Isolation and Characterization of Aortic MSCs

MSCs were isolated from healthy and diseased aortic tissues according to an established enzymatic method [15 (link), 21 (link)]. Before digestion, the thrombus and the peri-adventitial adipose tissue were removed from the aneurysm sac. The tissues were then cut into 2-cm² sections and incubated with 0.3 mg/ml Liberase type II (Liberase™ Research Grade; Roche) in serum-free Dulbecco’s modified Eagle’s medium (DMEM; Sigma Aldrich) at 37 °C o/n in a rotor apparatus. The digested tissue was filtered through decreasing diameter cell strainers and centrifuged at 400 x g. After cell viability testing, MSCs isolated from aneurysm (AAA-MSCs) and those obtained from healthy aorta (ha-MSCs) were cultured (37 °C incubator, 5% CO₂) in DMEM enriched with 20% fetal bovine serum (FBS; Sigma Aldrich) and expanded in vitro. MSCs at passage 3 were analysed by flow cytometer to detect the expression of mesenchymal markers CD44, CD90, CD73 and CD105, as described previously [15 (link)].