Spatial distribution and density of fibroblasts determine angiogenic response of endothelial cells

Fibroblasts play indispensable roles in orchestrating revascularization and angiogenesis of endothelial cells. However, it remains elusive the specific conditions under which the influence of fibroblasts becomes important in modulating angiogenic formation. Here, we extend a previously established microfluidic co-culture system to enable precise manipulation of the density of human lung fibroblasts (HLF) and their distance to human umbilical vein endothelial cells (HUVECs) in a wide range within fibrin hydrogels. Using live cell imaging and image analysis, we quantify the effects of fibroblast density, HLF-HUVEC cell-cell distance, and culturing time on cell migration, morphology, microvasculature, and endothelial sprouting. We find that, for fibroblasts to have a significant positive impact in the angiogenesis of endothelial cells, the fibroblast density should be no less than ~3×10⁶ cells/ml and the cell-cell distance should be no greater than ~2 mm. The experimental findings can be captured by a minimal model accounting for the concentration profile of pro-angiogenic factors secreted by fibroblasts. Our results provide insights into the remodeling of vasculature and the guidance for engineering vascularized tissue mimics.