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Table 1 Available assays to evaluate angiogenic potential

From: Angiogenic potency evaluation of cell therapy candidates by a novel application of the in vitro aortic ring assay

Assay References Advantages Limitations
In vitro
Endothelial proliferation assays Gomez and Reich 2003 [19]
Yu et al. 2004 [20]
• Reproducible and easy to set up
• Provide quantifiable data
• Measure proliferation and apoptosis
• Short window of analysis after culture due to endothelial cell senescence
• Sensitive to cell density
• Involve endothelial cells from macrovascular origin (HUVEC)
Endothelial cell migration assays
• Transwell systems
• Wound healing assays
Wong and Gotlieb 1984 [21]
Schor et al. 2001 [22]
Albini et al. 2004 [23]
• Reproducible
• Short duration (4–6 hours)
• Quantitative analysis of endothelial cell migration over time
• Sensitive to small alterations in concentration gradients
• Difficult to define and maintain transmembrane gradients
• Inability to observe cell migration in transwell
• Challenging to establish matching conditions between control and experimental groups
• Difficult to obtain accurate results with low cell counts
Endothelial tube formation assays Lawley and Kubota 1989 [24]
Kanzawa et al. 1993 [25]
Auerbach et al. 2003 [26]
• Useful to test angiogenic and anti-angiogenic effects of compounds
• More representative of in vivo angiogenesis than 2D assays
• Introduce ECM to culture system
• Measure proliferation and differentiation
• Lack of consistent lumen formation
• Homogeneous tubules
• Sensitive to uneven matrix coating of wells and cell density
• Time-consuming analysis (multiple parameters of analysis)
Ex vivo
Chick aortic arch model Staton et al. 2009 [18] • System includes nonendothelial cells (pericytes, smooth muscle cells) and ECM
• Easy set up, early cell outgrowth (48 hours)
• Embryonic endothelial cells resemble microvascular phenotype
• Endothelial cells are in a proliferative state in the embryo (not representative of true in vivo scenarios)
In vivo
Chick chorioallantoic membrane assay Ribbati et al. 1995 [27]
Ejaz et al. 2004 [28]
• Simple and inexpensive
• Ideal to implant tissue or organ grafts
• CAM membrane is immunoprivileged enabling xeno-graft studies
• Ideal for large-scale screening
• Noninvasive observation
• CAM has endogenous vasculature, difficult to distinguish pre-existing and newly formed vasculature
• 11-day incubation time prior to implantation of test reagents
• Incision in shell may induce inflammation and a specific angiogenic response
• Sensitive to oxygen tension
Matrigel™ plug assay Passaniti et al. 1992 [29]
Baker et al. 2006 [30]
• Quantitative histological analysis
• Matrigel™ provides natural environment for angiogenesis
• Time consuming, including 2 weeks of plug incubation in host, isolation, sectioning, analysis
• Costly
Sponge/matrix implant assay Salvatore et al. 1961 [31]
Dellian et al. 1996 [32]
• Include defined polymers to study angiogenesis
• Angiogenic response can be monitored over time in live animals
• Ideal for studying tumor-induced angiogenesis
• Implants can become encapsulated with cytokine-secreting macrophages
• Inflammatory response may interfere with angiogenic response
• Undesirable fibrosis
• Variable retention of test compounds in different substrates
Corneal assay Gimbrone et al. 1974 [33]
Muthukkaruppan and Auerbach 1979 [34]
• New blood vessels easily observed due to the absence of background blood vessels
• Executed in mice, rats, rabbits
• Noninvasive monitoring and data easily quantifiable
• Challenging surgical procedure
• Limited space of test substance injection
• Inflammation difficult to avoid
• Atypical angiogenesis because cornea is avascular
• Costly
Dorsal air sac model Selye 1953 [35]
Oikawa et al. 1997 [36]
• Adaptable to various applications
• Allow continuous non invasive monitoring of endothelial networks
• Difficult to distinguish pre-existing and newly formed vasculature
• Delicate procedure (irritation to dorsal skin)
Zebrafish assay Rubinstein 2003 [37]
Isogai et al. 2001 [38]
• Enables large-scale projects
• Shared angiogenic genes and mechanisms
• Easily monitored/quantified
• Ideal for testing of anti-angiogenic compounds
• Suitable for genetic studies of angiogenesis
• Relevance of fish endothelial cell angiogenesis is under debate
• Nonmammalian cell types and involves embryonic cells
• Costly to maintain breeding conditions
  1. CAM Chick chorioallantoic membrane assay, ECM extracellular matrix, HUVEC Human umbilical vein endothelial cells