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Objective: To improve the understanding of the progression and treatment of cancer for the development of more accurate and physiologically relevant in vitro tumor models that serve as important tools in cancer research. Methods: Hydrogels for 3D cell culture were prepared as previously described by our group. To measure the cell proliferation and chemotherapeutic sensitivity, the WST-1 colorimetric assay was performed and colony-forming assay was performed to compare colony growth. Wound-healing assay was performed by making scratch wounds when cells reached confluency and scratch closure was monitored. Hydrogel invasion assay was performed and the depth of cell invasion was observed by cell fluorescence using a confocal laser scanning microscope. Western blot was performed for the detection of apoptotic proteins and key EMT and stemness markers were observed by RT-PCR. Flow cytometry was performed for the detection of cancer stem cell (CSC) population and CSC isolation. Results: 3D ovarian cancer (OC) exhibited biochemical and physiological features, including (1) enhanced cell proliferation, migration and invasion, colony formation, and chemoresistance; (2) suppressed apoptosis with altered expression levels of apoptosis-regulating molecules; (3) upregulated expression of crucial multidrug resistance-related genes; (4) accentuated expression of key molecules associated with malignant progression, such as epithelial--mesenchymal transition transcription factors, Notch, and pluripotency biomarkers; and (5) robust enrichment of ovarian CSCs. Conclusion: An effective 3D in vitro culture method was developed and optimized for growing multicellular OC spheres derived from different OC cell lines. And we demonstrated its usefulness in the isolation and enrichment of ovarian CSCs. [ABSTRACT FROM AUTHOR]