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The differences between cell behaviors in 2-dimension culture in vitro and 3-dimension (3-D) microenvironment in vivo have constituted a major obstacle for tissue engineers. In our research, we study the 3-D culture of ECV304 cells in thermosensitive hydrogel. Human umbilical vein endothelial cells were encapsulated in hydrogel, and then incubated. To determine whether endothelial cells were capable of surviving in Dex-PCL-HEMA/PNIPAAm gels, acridine orange/ethidium bromide (AO/EB) double staining was used to label live and dead cells after two days in culture. 100 muL supernatant was aspirated from each well after 48 hours for the measurement of NO concentration. Cells in gels exhibited mostly rounded morphology or spindlelike shape. After 2 days, gels were degradated as about 70% as the initial, adhered cells were found at the bottom of petri dishes, which escaped from the gels after their degradation. There was little cell apoptosis in 3-D culture. Endothelial cells had greatly proliferated and were at a high density within the matrix after culture for 2 days in growth media. No statistical significance of NO secretion were found between 2-D and 3-D culture.