Atherosclerosis, the leading cause of cardiovascular diseases [1–3], is a chronic inflammatory disorder characterized by deposition of cholesterol-containing low-density lipoproteins (LDL) in arterial walls, resulting in blood vessel narrowing and impaired perfusion, and increased leukocyte recruitment [4,5]. Current atherosclerosis in vitro models are 2-dimensional (2D), and fail to reproduce important features of atherogenesis including blood flow-induced shear stress and leukocyte-endothelial interactions [6–7]. Herein, we report a novel biomimetic blood vessel model to study the hemodynamics and leukocyte-endothelial interactions using a tunable, 3-dimensional (3D) endothelial barrier to mimic stenotic plaque. We first characterized the effects of THP-1 monocyte adhesion on activated endothelial monolayer, followed by whole blood perfusion at different channel constrictions to study leukocyte rolling phenotype.
A schematic of the polydimethylsiloxane (PDMS) microfluidic device is shown in Figure 1A. It consists of 3 layers, with a top cell culture chamber (800×100 µm (H×W)) for endothelial (HUVECs) cell culture, and a bottom pneumatic channel (1000×100 µm (H×W)), separated by a thin PDMS membrane (10 µm thick). By varying air flow into the pneumatic channel, the membrane is deflected upwards, thereby creating tunable constrictions in the cell culture chamber (Figure 1B). Endothelial inflammation is mimicked by growing HUVECs to confluency and treating them with tumor necrosis factor-α (TNF-α) to study leukocyte-endothelial interactions in cell culture media (RPMI) and whole blood flow.
We first performed confocal imaging to visualize channel constriction using FITC dye and 3D endothelial “stenotic plaque” (Figure 1B, C). The laminar flow profile over the 3D stenosis was studied from the rolling velocities of 10 µm beads, which varied significantly in the stenosis region (Figure 1D). As observed from Figure 2A, HUVECs expressed higher ICAM-1 due to TNF-α-treatment which facilitated binding of THP-1 cells. Interestingly, distinct adherence patterns of THP-1 for 50% and 80% constrictions was observed under flow (1 dyne/cm2) with increased adherence at the top of the constriction (Figure 2B, C). Upon increasing flow rate to 10 dyne/cm2, THP-1 adhesion was reduced in 80% constriction while completely eliminated in 50% constriction. (Figure 2D, E). Finally, whole blood was perfused through the device to study blood flow under stenosis conditions (Figure 3). As expected, significant leukocyte (mostly neutrophils) rolling and adhesion were observed, and average leukocyte rolling velocity was lowest with an 80% constriction (Figure 3D), possibly due to low shear at the top of the stenotic “plaque”. On-going studies are performed to gain better insights into the stenosis-induced hemodynamics effects on leukocyte-endothelial interactions.
In conclusion, the developed atherosclerosis-on-a-chip mimics a physiologically-relevant 3D stenotic blood vessel which enables long-term perfusion cell culture and on-chip visualization of leukocyte-endothelial interactions (rolling, adhesion). This model can also be further developed to study thrombus formation and other endothelial-related dysfunctions in cardiovascular diseases.
