Long-term culture of gastric tumor-PDOs needs a generic basal medium composed of ADMEM/F12 (advanced Dulbecco’s modified Eagle medium with nutrient mixture F12 Hams), antimicrobial reagent, B27, N2, HEPES and L-glutamine (or Glutamax), supplemented with EGF, noggin, R-spondin 1, gastrin, FGF-10, FGF-basic, Wnt3A, prostaglandin E2 (PGE2), Y-27632 (Rock inhibitor), nicotinamide (and/or N-acetylcystenine), A83-01 (TGF-β type I receptor inhibitor), SB202190 (p38 MAPK inhibitor) and HGF (exclusively for cholangiocarcinoma organoids) [27,70]. The gene discussed is RSPO1; the disease is cholangiocarcinoma.