Abstract
Angiogenesis, the formation of new blood vessels from pre-existing ones plays an important role during organ development, regeneration and tumor progression. The spheroid-based sprouting assay is a well-established and robust method to study the influence of genetic alterations or pharmacological compounds on capillary-like tube formation of primary cultured endothelial cells. A major advantage of this assay is the possibility to study angiogenesis in a 3D environment. Endothelial cells are cultured as hanging drops to form spheroids. Those spheroids are embedded into a collagen matrix and tube formation is analyzed 24 h later. By analyzing sprout number and sprout length the effects of genetic manipulation or drug treatment on angiogenesis can be investigated.
Keywords: Endothelial cells, Angiogenesis, 3D cell culture, Capillary sprout formation, Spheroids, Vascular
Background
Blood vessels supply organs with oxygen and nutrients. In cases the local demands are not met anymore, cells secrete vascular endothelial growth factor (VEGF) to induce the formation of new blood vessels. The new vessel sprout is composed of a leading tip cell which is trailed by stalk cells (Potente and Makinen, 2017). Angiogenesis occurs under physiological conditions (e.g., growth of muscle and adipose tissue) as well as pathological conditions (e.g., wound healing, macular degeneration, and tumor growth). As such, there is a great need to decipher the basic mechanisms coordinating angiogenesis and to test compounds that interfere with pathological angiogenesis.The spheroid-based sprouting assay, which was developed by Dr. Thomas Korff and Dr. Hellmut Augustin in the late 90s (Korff and Augustin, 1999), enables researchers to investigate the effects of drugs or genetic manipulations on sprouting angiogenesis in a fast and robust manner (Heiss et al., 2015). One great advantage of the spheroid-based sprouting assay is the analysis of sprout formation in a 3D environment. This promotes cell-cell signaling between endothelial cells. Upon stimulation with VEGF endothelial cells degrade the surrounding matrix and invade it. This mimics the situation in vivo. Thereby, this assay better reflects in vivo angiogenesis than other well known in vitro angiogenesis assays such as 2D tube formation on Matrigel (Nowak-Sliwinska et al., 2018).Sprout number and length are read-outs for the angiogenic potential. In addition, this assay can be used to analyze competition for the tip cell position. Therefore, genetically modified endothelial cells, which are labeled with different fluorophores, are mixed before spheroid formation (Figure 1). Thereby, it can be analyzed, which genetic manipulation leads to a preference for the tip or the stalk position (Tetzlaff et al., 2018). With live-cell imaging it is possible to investigate the migration of endothelial cells and the dynamic competition of endothelial cells for the tip position.Figure 1. Endothelial cells expressing either mCherry or GFP mixed before spheroid formation. Spheroid was embedded in a collagen matrix and sprouting was analyzed 24 h later by using a fluorescence microscope. Scale bar = 100 µm.
Materials and Reagents
Equipment
Software
Procedure
Note: Hereon prepare the methocel solution containing 20% FBS which is needed on the next day. Mix carefully the methocel stock solution with 20% FBS. Avoid air bubbles. In case air bubbles are formed, check whether they disperse on the next day. If not, use another batch.
Figure 2. Preparation of hanging drops for spheroid formation. A. Petri dish with hanging drops. B. Representative image of a spheroid in a hanging drop 24 h after upside-down incubation. Scale bar = 100 µm.
Data analysis
Notes
Recipes
Acknowledgments
The assay was originally developed by Dr. T. Korff and Dr. H.G. Augustin (Korff and Augustin, 1999; Pfisterer and Korff, 2016). This work was supported by the Deutsche Forschungsgemeinschaft (SFB-TR23, project A7) and the Helmholtz society to A.F.
Competing interests
The authors declare that they have no conflict of interest.
References
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