Huvecs

Prior to cell seeding, the surface of the microfluidic device was coated using 100 μg/ml human fibronectin (Sigma Aldrich, Israel) in PBS. Human umbilical vein endothelial cells (HUVECs), (Lonza, Israel) were used in all assays. HUVECs were cultured in T75/T150 flasks with ECM media supplemented with FBS, Endothelial Cell Growth Supplement, penicillin/streptomycin solution (ScienCell, Switzerland). HUVECs were trypsinized using trypsin B (Biological Industries, Israel), and 10⁶ cells were seeded in each microfluidic channel. The cells were then incubated at 37°C and 5% CO² with standard media for 2 hr to enable a stable cell adhesion.
Inflammation was induced by activating the HUVECs using 0.1 μg/ml TNF‐α (R&D Systems, MN) in a serum free media, which resulted in E‐selectin and ICAM‐1 over‐expression. The activation was induced under static conditions at 37°C and 5% CO₂ over different durations (0.5–6 hr). Then the microfluidic channels were washed twice with PBS. Cells were then fixed using 4% paraformaldehyde (Sigma Aldrich, Israel) for 10 min at room temperature, washed three times and stored at 4°C.

Human umbilical vein endothelial cells (HUVECs) (PromoCell GmbH, Heidelberg, Germany) were used to test the effect of the bacteria on vascular properties. After thawing, the frozen HUVECs were expanded in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO₂ in a humidifying incubator and used at passages p3 to p6 (30 (link), 44 (link)). Cells were grown to 80 to 90% confluence before being transferred to transparent polyethylene terephthalate (PET) Transwell supports (0.4 μm pore size, Greiner Bio-One, Austria), to a plastic well plate (Corning), to a glass-bottom well plate (Cellvis, Mountain View, CA), and to the insert chip (30 (link)). Before seeding, the uncoated substrates were treated with entactin-collagen IV-laminin (ECL) cell attachment matrix (Merck) diluted in Dulbecco’s modified Eagle’s medium (DMEM) (10 μg/cm²) for 1 h in the incubator. Then, the HUVECs, harvested with trypsin/EDTA solution (Biological Industries), were seeded inside the culture platforms at a density of 250,000 cells/cm² and grown for 48 h. Then, bacteria were added and their effect on cell behavior was tested after 1 h, 4 h, and 24 h.

Human vascular endothelial cells (HUVECs) and L929 mouse skin fibroblasts were purchased from the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Biological Industries, USA) and 1% penicillin–streptomycin (Thermo Fisher Scientific, USA). Cells were incubated at 37 °C with 5% CO₂.