Background The disturbed flow contributes to juxta-anastomotic intimal hyperplasia (IH) in arteriovenous fistulas (AVFs). This study developed an in vitro method aiming to understand the hemodynamic impact on endothelial cells (ECs) in AVFs. Methods A tubular bifurcation AVF model was constructed, and the disturbed flow was induced near the bifurcation by pulsatile flow. Hemodynamics was simulated using computational fluid dynamics (CFD) and visualized as 2D contour plots. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured on a tailored polycarbonate membrane (PCM) and placed in the model. HUVECs on the PCM allowed precise mapping to the hemodynamic plots. Results CFD identified four regions: the outer wall with high time-averaged wall shear stress (TAWSS MAX) and transverse wall shear stress (TransWSS MAX), the inner wall with low and oscillatory wall shear stress (L/O), and the pulsatile flow (PF). HUVECs in PF were aligned in the direction of flow. The cells in other regions showed more focal adhesion junctions and fewer glycocalyces. HUVECs on inner wall had the lowest expression of Krüppel-like factor 2 and endothelial nitric oxide synthase, while the outer wall showed the highest expression of platelet-derived growth factor and transforming growth factor-β. Conclusions We developed an in vitro AVF model and validated the effects of different hemodynamic profiles on ECs by matching CFD plots with cell positions on a tailored PCM. This study shows that the in vitro AVF model can be a promising tool to assess the impact of interventions aimed at improving ECs function in AVFs. Statement of Significance In Vitro Model Development : An innovative in vitro model was developed to simulate arteriovenous fistula conditions, allowing for direct assessment of endothelial cell behavior under varied hemodynamic conditions. Linking Hemodynamics to Cell Response : The research successfully correlated computational fluid dynamics results with specific endothelial cell positions, facilitating a clearer understanding of the impact of hemodynamics on cell morphology and function. Arteriovenous Fistula Failure Understanding : The study enhances the understanding of arteriovenous fistula failure mechanisms, specifically the role of intimal hyperplasia caused by disturbed flow.

Disturbed flow is one of the pathological initiators of endothelial dysfunction in intimal hyperplasia (IH) which is commonly seen in vascular bypass grafts, and arteriovenous fistulas. Various in vitro disease models have been designed to simulate the hemodynamic conditions found in the vasculature. Nonetheless, prior investigations have encountered challenges in establishing a robust disturbed flow model, primarily attributed to the complex bifurcated geometries and distinctive flow dynamics. In the present study, we aim to address this gap by introducing an in vitro bypass flow model capable of inducing disturbed flow and other hemodynamics patterns through a pulsatile flow in the same model. To assess the model's validity, we employed computational fluid dynamics (CFD) to simulate hemodynamics and compared the morphology and functions of human umbilical venous endothelial cells (HUVECs) under disturbed flow conditions to those in physiological flow or stagnant conditions. CFD analysis revealed the generation of disturbed flow within the model, pinpointing the specific location in the channel where the effects of disturbed flow were observed. High-content screening, a single-cell morphological profile assessment, demonstrated that HUVECs in the disturbed flow area exhibited random orientation, and morphological features were significantly distinct compared to cells in the physiological flow or stagnant condition after a two days of flow exposure. Furthermore, HUVECs exposed to disturbed flow underwent extensive remodeling of the adherens junctions and expressed higher levels of endothelial cell activation markers compared to other hemodynamic conditions. In conclusion, our in vitro bypass flow model provides a robust platform for investigating the associations between disturbed flow pattern and vascular diseases.