ObjectiveTo investigate the most appropriate culture time with the action of EGF in colon cancer stem cells enrichment by suspension culture.MethodsDLD-1 cells were cultured in serum-free medium containing 20 ng/mL EGF to generate spheroid cells. The time gradient was set to 10 d, 20 d, 30 d and 40 d, the cell proportion of CD133+, CD44+ and CD133+CD44+ were confirmed by flow cytometery. The ability of self-renewal was detected by the sphere forming assay and the limited dilution assay, and the in vitro tumorigenicity of the cells was detected by the colony formation assay.ResultsIn the 30 d group, the proportion of CD133+ and CD133+ CD44+ cells were significantly higher than those in the other groups (allP<0.05), the CD44+ cell was higher than that in the 20 d group (P<0.05), but there was no significant difference with the other two groups (P>0.05). The results of the limited dilution assay and the colony formation assay, the number of spheres in the 30 d or 40 d group was the highest among the 4 groups, and there was no statistical difference between the 30 d group and 40 d group (P>0.05), with statistically significant difference between the 30 d, 10 d and 20 d groups (all P<0.05). The results of the sphere forming assay and the self-renewal ability of 30 d group was significantly higher compared with other groups (all P< 0.05).ConclusionThe cancer stem cells could be enriched more efficiently by suspension culture using 20 ng/mL EGF for 30 days.