Dynamically Stretchable Vasculature-on-a-Chip Model by 3D-Printed Porous Molds to Mimic Coronary Arteries During the Cardiac Cycle

<b><i>Background.</i></b> Coronary arteries are not stationary but experience cyclical stretching as the heart expands and contracts. The cyclical stretching and relaxing of the coronary arteries during each cardiac cycle not only influences the vessel on a cellular level but also inevitably affects hemodynamics. Changes in mechanical stretching and hemodynamics of blood vessels have been shown to play key roles in developing cardiovascular diseases (<i>i.e.,</i> atherosclerosis), especially in regions of arterial branching. Despite advances in organ-on-a-chip and 3D bioprinting, however, there is yet a single fabrication technique that enables the fabrication of freestanding, branching vasculature-on-a-chip models to mimic the dynamic stretching movement of <i>in vivo</i> coronary arteries during the cardiac cycle such as simultaneous radial and angular stretching.<br/><b><i>Contribution.</i></b> To bridge this gap, we aim to fabricate a cell-laden vasculature-on-a-chip model to mimic the dynamical movement of a coronary artery. To achieve that, we developed a novel fabrication method to create freestanding, branching, and multilayered vascular constructs using 3D-printed porous molds. We demonstrated that this freestanding vascular model permitted recapitulating physiologically relevant motions (<i>e.g.</i>, linear, radial, and angular stretching) that can be externally controlled.<br/><b><i>Technical achievement. </i></b>To fabricate vascular models, two-part molds were made of porous hydrogels (<i>i.e.,</i> poly(ethylene glycol) diacrylate (PEGDA) hydrogel) that were 3D-printed to bear the shape of interest (<i>i.e.,</i> linear and bifurcating vessels). The molds were immersed in calcium chloride solution prior to casting. When an alginate-containing precursor solution was perfused into the mold cavity, the calcium ions in the mold diffused inwards, prompting ionic crosslinking of the alginate in the precursor, resulting in the formation of a tubular construct. After evacuating the uncrosslinked precursor, the vessel construct was subjected to ultraviolet (UV) radiation to crosslink the photocurable monomer contained in the precursor (<i><u>e.g.</u></i>, gelatin-methacryloyl (GelMA), PEGDA). Using this approach, we successfully fabricated freestanding, branching vascular constructs that were laden with relevant vascular cells (<i>i.e.</i>, smooth muscle cells, endothelial cells). Crucially, our technique permitted the fabrication of multilayered constructs, where the outer core was mechanically reinforced by blending PEGDA, while the inner core was incorporated with bioactive materials (<i>i.e.,</i> GelMA) to improve cell compatibility. This mechanically reinforced outer core was critical for sustaining the cyclical stretching over an extended length of time.<br/>Due to the freestanding and compliant nature of our created vascular constructs, the fabricated vascular models were readily attached to external surfaces that exhibited the desired motions. We fitted the vascular construct on a specially designed chip with an expandable balloon that simultaneously offered radial and angular stretching, which mimics the dynamic movement of the heart during the cardiac cycle. By performing particle image velocimetry (PIV), we observed that stretching and relaxing of the vascular construct drastically altered the fluid flow profile through a bifurcating vascular construct. The developed vascular models were also compatible with vascular stents, which offered a capability to test and observe the device performance under physiologically relevant motions <i>in vitro</i>. <br/><b><i>Significance. </i></b>Overall, we developed a technique to fabricate freestanding, branching, cell-laden vascular constructs that can mimic the dynamic stretching of <i>in vivo</i> coronary arteries during the cardiac cycle. The successful fabrication of a biomimetic vasculature-on-a-chip model is the first step toward a better understanding of CVDs, and we believe that the developed tool has the potential to accelerate future mechanobiological research that advances therapeutic interventions.