The three-dimensional culture of blood vessel wall cells now permits the construction of a human blood vessel mimic (BVM). Previous studies have used the human BVM as a tool to perform in vitro testing of medical devices and imaging instrumentation. The purpose of the current study was to enhance this technology through both the elimination of animal serum and the modification of scaffold properties in human BVM preparation. Additionally, BVMs were implanted with vascular stents to observe a potential cellular response to the devices in a serum-free environment. Serum-free culture of human adipose-derived stromal vascular fraction (SVF) cells was accomplished through sequential adaptation from a serum-supplemented medium. The adipose-derived SVF serves as a source of both human endothelium and human smooth muscle cells. Utilizing established pressure-sodding technologies, these cells were incorporated into the luminal surface of either expanded polytetrafluoroethylene (ePTFE) tubular scaffolds or electrospun poly(l-lactide-co-caprolactone) scaffolds, and the resulting constructs were cultivated in a perfusion bioreactor using a serum-free medium. Histological analysis of BVMs created using ePTFE scaffolds indicated that a complete lining of cells had formed on the inner surfaces of the grafts. Vessel mimics were also established under serum-free conditions on the highly porous electrospun tubes, resulting in cellularization throughout the scaffold wall in addition to inner and outer surfaces. Neither endothelial cells nor smooth muscles cells were identified among the mesenchymal cells present in each type of BVM. Bare metal stents were deployed within the electrospun BVMs, and after bioreactor perfusion, scanning electron microscopy and nuclear-specific bisbenzimide staining confirmed the presence of cells on stent surfaces. The outcomes of this study support the hypothesis that BVMs developed using serum-free conditions are affected by scaffold variations and exhibit tissue growth over implanted medical devices. Ultimately, employing serum-free methods could lead to controlled, reproducible BVM production and interpretable, human-specific results in studies of device-tissue interaction, toxicity, and other vascular phenomena; however, comparisons to in vivo biological responses and incorporation of defined blood vessel cells will be critical to validating the serum-free BVM as an appropriate device testing alternative.