Endothelial Cell Culture and Bacterial Interaction

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.