Spheroid-based human endothelial cell microvessel formation in vivo

The study of angiogenic endothelial cells (ECs) has in recent years greatly stimulated multiple fields of vascular biology research. A number of cellular models and numerous complex developmental, manipulatory and tumor animal models have been developed to study angiogenesis in vitro and in vivo. To connect the versatility of cellular assays with the complexity of readouts of in vivo experimentation, we have developed an endothelial transplantation assay. This assay is based on grafting ex vivo generated EC spheroids (2 d) in a suitable matrix in immunocompromised mice, to give rise to a 3D network of capillaries (20 d). This vasculature connects to the mouse vasculature, is perfused and matures by recruiting mouse mural cells. Here we describe the detailed protocol for this assay, including generation of spheroids, injection into mice, excision and processing of resulting plugs, and quantification by immunohistochemical analysis of the resulting vasculature.