Table 1.

Comparison of in vitro assays to study angiogenesis

Type	Assay	Strengths	Weaknesses	References
2D	Scratch	Easy to perform Easy to quantify	Lacks soft substrate for the cells Migration is in 2D	[12]
	Tube formation	Cells adhere to soft substrate Self-organization into cords Reasonable throughput Tools are available for quantification	No distinct tip/stalk cell phenotype Basement membrane extracts contain significant levels of growth factors and have a high batch-to-batch variability Limited tube survival (< 2 days) High use of reagents compared to microfluidic assays Lumens not accessible nor perfusable	[13]
3D	Spheroid	Cells grow in 3D in a soft supportive matrix Endothelial cells differentiate into tip and stalk cells Clear lumen formation Fusion of sprouts is observed Laser dissection allows capture of cells Tools available to quantify the angiogenic sprouts	Lacks spatial control over gradients Higher use of reagents compared to microfluidic assays Spheroids are randomly distributed throughout gel/matrix Lumens are not accessible nor perfusable	[14, 15, 40]
	Microfluidic	Biochemical gradients can be created and maintained Lumen formation occurs early (more comparable  to in vivo) Angiogenic sprouts can be perfused Spatial control over multiple cells (e.g., fibroblasts, pericytes)	Some devices require for pumps to supply flow and maintain gradients Handling and scalability issues due incompatibility with other equipment Some devices need to be manufactured by the end-user Biocompatibility of the used materials Lack of standardization Limited possibilities to extract a subset of cells	[18–30, 32, 33]