Generating Mouse Pancreatic Tumor Organoids

Pancreas tissue from Pdx1^Cre;LSL-Kras^G12D mice were digested with 125 μg/ml collagenase type XI (Sigma, St. Louis, Mo) and 125 μg/ml Dispase II (ThermoFisher Scientific, Waltham, Mass) at 37°C for 1 hour.^{27 (link)} Digested cells were collected by centrifugation and be incubated with rat monoclonal EPCAM antibody (G8.8, DSHB, Iowa City, Iowa) at 4°C for 30 min. After washing, cells were incubated with anti-rat IgG microbeads (Miltenyi Biotech, Auburn, Calif) and EPCAM+ tumor cells were separated by MACS Pro separator (Miltenyi Biotech). EPCAM+ cells (2 × 10⁴ cells) were suspended in 20 μl of matrigel (Corning, Tewksbury, Mass), plated on a round bottom 96-well plate, and incubated in a CO₂ incubator at 37°C for 30 min. After polymerization of the matrigel, the cells were cultured in 100 μl of advanced DMEM/F-12 glutamax (ThermoFisher Scientific) supplemented with 1X penicillin/streptomycin, 1x B27 supplement, 10 mM HEPES (Sigma), 1.25 mM N-acetylcystein, 10 mM nicotinamide, 10 nM Gastrin I, 0.5 μM A83–01 (R&D Systems, Minneapolis, Minn), 0.1 ng/ml FGF-10, 100 ng/ml Noggin, 50 ng/ml EGF, 1 μg/ml R-Spondin-1 (PeproTech, Rocky Hill, NJ), and 10 nM Y-27632 (Tocris, Minneapolis, Minn).^{28 (link)} Tumor organoids formed in matrigel were disrupted by pipetting and were passaged from 1 well to 3 wells.