A Microcarrier Bead-Based Three-Dimensional Angiogenesis Model Using Human Umbilical Vein Endothelial Cells.

Angiogenesis is a multistep biological process involving endothelial cell activation, migration, elongation, lumen formation, branching, and anastomosis. Conventional two-dimensional culture systems are useful for the preliminary evaluation of drugs, genes, or culture conditions, but they do not adequately recapitulate the spatial organization of endothelial morphogenesis within a three-dimensional extracellular matrix. The present protocol describes a microcarrier bead-based three-dimensional angiogenesis assay using human umbilical vein endothelial cells (HUVECs). The HUVECs are first seeded onto microcarrier beads bearing chemically coupled acid-denatured porcine type I collagen and then embedded in a basement membrane matrix. Oral mucosal fibroblasts are subsequently seeded on top of the gel to provide paracrine support for endothelial sprouting and vascular-like structure formation. The culture medium is replaced every 2 days, and sprouting behavior and angiogenic phenotypes are monitored by inverted microscopy. Under appropriate culture conditions, endothelial sprouts emerge within several days, followed by the formation of lumen-like structures and inter-sprout connections. This method provides a convenient and reproducible platform for in vitro studies of endothelial morphogenesis and can be further applied to investigations of gene manipulation, pharmacological intervention, and disease modeling.