In the past 25 years, several efforts have been focused on developing vascular substitutes showing long-term patency when implanted in humans. In this study, we present our last findings in an ongoing project aiming to develop a tissue-regenerated blood vessel from collagen-based scaffolds. A collagen-based scaffold with adequate biological properties was developed by directly assembling collagen and cells in a cylindrical geometry. The technique mainly involves the use of a ventilated rotating tube allowing smooth muscle cells to grow in a cylindrical collagen matrix. Scanning electron microscopy (SEM), histology, and cell cycle analyses were carried out in order to assess the biological potential of the cell-based scaffold for further maturation. Compliance results showed that this technique allows cells to assemble in the collagen matrix, thus providing enough rigidity to the structure to be handled and mounted in a perfusion bioreactor for further growth and maturation.