Pancreatic Organoid Culture Protocol

Pancreatic organoids were derived and cultured as previously described^{20 (link),45 (link),46 (link)}. Briefly, adult pancreatic tissue was digested for 45–60 minutes at 37 °C with the collagenase/dispase dissociation Medium (1% in DMEM media (Gibco), supplemented with 1% FBS (Gibco) and Collagenase type XI 0.012% (w/v) (Sigma), dispase 0.012% (w/v) (Gibco)). Single ducts were manually picked and seeded in Matrigel (BD Bioscience). Ducts were cultured in AdDMEM/F12 (Invitrogen) supplemented with N2 and B27 (Lifesciences), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma) and the growth factors: 50 ng/ml EGF (Peprotech), 5% RSPO1-conditioned media, 5% Noggin-conditioned media (in-house prepared), 100 ng/ml FGF10 (Peprotech) and 10 mM Nicotinamide (Sigma). Differentiation medium consisted in removal of EGF, RSPO1-conditioned media and addition of the Wnt Inhibitor IWP-2 (0,25 μM, Stemgent^{47 (link)}) for the last 2 days of induction, with or without doxycycline, as indicated.