Tissue engineering of human blood vessels is pursued as a clinical revascularization therapy as well as to develop in vitro human blood vessel models with native-vessel like organization. Clinical revascularization strategies that use autologous vessels are hampered by poor quality and limited availability. Synthetic vascular scaffolds may be used as alternative, but for replacement of smaller vessels (<4 mm range) this is associated with high thrombogenicity and patency loss. Pre-cellularization of the polymer constructs with autologous vascular cells could protect smaller diameter grafts against patency loss and improve long term graft survival upon implantation. In vitro seeded vascular grafts using patient-derived induced pluripotent stem cells (iPSCs) could be used for disease modeling or therapeutic screening. Here, we use iPSCs vascular Organoid Derived Endothelial Cells (ODECs) and Mural Cells (ODMCs) as a (autologous) vascular cell source for in vitro tissue engineering of small diameter biodegradable vascular grafts.
The 3D vascular organoid culture method described by Wimmer et al.  was further developed to isolate pure populations of ODECs and ODMCs, which can be cryopreserved and expanded in traditional 2D culture without loss in cell pool purity, viability, and proliferative capacity. 2D ODEC culture was used in dynamic flow and TEER experiments to determine the shear stress responsiveness of the cells. Both ODECs and ODMCs were subsequently seeded on 3 mm diameter degradable solution electrospun polycaprolactone-bisurea (PCL-BU) scaffolds and exposed to flow for 48 hours.
The 2D dynamic flow experiments and TEER experiments showed that ODECs are shear stress responsive and were able to establish and restore the endothelial barrier after thrombin stimulation. Additional experiments demonstrated that ODECs and ODMCs could be successfully seeded in bilayer configuration on the PCL-BU scaffolds, forming the luminal endothelium and underlying medial layer that partially mimicked the layered structure of a human native vessel. Exposure of the resulting human organoid derived tissue engineered vascular graft (TEVGs) to lumen perfusion in a flow-bioreactor setup showed integrity preservation of the bilayer configuration and endothelial attachment to scaffold substrate after subjection to flow.
In conclusion, iPSC derived vascular organoid cells can be successfully used as a source of functional, flow-adaptive vascular cells for tissue engineering of perfused macrovascular grafts. Therefore, our protocol offers a TEVG based solution for replacement of small caliber native human macrovessels using patient derived cells.
 Wimmer, R.A., Leopoldi, A., Aichinger, M. et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565, 505–510 (2019)."