Fig 2: Effect of GSK inhibitor in gastric organoid culture. The gastric organoids were fragile and damaged easily during the passaging process. (a) Organoids without GSK inhibitor resulted in apoptotic deaths and damaged the formation of the 3-D structure. (b) The addition of GSK inhibitor (2 µM) supplied in every 2–3 days dramatically reduced and protected the cells-damage and stimulated the cell growth 2-threefold faster than in the control culture of the organoids. Human adenocarcinoma of stomach organoid lines, HCM-BROD-0208-C16 cancer model primary adenocarcinoma of stomach was purchased from American Type Culture Collection (PDM-146™; ATCC, USA) and cultured as its recommended protocol [5] (bright view images, scale bar = 100 µm). (c) The stomach organoids were generated by seeding in 96-well plates and grown 2 days GSK inhibitor (n = 6): Th organoids were incubated with CellTiter-Glo®3D reagent (G9681, Promega, Madison, WL, USA) prior to culture medium addition, and results were acquired to assay (*** P < 0.001)

Fig 3: Effect of niche factors in gastric organoid culture. The concentration of R-SPONDIN 1 in the gastric cancer organoids culture was 10%, and the concentration of Wnt3a in the cancer organoids was 20% (panel a) and 10% (panel b). Human adenocarcinoma of stomach organoid lines, HCM-BROD-0208-C16 cancer model primary adenocarcinoma of stomach was purchased from American Type Culture Collection (PDM-146™; ATCC, USA) and cultured as its recommended protocol [5] (bright view images scale bar = 50 µm). (c) The stomach organoids were generated by seeding in 96-well plates and grown 2 days at different percentages of Wnt (n = 6). Th organoids were incubated with CellTiter-Glo®3D reagent (G9681, Promega, Madison, WL,USA) prior to culture medium addition, and results were acquired to assay (*** P < 0.001),