A 3D angiogenesis model in vitro has been developed to study migration, survival, proliferation and differentiation of human microvascular endothelial cells (HMVEC) in a fibroblasts (or tumor cells) and collagen environment. The branching three-dimensional capillary-like structures are directly dependent on fibroblast-endothelial cell contact and are not achieved when fibroblasts (FF) are replaced by the different stages of melanoma cells that include radial growth, vertical growth and metastatic stages. Vascular endothelial growth factor (VEGF), when overexpressed in fibroblasts, can stimulate HMVEC proliferation. Neutralizing antibodies against VEGF and blocking antibodies for VEGF-receptor 2 could inhibit but not completely obliterated capillary network formation, suggesting that the VEGF signaling pathway is important but not exclusive. The other fibroblast-derived soluble factors and fibroblast-endothelial cell contact are essential for endothelial cell survival and differentiation.
Day 1: HMVEC cells are plated on bovine collagen I coated 24-well-plate and grow to about 80% confluence.
Day 2: Add 150ul/well collagen I mixture as the first acellular layer on a HMVEC monolayer. 450ul/well collagen I mixture with 2.5-5X105/ml fibroblasts are overlaid as the second cellular layer. 1ml/well EBM-2 media is added on top and media is changed every 2 days.
Day 5-7: Harvest reconstructs and stain HMVEC cells with anti-CD31 or anti-vWF VIII antibody.
1.Enriched 10x M199 media is 10xM199 with vitamin C (50ug/ml), heparin (100U/ml) and FBS (1%).
2. Keep all reagents on ice.
3. Change the amount of Sodium Bicarbonate according to the color of collagen mixture (like straw yellow).