In vitro feeder-free organoid culture and passages

Freshly sorted lineage-labelled cells from Scgb1a1-CreER^TM/+;R26R^tdTomato/+ or Sftpc-CreER^T2;R26R^tdTomato/+ mice were resuspended in basic medium (AdDMEM/F12 (Invitrogen) supplemented with B27 (Invitrogen), 1mM N-Acetylcysteine (Sigma), 10 mM Nicotinamide (Sigma)). Cells were mixed with growth factor-reduced (GFR)-Matrigel (BD Biosciences) at a ratio of 1:1. A 100 μl mixture was placed in a 24-well Transwell insert with a 0.4 μm pore (Corning). Approximately 10×10³ epithelial cells were seeded in each insert. After GFR-Matrigel formed a gel, 500 μl of culture medium (basic medium supplemented with the growth factors: 50 ng/ml murine EGF (Life Technology), 100 ng/ml human FGF7/KGF (Peprotech), 100 ng/ml human FGF10 (Peprotech), 50% WNT3A-conditioned media (provided by Tissue Core Facility of Cambridge Stem Cell Institute), 10% RSPO1-conditioned media (provided by Tissue Core Facility of Cambridge Stem Cell Institute), 100 ng/ml Noggin (Peprotech)) was placed in the lower chamber. Medium was changed every other day and ROCK inhibitor Y27632 (10 μM, Sigma) was added in the medium for the first 2 days of culture. Passage was performed once per 2 weeks. For passages, organoids were removed from the Matrigel by incubation with dispase (1 mg/ml) for 40 mins at 37 °C, followed by dissociation into single cells using trypLE (Gibco) treatment for 5min at 37°C. 5~10×10³ cells were transferred to fresh GFR-Matrigel in 24-well Transwell insert. For organoid culture from a single cell with limiting dilution, FACS-sorted cells were plated into 48-well plates (Corning) with one cell per well. Every dilution was replicated in 48-well plates (Corning) for two independent experiments. Single cells imbedded in Matrigel were monitored at microscope with RFP channels to check the expression of Tomato expression.

For human KDR⁺ cell culture in organoids, approximately 10×10³ epithelial cells were resuspended in a 20 μl of 100% GFR-Matrigel and seeded in 48 well plates, followed by 30 min incubation at 37 °C for solidification. Then, 250μl of airway cell culture medium (basic medium supplemented with the growth factors: murine EGF (50 ng/ml, Life Technology), human FGF7/KGF (100 ng/ml, Peprotech), human FGF10 (100 ng/ml, Peprotech), Noggin (100 ng/ml, Peprotech), A83-01 (1μM, Tocris), SB202190 (1μM, Tocris) was added to each well. To avoid the growth of fungal and bacterial infection, 250 ng/mL Amphotericin B and 50 μg/mL gentamicin were added to culture medium. For culture with DAPT treatment in Fig.7g, organoids cultured for 8 days with airway cell culture medium were followed by inclusion of CHIR99021 (2μM, Tocris) for additional 10 days with or without DAPT (20μM). Medium was changed every 3-4 days and ROCK inhibitor Y27632 (10μM, Sigma) was added in the medium for the first 4 days of culture. Passage was performed once per 3 weeks.