Organoid modeling of the tumor immune microenvironment
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Supplemental information for "Organoid modeling of the tumor immune microenvironment", Neal et al., Cell. Please see manuscript for further information.
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Human PDO culture 14 Tumor tissues were minced finely, washed twice in ADMEM/F12 (Invitrogen) containing 1X Normocin 15 (InvivoGen), resuspended in Type I collagen gel (Trevigen), and layered in a double dish air-liquid 16 culture system as previously described8. Organoids were cultured in human organoid medium 17 (ADMEM/F12 supplemented with 50% Wnt3a, RSPO1, Noggin-conditioned media (L-WRN, ATCC), 18 HEPES (1mM, Invitrogen), Glutamax (1X, Invitrogen), Nicotinamide (10 mM, Sigma), N-Acetylcysteine 19 (1 mM, Sigma), B-27 without vitamin A (1X, Invitrogen), A83-01 (0.5 μM, Tocris), Pen-Strep Glutamine 20 (1X, Invitrogen), Gastrin (10 nM, Sigma), SB-202190 (10 μM, Sigma), and EGF (50 ng/mL, Invitrogen). 21 Organoids were passaged every 14-30 days by dissociation with 200 units ml−1 collagenase IV 22 (Worthington) at 37 °C for 30 min, followed by three 5-min washes with 100% FBS and replating at a 23 1:4 split into new air-liquid interface collagen gels. Additionally, in some cases, media was 24 supplemented with recombinant human IL-2 (Peprotech) at 600 or 6000 IU/ml.