubation for 30 mins at 37°C in an incubator, 500 ml of 3D media was placed in the bottom chamber to generate the liquid-air interface. Media were exchanged every other day.