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There is still a pressing need to develop small-diameter vascular vessels for vascular reconstructive procedures. Tissue Engineering offers the prospect of being able to meet this medical demand, as it allows the development of structurally complex blood vessels substitutes 1. Accordingly, the ultimate aim of this work is to develop small diameter vascular substitutes based on layering multiple cell types. Co-culture systems of human endothelial-smooth muscle cells and fibroblastic-smooth muscle cells were initially established. These co-cultures were then assembled to develop a tri-culture system, which mimics the structural organization of a blood vessel. Electrospun nanofibrous meshes were used as culturing substrates, which restrict cell migration although enabling biochemical communication 2. All the established culture systems presented viable and proliferive cell populations over time. Interestingly, the tri-culture system presented protein synthesis values much higher than the co-cultures, mostly of collagen. On the immunofluorescence micrographs were observed the maintenance of cell type-specific proteins expression, even in the presence of another cell type. Quantification of Growth Factors (GFs) on conditioned media of the co- and tri-culture systems demonstrated a synergistic interplay between Vascular Endothelial GF (VEGF) and basic Fibroblast GF (Bfgf). The VEGF was mainly expressed by smooth muscle cells, which leads to increasing levels in the co- and tri-culture systems. A similar trend is observed for Bfgf, expectedly produced by the fibroblastic cells. By its side, the platelet derived GF levels remain unaltered among conditions. This study demonstrated the fundamental importance of the intercellular crosstalk between endothelial, smooth muscle and fibroblastic cells. It reinforces the potential of a tri-culture system in the development of tissue engineered blood vessel substitutes.