In vitro culture of patient‑derived tumor cells offers many advantages in the development of novel therapies for colorectal cancer. Although various culture systems have been developed, the long‑term expansion of patient‑derived tumor cells remains challenging. The present results suggested that tumor cells isolated from colorectal cancer patient‑derived xenografts can be efficiently immortalized in conditioned medium from irradiated feeder cells containing Y‑27632, a rho‑associated coiled‑coil containing protein kinase (ROCK) inhibitor. Patient‑derived tumor cells proliferated rapidly, reaching 90‑95% confluence in ~6 days. Short tandem repeat analysis suggested that these tumor tissues and cultured cells presented 13 identical short tandem repeat loci, including Amelogenin, Penta E, Penta D, D2S1338 and D19S433. Their epithelial phenotype was confirmed by staining for epithelial cell adhesion molecule and cytokeratin 20, whereas vimentin was used as a mesenchymal marker. When cells were transferred to 3D cultures, they continued to proliferate, forming well‑defined tumor spheroids. Expression levels of human telomerase reverse transcriptase and C‑Myc mRNA were increased in cultured cells. Finally, immortalized cells were used for the screening of 65 anticancer drugs approved by the Food and Drug Administration, allowing the identification of gene‑drug associations. In the present study, primary culture models of colorectal cancer were efficiently established using a ROCK inhibitor and feeder cells, and this approach could be used for personalized treatment strategies for patients with colorectal cancer.