A self-assembled fibroblast-endothelial cell co-culture system that supports in vitro vasculogenesis by both human umbilical vein endothelial cells and human dermal microvascular endothelial cells

The construction of vascularized connective tissues is an important goal in tissue engineering in that the presence of a patent bio-engineered vasculature should facilitate vascularization of an implant. Fibroblasts play an essential role in the angiogenic process through their production of extracellular matrix molecules and through their release of essential growth factors. Therefore, the aim of this study is to develop a thin 3-dimensional model in which fibroblasts support endothelial cells in the formation of tube-like structures. Macro- and microvascular endothelial cells were seeded onto confluent lawns of human fibroblasts and were cultured in the presence of high levels of ascorbate 2-phosphate to create a tissue-like structure in which endothelial cell organized into tube-like structures. The process was visualized in the culture dish through labeling of cells with a long-lasting fluorescent vital dye. Intact sheet-like structures were created in which endothelial cell tube-like structures were encased by fibroblasts and were surrounded by a basement membrane. These structures appeared to contain a lumen and remained stable for up to 5 weeks in culture. This culture system provides an in vitro method to study fibroblast-endothelial cell interactions and to study the effects of pro- and anti-angiogenic factors on endothelial cell differentiation. This system also provides an experimental basis for developing vascularized tissue-engineered connective tissue.