FRESH 3D Bioprinted Collagen‐based Resistance Vessels and Multiscale Vascular Microfluidics

The pursuit of an ideal model system to study the pathophysiology of vascular disease has included the use of tissue engineered blood vessels (TEBV), vascularizing engineered volumetric tissue, silicone-based "organ-on-a-chip" platform, and tissue decellularization; yet we are still far from generating engineered vascular tissue that recapitulate native physiology. Despite advances such as 3D bioprinting that have led to new approaches for creating fluidic channels, it remains challenging to produce continuous networks with vessels ranging from large (>6 mm) to small (<1 mm) using native-like extracellular matrix (ECM) that recapitulates the mechanical properties, geometric organization, and complexity of the cellular and extracellular microenvironment. An ideal engineering strategy would combine the controlled fluid flow achieved by organ-on-a-chip platforms with the ECM structure, composition, and biomechanics of decellularized tissue. Here, using Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting, we spatially define ECM composition and vascular geometry to create collagen-based microfluidics and an engineered resistance artery scaffold to study how ECM composition, mechanical properties, and fluid flow contribute to vascular disease. A 3D vascular microfluidic CAD model was designed with a hierarchically branching network containing a single inlet and outlet of 1 mm that branched down to 250 µm channels embedded within a collagen scaffold. This model was FRESH printed with 24 mg/mL collagen-I and yielded a high-fidelity scaffold with patent internal vasculature. 3D imaging and gauging analysis of the CAD model compared to the collagen scaffold revealed an average positive deviation of 9.9 µm and an average negative deviation of 56.3 µm. To interface with and perfuse the soft collagen microfluidic scaffold we 3D printed a custom bioreactor chamber for long-term culture capable of >1-week perfusion under physiological conditions. In addition to embedded vascular networks, FRESH bioprinting allows for direct fabrication of suspended vessels. We designed an ECM scaffold scaled to match a resistance vessel consisting of a collagen-I frame and suspended vessel (inner diameter of 300 µm, outer diameter of 500 µm). The collagen frame provides overall scaffold stability and allows interfacing to the custom perfusion bioreactor. Using multimaterial FRESH printing, we varied the ECM composition by patterning fibronectin along the inner lumen and collagen-I within the outer adventitial wall. Biomechanical evaluation using pressure and wire myography revealed a burst pressure >140 mmHg and an average peak tension of 600 mg, comparable to mouse mesenteric resistance arteries. Together, this work highlights the exciting potential for ECM-based microfluidic scaffolds and vascular tissue engineering that will set the foundation for future work to replicate a healthy and hypertensive vessel microenvironment. In addition to vascular biology, the collagen-based microfluidics are amenable to other organ systems, and can be utilized to study cell proliferation, metastasis, and ECM remodeling in a 3D environment.