Pancreatic Ducts Organoid Isolation and Culture

Pancreatic ducts were isolated from the whole organ of 20 weeks old KP, KPC, KPC;KhkC^–/–, KPC;KhkA^–/–, KPC;KhkA/C^–/– mice as previously described.⁵⁴ (link) Each organoid line was isolated from an individual mouse. Isolated ducts organoids were embedded in growth-factor reduced (GFR)-Matrigel (Corning), and cultured in organoid medium (OM), which is composed of AdDMEM/F12 (Gibco) supplemented with GlutaMAX (Gibco), HEPES (Gibco), Penicillin-Streptomycin (Invitrogen), B27 (Gibco), N-2 (Gibco), 1.25 mM N-Acetyl-L-cysteine (Sigma), 10 nM Gastrin I (Sigma) and the growth factors: 100 ng/ml FGF10 (Peprotech), 50 ng/ml EGF (Peprotech), 100 ng/ml Noggin, 100 ng/ml RSPO-1 (Peprotech), and 10 mM Nicotinamide (Sigma). For the first week after duct isolation the culture medium was supplemented with 100 μg/ml Primocin (InvivoGen).