Abstract: Objective：To investigate the feasibility of preparing ABCG2 positive gastric cancer cell line MKN45 by spheroid cell suspension culture method. Methods：The human gastric cancer cells MKN45 were cultured in the serum free medium which contained fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The formation of spheroid was investigated and the expression levels of stem cell marker ABCG2 in the spheroid cells and primary adherent cells were detected. Results：Under the condition of serum free culture, the gastric cancer cell line MKN45 could form spheroid. The expression level of ABCG2 in the spheroid cells was higher than that in the parental cells, the difference was statistically significant (P<0.05). Conclusions：The spheroid cells formed by suspension culture of gastric cancer cell line MKN45 possess the characteristics of cancer stem cells.