Ex-ovo cultured chorioallantois membrane as a biointerface platform for extended angiogenesis studies

Angiogenesis modeling in a physiologically relevant, ethically acceptable, and versatile system remains a critical need in cancer biology, regenerative medicine, and preclinical drug testing. The chick chorioallantoic membrane (CAM) is a well-established model for studying angiogenesis, tumor growth, tissue grafting, and therapeutic responses; however, conventional in-ovo approaches are limited by a short experimental window (E7-E14.5), immune-related constraints, and restricted imaging accessibility. In this review, we critically assess the CAM current biomedical applications, including tumor angiogenesis and metastasis, biomaterial integration, immune-oncology modeling, and high-content drug screening. We present a temporal profiling of angiogenesis-associated genes during CAM development, revealing a coordinated sequence of pro-angiogenic, remodeling, and inhibitory phases, and identifying E10-E11 as the optimal harvest window for maximal sprouting and vascular responsiveness. To overcome in-ovo limitations, we propose and validate an ex-ovo cultured CAM model, surgically isolated from the embryo and maintained as a viable, vascularized scaffold. Proof-of-concept assays illustrate feasibility, including preserved endothelial integrity (CD31, CD34), metabolic activity (MTT), oxidative responses (DHR123), and nitric oxide secretion, supporting the conceptual framework developed in this review. With strong potential for standardization, this ex-ovo CAM system could bridge the gap between simple in-vitro assays and complex in-vivo models, enabling longer-term experimentation, precise spatial control, and compatibility with tumor organoids, immune cell co-cultures, and engineered tissues. STATEMENT OF SIGNIFICANCE: The chick chorioallantoic membrane (CAM) is widely used to study angiogenesis and tumor biology, but its in ovo format is limited by a short experimental window and ethical constraints. This review introduces an ex-ovo cultured CAM system that preserves vascular and extracellular matrix integrity while extending experimental usability. By positioning the CAM as a biologically derived biointerface, we highlight its capacity to model biomaterial-cell interactions, vascular remodeling, and tissue integration. This platform provides a cost-effective, ethically refined, and translational tool that bridges in vitro assays and mammalian models, directly advancing biomaterials science and regenerative applications.