The technique of gently harvesting cells and cell sheets using stimuli-responsive cell culture carriers was applied to human umbilical vein cord endothelial cells (HUVEC). To meet the particular requirements of this demanding cell type, a copolymer consisting of N-isopropylacrylamide and diethyleneglycol methacrylate (poly(NiPAAm-co-DEGMA)) was combined with a fine-tuned protein pre-coating. Using this approach the detachment of HUVEC sheets was studied. Furthermore, the behavior of the extracellular matrix upon cell detachment was followed by protein staining. The results demonstrate the feasibility of harvesting HUVEC sheets from stimuli-responsive polymer layers and provide valuable options for the advanced engineering of vascular structures.