Table 1.

In vitro, ex vivo and in vivo 3D models/techniques to study angiogenesis.

Assay	Application	Considerations	References
In vitro assays
Cell culture wound closure assay	Assessing EC migration		(71, 72)
Boyden chamber	Assessing EC migration		(73)
Collagen lumen assay	Assess tube and lumen formation	Limited useful time frame (72 h), due to contraction of gels	(74)
Fibrin bead assay	Assessing sprouting and lumen formation		(75)
Vascularised micro-organ platform (VMO)	Assessing all steps of vascular formation. In VMO, endothelial cells form a luminised, perfusable, vascular network that respond to shear stress		(76)
EC-coculture spheroids	Assessing the interplay between endothelial cells and mural cells and special growth of vascular structures, in physiological and pathological conditions. Drug screening		(58, 77, 78)
Ex vivo (organotypic assays)
Aortic ring assay	Assessing the outgrowth of endothelial cells (and other cells) from aortic explants. This system allows to study sprouting from a native tissue, and to test the efficacy of pro- and anti-angiogenic molecules in a physiological environment	Lack of flow. High results variability depending on background and age of the animal or donor	(79, 80, 81)
Retinal explant assay	Studying microvessel formation and its remodelling		(82, 83)
In vivo
In-ovo chorioallantoic membrane assay	Assessing the vascularisation of allografts or xenografts. Testing the effect of pro-angiogenic and anti-angiogenic treatments	The chicken embryos hatch around developmental day 21 and experiments cannot exceed day 18. Limited quantification to evaluate the results	(84)
Corneal angiogenesis assay	Studying molecular and cellular mechanisms of angiogenesis. Testing the effect of pro-angiogenic and anti-angiogenic treatments	It requires considerable technical skills	(85, 86)
Matrigel plug assay	Assessing the effect on vascularisation of cells and substances mixed with Matrigel and injected subcutaneously		(87)