Recent reports indicate that adipose tissue is a novel source of multipotent stem cells that can be used in cell therapy and tissue engineering. However, using the traditional cultivation of adipose tissue-derived stem cells (ADSCs), it is hard to meet the needs of clinical applications. To obtain a large number of ADSCs while retaining their stemness, we seeded ADSCs in collagen/chitosan scaffolds and compared the proliferation of ADSCs in a 3-D static environment in dishes and a 3-D dynamic environment in spinner flask. The growth dynamic parameters of ADSCs were examined using a CCK-8 kit every other day, and the variations of glucose and lactic acid concentrations were analyzed every day. After 14 days, the cells were observed under a scanning electron microscope. The surface markers (CD29, CD34, CD44, CD45, CD73, CD105, CD166 and HLA-DR), the specific transcription factors (Nanog, Oct-4, Sox-2 and Rex-1) and the multi-differentiation potential (adipogenic, osteogenic and chondrogenic) were also assayed to identify the stemness of expanded cells. The results showed that the cells in scaffolds in spinner flask could be expanded by more than 26 times, and they presented better morphology and vitality and stronger differentiation ability than the cells cultivated in scaffolds statically. All the cells maintained stem cell characteristics after proliferation. Therefore, spinner flask cultivation is an easy-to-use, inexpensive system for expanding ADSCs in 3-D scaffolds.