Y 27632

Under informed consent as approved by the Institutional Review Board (HS-2832) of National Jewish Health, human nasal epithelial (HNE) cells were obtained by nasal brushing from three CF individuals homozygous for the F508del CFTR mutation. HNE cells were cultured as previously described [21 (link),24 (link)]. Briefly, HNE cells captured by nasal brushings were expanded on an irradiated NIH 3T3 feeder layer in a complete F-media in the presence of the RhoA kinase inhibitor Y-27632 (ApexBIO). Once expanded, cells were then plated on Type I bovine collagen-coated Transwell inserts (Corning) for differentiation in the absence of Y-27632. After initial seeding on inserts (2.5 × 10⁴ cells cm⁻²), cells remained submerged in PneumaCult^™-Ex Plus (STEMCELL Technologies) media for 2 d, then the apical media was removed and the basal media was replaced with PneumaCult^™-ALI media (STEMCELL Technologies). Cells remained at ALI for 21–28 d prior to electrophysiological analysis, with basal media changes every 2–3 d. Well-differentiated cultures were defined by the presence of beating cilia and secreted mucus.

Freshly purified cells were plated into single wells of 12-well plates with 50% Wnt3a conditioned medium in Advanced DMEM/F12 medium supplemented with 50 ng/ml EGF, 5 ng/ml Noggin (120-10C, Peprotech), 10 μM Y27632 (S1049, Selleckchem) and 2% B27 minus vitamin A. At day 4 single cells were prepared using Accutase (A6964, Sigma) and resuspended at 5 × 10^{4 (link)} cells in 50 μl of 10% RSPO1 conditioned medium plus 50 ng/ml EGF, 5 ng/ml Noggin, 10 μM Y27632 and 2% B27 minus vitamin A, with 150 μl of Matrigel (356231, Corning) on ice. The cell-Matrigel mixture was transferred into tissue culture plates (40 μl/well in 24-well plates or 25 μl/well in μ-Slide 8-well chamber (80826, ibidi)). After 36-48 h, the RSPO1/EGF/Noggin conditioned medium was replaced by organoid differentiation medium composed of 10% RSPO1 conditioned medium supplemented with 100 ng/ml FGF10 (100-26, Peprotech), 50 ng/ml EGF and cultured for 15 days, with a medium exchange every 2-3 days. For long-term culture, the tubuloids were grown in maintenance medium consisting of Advanced DMEM/F12 supplemented with 50 ng/ml EGF, 50 ng/ml FGF2, 100 ng/ml IGF1, 500 ng/ml of Insulin, 2 % B27 minus vitamin A. For full details see Supplementary Methods.

CLR28 PDX (Yamaguchi et al., 2019 ) was harvested at a size of 100 mm³ from NSG mice. Grossly necrotic tissue was trimmed out and the remaining tumor was washed in ice-cold PBS and minced with a #10 scalpel. The tumor fragments were incubated with 5 ml Collagenase type XI (Sigma) in DMEM at 5 mg/ml supplemented with 10 μg/ml DNAse1 (Sigma) and Y-27632 (Sigma) 10μM at 37°C for 1H. The digested tumors were triturated three times and allowed to sink by gravity, and 3 ml of supernatant was removed (fraction 1). Fresh DMEM (3 mL) was added and triturated 3 times to repeat the process for fractions 2–4. The fractions were examined under a bright-field microscope and the fraction most enriched with colonic epithelium was chosen for downstream processing. Tubes were centrifuged at 200G for 5 minutes at 4°C, the supernatant was discarded, the pellet was resuspended in Matrigel (Corning), 30 μl of the Matrigel-colonic epithelium mix was plated in a well of a 24-well plate on a heating pad and allowed to solidify, and then 500 μL of organoid growth media (Advanced DMEM/F-12, HEPES 10 mM, GlutaMAX supplement, BSA 0.1%, Wnt3a, R-spondin, B27, nicotinamide 1.25 mM, N-acetylcysteine 1.25mM, primocin 100 μg/mL, mNoggin 100 ng/mL, hEGF 50 ng/mL, hFGF 100 ng/mL, hGastrin I 10nM, A 83–01 500 nM, Y-27632 10.5 μM, PGE2 1μM) was added.