Endothelial cell migration is a crucial step in the process of new blood vessel formation-a necessary process to maintain cell viability inside thick tissue constructs. Here, we report a new method for maintaining cell viability and inducing cell migration using a perfused microfluidic platform based on collagen gel and a gradient hydrogel sheet. Due to the helpful role of the extracellular matrix components in cell viability, we developed a hydrogel sheet from decellularized tissue (DT) of the bovine heart and chitosan (CS). The results showed that hydrogel sheets with an optimum weight ratio of CS/DT = 2 possess a porosity of around 75%, a mechanical strength of 23 kPa, and display cell viability up to 78%. Then, we immobilized a radial gradient of vascular endothelial growth factor (VEGF) on the hydrogel sheet to promote human umbilical vein endothelial cell migration. Finally, we incorporated the whole system as an entirety on the top of the microfluidic platform and studied cell migration through the hydrogel sheet in the presence of soluble and immobilized VEGF. The results demonstrated that immobilized VEGF stimulated cell migration in the hydrogel sheet at all depths compared with soluble VEGF. The results also showed that applying a VEGF gradient in both soluble and immobilized states had a significant effect on cell migration at limited depths (<100 μm). The main finding of this study is a significant improvement in cell migration using an in vivo imitating, cost-efficient and highly reproducible platform, which may open up a new perspective for tissue engineering applications.