Establishment and Propagation of Pancreatic Organoids

hPDOs were established from fine-needle biopsy (FNB) or resected pancreatic cancer specimens^{10 (link)}. Tumour tissue was minced and enzymatically digested with 5 mg/mL Collagenase-II (ThermoFisher) in AdF Base medium for 45 minutes at 37°C, shaking (700 rpm). Upon removal of red blood cells using red-blood cell lysis buffer (Merck), cells were spun, washed and seeded in 20 μL droplets of Matrigel and grown in hPOCM. For passaging, following removal of the growth media, Matrigel domes were depolymerised in ice-cold PBS. hPDOs were mechanically dissociated into fragments using a 200 μL pipette tip and re-plated at a 1:2 split into new 20 μL Matrigel droplets. Frozen organoid stocks were established by mixing dissociated organoid fragments with RCFM followed by gentle cryopreservation. Organoids were rapidly thawed and washed twice in warm AdF base medium. hPDOs were seeded in 20 μL droplets of Matrigel in enriched hPOCM (enrichment consisted of addition of 50 ng/mL EGF (Invitrogen) and increasing concentrations of Wnt3a [200 ng/mL] and RSpondin 1 [1 μg/mL]). hPDOs were passaged at least once before usage for experimental procedures.