Table 1.

In vitro, In vivo, and Ex vivo models of angiogenesis

	Assays	Technique	Scientific readout	Advantages	Disadvantages
In vitro	Endothelial cell proliferation assay	Cell counting	Percentage of viable cells	Quantification of proliferating cells, apoptotic cells, and DNA content of the cell	Prone to manual error
		MTT assay	Measuring succinate dehydrogenase activity	Less expensive	Time-consuming
		3H-Thymidine /Bromouridine incorporation assay	DNA/RNA synthesis
	Endothelial cell migration assay	Boyden chamber	Cell migration and invasion	Determination of migratory capacity of cells	Technical difficulties
		Matrix degradation assay	Molecular factors influencing migration	Quantification of the rate of migration	Time-consuming
		Wound healing	Directional cell migration		Low rate of reproducibility
					Unable to accurately determine differences between proliferation and migration
	Endothelial cell differentiation assay	Matrigel assay	Rearrangement of cells to form tubules	Quantification of pro-angiogenic factors	Technical difficulty
		3D spheroid assays	Paracrine interactions and modulated pathways	Influence of biomolecules on ECs	Time-consuming
		Co-culturing ECs with other cell types
In vivo	Matrigel plug assay	Immuno-histochemistry staining	Quantification of newly formed blood vessels	Ideal model to study tissue regeneration	Expensive
					Time-consuming
	CAM assay	Immuno-histochemistry staining	Formation of new blood vessels	Evaluation of angiogenic response	Sensitivity of the membrane to oxygen tension
	Corneal angiogenesis assay	Microscopic observation	Vessel length and vascular sprouts	The reliable method as the cornea is devoid of pre-existing vasculature	Inappropriate for large scale studies
		Immuno-histochemistry staining
	Rodent mesentery angiogenesis assay	Immuno-histochemistry staining	Percentage of vascularized area	Extremely thin tissue enables easy visualization	Difficulty in quantification of angiogenesis
				High sensitivity
Ex vivo	Rat aortic ring assay	Microscopic observation	Angiogenic sprouts and vessel length	Mimics in vivo conditions	Vessel growth is influenced by surrounding tissue
	Chick aortic arch assay	Microscopic observation	Cellular proliferation, migration, tube formation and vessel branching	Less expensive and less experimental time	Vessel growth is influenced by surrounding tissue
	Rodent ear angiogenesis assay	Intravascular staining with biotinylated lectin	Vessel growth and branching	Easy visualization	Vessel growth is influenced by host cell interactions
				Mimics in vivo conditions
	Mouse fetal metatarsal angiogenesis assay	Immuno-histochemistry	Vessel sprouts, molecules influencing angiogenesis	Better representative of in vivo sprouts	Devoid of biomechanical force influencing the phenotype
				Employs microvascular cells	Requires technical precision