Birb796

After 21 days of differentiation in CK + AB or CK + DCI organoids were passaged and fed with SFFF or SFFF without FGF10 consisting of: Advanced DMEM/F12, 2 mM Glutamax, 1× B27 supplement, 100 U/mL penicillin-streptomycin, 15 mM HEPES, 0.05% BSA, 10 µM TGFβ inhibitor SB43152 (APExBIO, Cat#A8249), 1 µM p38 MAP kinase inhibitor BIRB796 (APExBIO, Cat#A5639), 3 µM CHIR99021, 50 ng/mL rhEGF (R&D Systems, Cat#236-EG) with (SFFF) or without the addition of 10 ng/mL FGF10 (produced in-house). Organoids were passaged every 7–14 days at a ratio of 1:6 by needle sheering⁹³ .

Distal lung sections from a single patient were minced using a scalpel. Minced lung was enzymatically dissociated to a single cell suspension using 1 mg/mL collagenase A (Roche, Cat#10103578001), 2–4 U/mL elastase (Worthington, Cat#LS002274), and 0.1 mg/mL DNAse (Roche, Cat#10104159001), filtered through a 100 µM cell strainer, subjected to red blood cell lysis (Roche, Cat #11814389001), washed with PBS, and seeded into Matrigel. After two passages, cultures were subjected to FACS (see below: Fluorescence-activated Cell Sorting). AT2 cells were isolated on the basis of positive HTII-280 staining and reseeded into Matrigel with primary AT2 organoid media consisting of: Advanced DMEM/F12, 2 mM Glutamax, 1× B27 supplement, 100 U/mL penicillin-streptomycin, 15 mM HEPES, 0.05% BSA, 10 µM SB43152 (APExBIO, Cat#A8249), 1 µM BIRB796 (APExBIO), 3 µM CHIR99021, 50 ng/mL rhEGF and 10 ng/mL rhFGF10 (Cite Katsura). Organoids were expanded in primary AT2 organoid media and passaged every 3–4 weeks at a ratio of 1:2–3 by TrypLE-mediated dissociation.