Cell Culture and Seeding Into the AFS Channel

Human Umbilical Vein Endothelial cells (HUVECs) and Human Aortic Endothelial Cells (HAECs) were purchased from Lonza (Cat. No. CC-2517, Cat. No. CC-2535). The culture medium was the endothelial basal medium-2 (EBM-2) supplemented with endothelial growth medium (EGM-2) BulletKit from Lonza (Cat. No. CC-3162). Cells were grown in tissue culture flasks and maintained in humidified atmosphere at 37°C and 5% CO₂. The culture medium was changed every 2 days and cells were used up to the 5th passage to ensure the expression of key endothelial protein components. The procedure to functionalize the microfluidic chip, adapted from Silvani et al. (2019), is here described in detail. Prior to cell seeding, the AFS chip was functionalized with fibronectin (100 μg/mL in EGM, Sigma-Aldrich, Cat. No F1141) using Tygon tubing (John Morris Scientific, Cat. NO ND-100-80), followed by incubation for 45 min at room temperature (RT). Culture flasks, with 80–90% cell confluence, were washed with Dulbecco Phosphate Buffered Saline (PBS) (Sigma Aldrich, Missouri, USA), detached using TrypLE™ solution (Gibco, Cat. No 12604-013) for 2 min at 37°C in 5% CO₂ and blocked with growth media. Cell suspension was harvested and centrifuged at 1,400 rpm for 7 min and the supernatant was discarded. Cells were resuspended in EGM at an average concentration of 10⁸ cells ml⁻¹ and transferred into a 1.5 ml Eppendorf tube. Using Tygon tubing, cells were then pulled through the channel up to the desired confluence of 60–70% and incubated at 37°C + 5% CO₂ for 4 h, to let the cells attach to the bottom of the AFS channels under static conditions. After incubation, the endothelialized channel was ready either for acoustic measurements or to be connected to a peristaltic pump for growth media perfusion. Initially set to 66 μL/min, the flow rate was then increased to 120 μl/min (corresponding to a shear stress of 6 dyn/cm²) until completed 48 h of culture to achieve full junction maturation. All perfusion experiments were performed within a dry incubator to avoid damaging the AFS chip.