Unlike cancer cell lines, tumors and primary cultured cells exhibit phenotypic heterogeneity. Although methods for establishing organoids within three-dimensional (3D) gels are well-known, the growth is slower than that of two-dimensional (2D) cultured cells and many niche factors need to be added. In this study, we established primary cultured organoid in 2D culture (2D organoid; 2D0) in a reproducible manner and with clinical phenotypic heterogeneity. The 2D0 contained cancer stem cells (CSCs) expressing CD44 and CD133. The addition of basic fibroblast growth factor and transforming growth factor-beta as niche factors was necessary to establish the 2D0 and maintain the CSC population. The established 2D0 showed sufficient proliferation, and the culture could be transferred to the 3D culture. Morphological analysis of the xenograft induced by 2D0 reflected parental tumor differentiation, and gene expression in the 2D0 was similar to that in the parental tumor. In vitro drug sensitivity analysis using the 2D0 reflected individual clinical courses. The 2D0 is an in vitro personal cancer model, and the results of the drug sensitivity assessment may be useful for introducing clinical chemotherapy agents in personalized medicine. (C) 2019 Elsevier Inc. All rights reserved.