4.1. Cell Culture

Porcine aortic endothelial cells were harvested from porcine aortas (collected from a local slaughterhouse), then isolated and cultured as previously described in [45] by gently scraping the intima of the descending part of the porcine aorta. After centrifugation at 1400× g for 15 min in Medium 199 (M-199), the pellet of PAECs was purified from this suspension, resuspended in M-199 medium with Earle’s salts, and supplemented with 100 IU/mL penicillin G, 100 µg/mL streptomycin, and 20% newborn calf serum. Cells were then seeded onto culture dishes or silicone dishes fixed on glass coverslips and incubated at 37 °C in 5% CO₂. This study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication, 8th edition, 2011). All experiments were performed in accordance with the regulations of the Animal Research Committee of the Hamamatsu University School of Medicine.