(J-365) 3D Bioprinted Collagen Microfluidics for Vascularization of Functional Volumetric Tissue

Introduction:: Microfluidic platforms and organ-on-chip devices attempt to create minimal functional units for experimentation and drug development; however, these rely on soft lithography and sequential layering of polydimethylsiloxane (PDMS) silicones to construct monolithic rectangular channels that lack the complexity and architecture of vascular tissue ECM¹. A key question is: Can we engineer an ECM-based multiscale microfluidic vascular scaffold with complex 3D geometry, accurate mechanical properties, and fluid flow patterns similar to native vasculature? Advances in our FRESH bioprinting platform provide new capabilities to 3D print multiscale vascular networks from ECM proteins (collagen-I, fibronectin, fibrin) at high spatial resolution², but the mechanical softness (E < 150 kPa) of printed hydrogels make interfacing with perfusion systems challenging. Here, we FRESH bioprint collagen-I as the base material to create an endothelialized vascular microfluidic platform that recapitulates the geometries of well-known organ-on-chip devices. A custom bioreactor system that directly interfaces with the printed chips enables continuous perfusion culture and in situ live imaging. By FRESH 3D printing collagen-I microfluidic ships, we transition away from laborious lithographic assembly of inert silicones and plastics to rapidly prototyping remodelable ECM-based chips. Additionally, the fully 3D geometric fabrication and multimaterial capability opens new possibilities for chip design to study diseases and tissue systems in ways previously not possible.


Materials and Methods:: Digital models of the collagen microfluidic chips (parallel channels, mixing, 3D helical, multiscale branching, and lung networks) and a custom perfusion bioreactor were created in Autodesk Fusion 360 and printed from collagen-I (Advanced Biomatrix, 23 mg/mL) and Biomed Clear SLA resin (Formlabs), respectively. Collagen-I was FRESH 3D printed on a custom multimaterial bioprinter equipped with optical coherence tomography (OCT, ThorLabs Vega 1300) for in-process monitoring and 3D geometric gauging (CloudCompare)^3. Perfusion was performed with a peristaltic pump (Ismatec) at 100 µL/min. Fluorescence imaging of perfused dextran was performed on a Leica M165 FC stereo microscope. HUVECs were cultured in EGM-2 media (Lonza), perfusion-seeded into the microfluidic chips, and imaged following immunofluorescence staining and optical clearing on a Nikon FN1 A1R MP confocal microscope.


Results, Conclusions, and Discussions:: Inspired by the success of the organ-on-chip field, we developed Collagen-based High-resolution Internally Perfusable Scaffolds (CHIPS) as FRESH-printed microfluidics to resemble well studied models such as parallel, mixing, and 3D-helical channels at high-fidelity (20x12.5x6 mm volume). 3D OCT imaging and gauging revealed rms error ± 24 µm rms error and observable patency of 500 µm internally perfusable channels. A custom bioreactor enabled perfusion of a red and blue dye into the collagen construct demonstrating the quality of the interface between the printed collagen and bioreactor. 24-hour continuous perfusion and real-time imaging of fluorescence dextran demonstrates rapid (3/10 kDa), gradual (40 kDa), and minimal (70 kDa) diffusion that correlates with the size of the dextran. Additionally, using multimaterial printing capabilities, we FRESH-print cellular channel linings (fibrin/fibronectin), regionally localized growth factors (VEGF), and alter material stiffness (collagen concentration from 6-35 mg/mL). Following 12-days of perfusion, HUVECs formed a confluent monolayer and extensive microvessel networks within the FRESH-printed CHIPS. Here we present FRESH CHIPS as a collagen-based microfluidics and perfusion bioreactor vascularization platform to mimic the cellular and ECM composition for tissue engineering of functional volumetric tissue.


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References (Optional): : [1] Bhatia SN, Ingber DE. Microfluidic organs-on-chips. Nat. Biotechnol. (2014); [2] A. Lee, et. al., 3D bioprinting of collagen to rebuild components of the human heart. Science. (2019); [3] Tashman JW, et. al. In Situ Volumetric Imaging and Analysis of FRESH 3D Bioprinted Constructs Using OCT. Biofabrication. (2022)