Tumor microenvironment has been implicated in tumor development and progression. As a three-dimensional tumor microenvironment model, air liquid interface (ALI) organoid culture system was recently produced. In our previous study, ALI organoid model from normal and tumor colorectal tissues of human patients was established. Both organoids were successfully generated and showed cystic structures containing an epithelial layer and surrounding mesenchymal stromal cells. Structures of tumor organoids closely resembled primary tumor epithelium. Tumor organoids were more resistant to toxicity of 5-fluorouracil and Irinotecan than colorectal cancer cell lines, SW480, SW620 and HCT116 (Usui et al., Stem Cells Int, 2016, Curr Protoc Toxicol, 2018). We also demonstrated that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer (Usui et al., Int J Mol Sci, 2018).