One of the key concepts in tissue engineering (TE) strategies is the need for mass transport of nutrients and oxygen supply. In this work, we propose the creation of hollow tubes, as blood vessel substitutes, able to support endothelial and smooth muscle cells adhesion and proliferation. Hollow tubes will be obtained by building-up multilayers on sacrificial tubular templates using layer-by-layer technology and template leaching . Tubes of chitosan and alginate multilayers were prepared with crosslinking and their physicochemical characterization was performed using different techniques. To optimize cell culture conditions, human endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were separately seeded on tubular structures. Since multicellular systems of ECs and SMCs are the main constituents of blood vessels, cell culture studies were then performed using a co-culture of both cell types. To perform the culture of HUVECs on the inner wall of the tubes and the HASMCs on the outer layer, we develop an home-made apparatus that was able to feed cells with their respective culture medium. Extensive biological characterization was assessed to evaluate the potential of these structures. The hollow tubes produced showed to be a suitable structure to promote cell adhesion and spreading, maintenance of phenotype, opening a new research field in order to develop innovative tubular structures for cardiovascular TE applications.