Advances and limitations in angiogenesis assays: Integrating in vitro, in vivo, and emerging technologies

The study of angiogenesis has rapidly evolved from fundamental morphometric observations to complex, multidimensional investigations. This comprehensive review synthesizes the expanding toolkit of angiogenesis assays, encompassing established in vitro models, ex vivo systems, and advanced in vivo platforms. Furthermore, we detail the integration of next-generation technologies, including artificial intelligence-based image analysis, spatial transcriptomics, and CRISPR-Cas9 screening, into modern vascular biology. Crucially, this review moves beyond the mere description of methodologies to provide a strategic, application-driven decision-making framework for researchers. By introducing a comprehensive decision matrix and an algorithmic selection guide, we empower investigators to navigate the complexities of assay selection. This framework carefully balances physiological fidelity and expected readouts against practical laboratory constraints and the ethical imperatives of the 3Rs principle (Replacement, Reduction, and Refinement). Ultimately, this strategic approach enables the design of highly reproducible, cost-effective, and translationally relevant experimental pipelines tailored to specific molecular, metabolic, or pharmacological research questions.