Expanding Mouse Pancreatic Duct Organoids

Pancreatic ducts were isolated from the bulk of the pancreas of mice older than 8 weeks by collagenase dissociation (Collagenase type XI 0.012% (w/v) (Sigma), dispase 0.012% (w/v) (Gibco), FBS (Gibco) 1% in DMEM media (Gibco)) at 37°C. Isolated ducts were mixed with Matrigel (BD Bioscience) and seeded and cultured as we described previously (Sato et al, 2009 (link); Barker et al, 2010 (link)). After Matrigel formed a gel, culture medium was added. Culture media was based on AdDMEM/F12 (Invitrogen) supplemented with B27 (Invitrogen), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma) and the growth factors: 50 ng/ml EGF (Peprotech), 10% RSPO1-conditioned media (kindly provided by Calvin Kuo), 100 ng/ml Noggin (Peprotech) or 10% Noggin-conditioned media (in-house prepared), 100 ng/ml FGF10 (Peprotech) and 10 mM Nicotinamide (Sigma). One week after seeding, organoids were removed from the Matrigel, mechanically dissociated into small fragments, and transferred to fresh Matrigel. Passage was performed in a 1:4–1:8 split ratio once per week for at least 9 months. To prepare frozen stocks, organoid cultures were dissociated and mixed with Recovery cell culture freezing medium (Gibco) and froze following the standard procedures. When required, the cultures were thawed using standard thawing procedures, embedded in Matrigel and cultured as described above. For the first 3 days after thawing, the culture medium was supplemented with Y-27632 (10 μM, Sigma-Aldrich).