Table 3 Aortic ring assay

• Cost-effective because the aorta is waste tissue from endpoint animal studies

• From one aorta, a high yield of replicates can be obtained (approximately 20 rings per adult animal)

• Rapid set up including aorta isolation and embedding

• Evaluates key steps of angiogenesis including matrix degradation, cell migration, proliferation and morphogenesis into tubular endothelial network

• In addition to endothelial cells, includes cell types important for angiogenesis such as resident pericytes and fibroblasts

• Endothelial cells have not been preselected by passaging and thus are in a quiescent state at starting point of the assay, reflecting in vivo conditions

• Quiescent endothelial cells respond by proliferating and differentiating into tubular networks

• Evaluates properties of cocultured candidate cell types including the ability to respond to signals of aortic tissue, induce migration, ECM processing and homing to endothelial networks

• Cell-to-cell connections can be observed using fluorescence microscopy

• Net effects on angiogenesis can be quantified using image analysis software to assess various network properties (network radial growth, loops, branches and nodes)

• Vessel outgrowths occur from a major vessel while in vivo angiogenesis occurs typically from micro vessels

• Takes 3–5 days for initial endothelial network to develop

• Variability in angiogenic response can also occur between animal’s due to strain, age and gender

• Lack of blood flow (limitation shared with other in vitro and ex vivo angiogenesis assays)

• Angiogenic vessel growth is in three dimensions, rendering imaging and quantification difficult

• Outgrowth vessels regress over time (2 weeks), thereby limiting long-term analysis