BEADS: An Interactive Semi-Automated Workflow for 3D Fibrin Angiogenesis Assays Enabling Co-Culture and Directionality Analysis

3D fibrin bead angiogenesis assays are widely used to study endothelial sprouting <i>in vitro</i>, yet current analytical approaches are either time-consuming or poorly adaptable to complex imaging conditions, limiting quantitative assessment of co-cultures, spatial interactions, and nearest-neighbor-dependent angiogenic behavior. In this study, we developed a semiautomated user-interactive image analysis pipeline, Bead-based Endothelial Angiogenesis Data Suite (BEADS), to provide standardized quantitative bead-centric metrics of sprouting, migration, and spatial orientation in 3D fibrin angiogenesis assays. BEADS integrates automated bead detection with manual correction, followed by guided sprout and migratory-cell annotation across multi-channel image z-stacks. Novel analytical capabilities include co-culture designation, nearest-neighbor pairing, and circular statistics for sprout-directionality quantification. Performance was evaluated in assays using co-cultured male and female human pulmonary microvascular endothelial cell (HPMEC)-coated beads. BEADS reduced hands-on analysis time approximately sevenfold compared with manual tracing while preserving sprout-length accuracy against manual ground truth. BEADS provides a standardized, extensible platform for microvascular image analysis, supporting co-culture experimentation, spatial endothelial-interaction metrics, migratory-cell quantification, and high-throughput adaptation. This semiautomated workflow enables quantitative microvascular research by integrating computational precision with endothelial behavior and is broadly applicable to angiogenesis assays that incorporate co-cultures, perturbations, or multi-label experimental designs.