Culture of pancreatic cancer patient-derived organoids (PDOs)

PDOs were generated from PDAC tissue harvested from cancer patients with GEM-sensitive or GEM-resistant PDAC. First, the tumor tissues were washed with phosphate-buffered saline (PBS) and cut into small pieces (2–3 mm3) with sterile surgical scissors. Second, the tissue samples were digested in MasterAim™ Tissue Enzyme Solution I (AIMINGMED, 100 − 051) in Advanced Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium (Thermo Fisher Scientific) for 1 h at 37 °C with intermittent shaking. Third, an additional Digestion™ Tissue Enzyme Solution I (AIMINGMED, 100 − 051) from MasterAim was applied. From the collected cell samples, aliquots of 600 cells resuspended in 30 µL Matrigel (356231, Corning, NY, USA) were added to individual wells of a 48-well plate and incubated at 37 °C for 5 min to allow solidification of the Matrigel. Cells were cultured in alkaline medium (advanced DMEM/F12, 10 mM HEPES, 1× GlutaMAX-I, 100 µg/mL Primocin, and 1× penicillin/streptomycin solution) or complete medium (advanced DMEM/F12, 10 mM HEPES, 1×GlutaMAX-I, 100 µg/mL Primocin, 1× penicillin/streptomycin solution, 500 nM A83-01, 10 µM Y-27632, 1.56 M N-acetylcysteine, 10 mM nicotinamide, 10 ng/mL fibroblast growth factor 10 [FGF10], 1× B27 supplement, 10 µM forskolin, 30% Wnt3A conditioned medium, 2% R-corresponding conditioned medium and 4% Noggin conditioned medium). The medium was changed every 3 days.