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The cancer stem cells(CSCs)from human osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs were isolated and identified.The primary cells derived from human osteosarcoma were digested by trypsin to prepare a single-cell suspension,and mixed homogeneously into 1.2% alginate gel.Single-cell alginate gel was cultured with serum-free DMEM/F12 medium.Epirubicin(0.8 μg/mL)was added to the medium to enrich CSCs.After cultured conventionally for 7 to 10 days,most of cells suspended in alginate gel were killed by epirubicin.But few cells survived and some single-cell cloning spheres formed.Immunofluorescent staining for Oct3/4 and Nanog was implemented to find cells with properties of self-renewal and multi-potential differentiation.Cells from cloning spheres were transplanted into BALB/c mice to detect the tumorigenicity in vivo.The results showed that some cells positive for Oct3/4(TRITC)and Nanog(TRITC)were found in single-cell cloning spheres,and most of positive cells were concentrated in the core of sphere.Cells from spheres could form osteosarcoma in the body of mice.It was concluded that cells from single-cell cloning spheres had the properties of the expression of parts of stem cell genes(Oct3/4 and Nanog),resisting anti-cancer drugs,and tumorigenicity in vivo.To sum up,it is believed that cells obtained from osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs are cancer stem cells.