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Background: Cancer stem cells (CSCs) are thought to be able to survive conventional chemotherapeutic treatments because the cells have more resistant to anticancer drugs than common cancer cells.Most in vitro studies in experimental cancer cells have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cancer cells behave differently when they are grown within a three-dimensional (3D) extra-cellular matrix.Purpose and Methods: In the present study, we isolated the CD44+CD117+CSCs from human epithelial ovarian cancer SKOV-3 cell line by magnetic associated cell sorting, analyzed the characteristics of CD44+CD117+CSCs and evaluated the effect of anticancer drugs 5FU, docetaxel, cisplatin and carboplatin on CD44+CD117+CSCs in a 3D versus a 2D in vitro environment as well as primarily investigate mechanism of the resistance to chemotherapeutic agents in CD44+1 17+CSCs.Results: The data showed that the CD44+CD117+CSCs possess faster grow trait than SKOV-3 cells in 3D culture.While anticancer drugs 5FU, docetaxel, cisplatin, and carboplatin inhibited CD44+CD117 +CSC growthby 50% in 2D culture in their IC5o values, this effect was only 34.4%, 40.8%, 34.8% and 21.9% at 3D one, respectively.Regarding the impact of paclitaxel on the cell viability of CD44+CD 117+CSCs detected by cell inhibitory and cell survival assays, fewer cells underwent apoptosis in a 3D culture than that in a 2D one.In the 3D culture model, cytotoxic drugs markedly affected the expressions of ABCG2 and ABCB1 of CD44+CD117+CSCs compared with the 2D system.Conclusion: Human epithelial ovarian cancer SKOV-3 CD44+CD117+ CSCs in 3D culture exhibit more chemoresistant than in 2D one, and the 3D culture provides a realistic model for study of the CSC response to anticancer drugs.