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Implantable 3D engineered vascular tissue constructs can be formed by co-culturing endothelial and fibroblast cells on macroporous scaffolds. Here we show that these constructs can be used for studying the dynamics of neovascular formation  in-vitro  by a combination of live confocal imaging and an array of image processing and analysis tools, revealing multiple distinct stages of morphogenesis. We show that this process involves both vasculogenic and angiogenic elements, including an initial endothelial multicellular cluster formation followed by rapid extensive sprouting, ultimately resulting in a stable interconnected endothelial network morphology. This vascular morphogenesis is time-correlated with the deposition and formation of an extensive extra-cellular matrix environment. We further show that endothelial network junctions are formed by two separate morphogenic mechanisms of anastomosis and cluster thinning.

Vasculogenic dynamics in 3D engineered tissue constructs