Senescent and Damaged VSMC Cultures

Human coronary smooth muscle cells from a young healthy donor were purchased from Promocell (C-12511) and cultured in growth medium (Promocell, C-22062), supplemented with 10% FBS and 1% penicillin-streptomicin (Sigma Aldrich). To achieve RS, cells were serially passaged (Bielak-Zmijewska et al., 2014 (link)). Cells were cultured until 70–80% confluence and re-seeded in T75 Flasks (Thermo Fisher Scientific, Nunc EasYFlask, #156499) at a density of 3.5 × 10³ cells/cm² until proliferative arrest (passage 6–8). Cumulative population doublings were counted using the formula cPD = X + 3.322^∗(log Y−log I), X representing the cumulative population doubling of the subculture used to initiate the culture, Y being the viable cell number on harvest, and I the cell number on inoculation. Low passage VSMCs (passage 2–3) were treated with bleomycin to induce DS, as previously reported (Gardner et al., 2015 (link)).