Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells

Abstract

The chick chorioallantoic membrane (CAM) assay model of angiogenesis has been highlighted as a relatively quick, low cost and effective model for the study of pro-angiogenic and anti-angiogenic factors. The chick CAM is a highly vascularised extraembryonic membrane which functions for gas exchange, nutrient exchange and waste removal for the growing chick embryo. It is beneficial as it can function as a treatment screening tool, which bridges the gap between cell based in vitro studies and in vivo animal experimentation. In this review, we explore the benefits and drawbacks of the CAM assay to study microcirculation, by the investigation of each distinct stage of the CAM assay procedure, including cultivation techniques, treatment applications and methods of determining an angiogenic response using this assay. We detail the angiogenic effect of treatments, including drugs, metabolites, genes and cells used in conjunction with the CAM assay, while also highlighting the testing of genetically modified cells. We also present a detailed exploration of the advantages and limitations of different CAM analysis techniques, including visual assessment, histological and molecular analysis along with vascular casting methods and live blood flow observations.

VEGF-A and FGF4 Engineered C2C12 Myoblasts and Angiogenesis in the Chick Chorioallantoic Membrane

“…We chose to use the CAM assay to investigate this pro-angiogenic potential. The CAM assay is a highly versatile, cost-effective model of angiogenesis with a vast vascular network of capillaries, veins, and arteries which can be easily manipulated and observed for experimental study [ 37 , 39 , 40 ].…”

“…Any chicks which had perished during the course of the experiment were excluded from the analysis. With the analyst blinded to the treatment used for each on-plant, angiogenesis as a result was scored by determining the vascular index of blood vessels which grew towards the on-plant, similarly, to as described previously [ 37 , 62 , 63 ]. In the case of the CAM filter disc assay, scores of 0, 1, or 2 were assigned to each blood vessel if the angle at which it approached the on-plant (⌀ 6 mm, A = 28.27 mm 2 ) was greater than 45°, and whether it branched within a ring (⌀ 12 mm) encircling each filter disc.…”

“…Several angiogenic assays, both in vitro and in vivo have been used in the past to study angiogenesis. These include in vitro assays such as the use of mouse and human endothelial cell lines (migration, proliferation, survival, and morphogenesis assays) [ 17 ] and in vivo assays including the mouse dorsal skin fold [ 35 , 36 ] and the chick chorioallantoic membrane (CAM) assay [ 37 ]. We have chosen to focus on using the CAM assay and an endothelial cell line to adequately assess the angiogenic potential of the hVEGF-A secreting stable cells.…”

“…We have chosen to focus on using the CAM assay and an endothelial cell line to adequately assess the angiogenic potential of the hVEGF-A secreting stable cells. The chick CAM is a highly vascularised membrane used for nourishment and gaseous exchange in fertilized chicken eggs [ 37 , 38 ]. With a vast vascular network of capillaries, veins, and arteries, the chick CAM can be easily manipulated and observed for experimental study [ 39 , 40 ].…”

A Novel Artificial Intelligence-Based Approach for Quantitative Assessment of Angiogenesis in the Ex Ovo CAM Model

“…In numerous CAM studies, different methods of quantifying vasculature have been used and described [ 6 , 7 , 8 ]. The manual count of the vessels dates back to Folkman’s research group’s first use of the CAM assay in 1974.…”