Background: Methods for the artificial three-dimensional (3D) culture of mouse and human small intestinal and large-intestinal stem cells have been established with CD24(+) or Paneth cell niches. In contrast, no studies have established stable 3D culture for rat colon stem cells. In this study, we established an advanced method for efficient rat colonic stem cell culture. Methods: Using various tissue homogenates, we investigated the colonic organoid forming capacity under the TMDU protocol immediately adjacent to Ootani's 3D culture assembly in the same culture dish. Next, we examined whether the supernatant from the colon could be replaced by a colon homogenate. Finally, we identified the bioactive substances that were indispensable for efficient organoid culture using protein purification by three-step column chromatography and proteomic analysis with a quantitative nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: By combining Ootani's method with the TMDU protocol, we established a refined culture method for Lewis rat colon organoids, which we refer to as the modified TMDU protocol. Furthermore, we confirmed that PGE(2) and galection-4 promoted rat colonic organoid formation. Conclusions: We established efficient rat colonic stem cell cultures in vitro. This success will contribute to the study of rat intestinal-disease models. 2017 Elsevier Inc. All rights reserved.