Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a crucial process in development, tissue repair, and various diseases such as cancer. To study angiogenesis in vitro, tube formation assays are widely employed as rapid and quantifiable models that recapitulate key steps of endothelial cell behavior during neovascularization. These assays enable researchers to investigate molecular mechanisms, screen pro- or anti-angiogenic compounds, and explore genetic pathways involved in vessel formation.
Tube Formation Assay Methodology
The endothelial cell tube formation assay involves seeding endothelial cells, such as human umbilical vein endothelial cells (HUVECs) or murine endothelial lines, onto a basement membrane matrix (e.g., Matrigel, collagen, or fibrin gels) that mimics the extracellular matrix environment. Cells rapidly adhere and align, forming capillary-like tubular networks typically within 2 to 16 hours.
Key steps include:
- Preparation of Basement Membrane Matrix: A thin layer of extracellular matrix is polymerized onto culture plates or wells, providing a scaffold.
- Cell Seeding: Endothelial cells are resuspended in tube formation media containing essential nutrients and growth factors and plated onto the matrix.
- Incubation and Monitoring: Cells begin to extend protrusions and migrate, forming interconnected tubular structures with lumen formation within hours.
- Visualization and Quantification: Tube networks are observed under phase contrast or fluorescence microscopy. Quantification includes measuring total tube length, number of branching points, loops, and tube area.
Applications
- Study molecular and genetic regulation of angiogenesis.
- Screen pharmaceuticals for pro- or anti-angiogenic properties.
- Evaluate effects of natural and synthetic compounds on vascular formation.
- Investigate disease mechanisms involving aberrant angiogenesis such as cancer, diabetic retinopathy, and chronic wounds.