Endothelial Cell Spheroid Sprouting Assay

Human umbilical vein endothelial cells (HUVEC) were cultured in the Endothelial Growth Medium-2 (EGM-2, Lonza), and cells in the passage 2–5 were used in this study. Some of the experiments were performed with HUVECs stably transduced with LifeAct-Green Fluorescent Protein (GFP). Spheroids (500 cells per spheroid) were formed with the use of an AggreWell 400 device (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. After a 24 h incubation, the spheroids were harvested and embedded in the growth factor-reduced Matrigel (Corning, New York, NY, USA) within a µ-Slide Angiogenesis chamber (Ibidi, Gräfelfing, Germany). EGM-2 medium containing microparticles was added to the samples after Matrigel gelation. Endothelial cell sprouting was monitored for up to 3 days. Alternatively, single cell HUVECs were seeded on top of the Matrigel and the effect of microparticles on capillary network formation in 2D was evaluated after 24 h. Image analysis was performed using ImageJ and the Sprout Morphology plugin [24 (link)]. We estimated the sprouting features including sprout number, length, and density of endothelial cells grown in a bead sprouting assay. Formation of cellular capillary-like networks was evaluated with the Angiogenesis Analyzer ImageJ plugin [25 ].