1. Human microvascular endothelial cells (HMVECs) with primary cell kits were cultured on collagen type I-coated dishes to 80% confluency, then overlaid with acellular collagen prepared in primary cell medium and supplemented with heparin, vitamin C, endothelial cell growth serum, and 10% FBS.
2. After polymerization, the collagen was overlaid with a second collagen layer containing 5 x 10⁵ cells/mL primary human dermal fibroblasts.
3. Endothelial cell growth medium was changed every 48 h.
4. Labeling of the endothelial cells with DiI and subsequent in situ observation of the reconstruct by inverted fluorescence microscopy showed that detachment of the endothelial cells from the substrate and migration into the acellular collagen layer began within 4 h.
5. After 24 h, the endothelial cells had migrated through the acellular collagen into the fibroblast-containing collagen layer and were found there throughout with increasing density over time.
6. Double staining for the proliferation marker Ki67 and endothelial-specific marker von Willebrand factor (vWF) demonstrated that endothelial cells proliferated in the matrix even after 5 days.
7. The endothelial cells came into close contact with fibroblasts and began forming vacuoles within 2 days and aligned into cords that structured into branching networks within 3–5 days.
8. Capillary networks formed by day 4 to 5 and increased through day 11.


 

Reference:
OMAIDA C. VELAZQUEZ, RUTHANNE SNYDER, ZHAO-JUN LIU, RONALD M. FAIRMAN and MEENHARD HERLYN. Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like 3-dimensional networks. The FASEB Journal. 2002; 16: 1316-1318.