Tunable Synthetic Hydrogels to Study Angiogenic Sprouting.

Angiogenic sprouting, the formation of new blood vessels from pre-existing vasculature, is tightly regulated by the properties of the surrounding tissue microenvironment. Although the extracellular matrix has been shown to be a major regulator of this process, it is not clear how individual biochemical and mechanical properties influence endothelial cell sprouting. This information gap is largely due to the lack of suitable in vitro models that recapitulate angiogenic sprouting in a 3D environment with independent control over matrix properties. Here, we present protocols for the preparation of endothelial cell spheroid-laden synthetic, dextran-based hydrogels, which serve as a highly tunable 3D scaffold. The adjustment of the hydrogels' adhesiveness, stiffness, and degradability is demonstrated in detail. Finally, we describe assays to elucidate how individual matrix properties regulate angiogenic sprouting, including their analysis by immunofluorescence staining and imaging. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of methacrylated dextran (DexMA) Basic Protocol 2: Generation of endothelial cell spheroids in microwells Basic Protocol 3: Endothelial cell sprouting in hydrogels of tunable stiffness Basic Protocol 4: Endothelial cell sprouting in hydrogels of tunable adhesiveness Basic Protocol 5: Endothelial cell sprouting in hydrogels of tunable degradability Basic Protocol 6: Imaging of endothelial cell spheroid-laden hydrogels Support Protocol 1: Preparation of pro-angiogenic cocktail for endothelial cell sprouting.