Liberase type 2

Segments of variously sized arteries with different embryological origin (epiaortic district and thoracic aorta) were obtained by postmortem human donors. The samples frozen for more than 5 years were dissociated by enzymic digestion with 0.3 mg/ml Liberase type II (Roche, Milan, Italy) in serum-free Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland) overnight at 37°C using a rotor apparatus. After digestion, the homogenate was filtered through a cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA), seeded at 1 × 10⁵/cm² on collagen-I coated T75 flasks plates with smooth muscle growth medium-2 (Sm-GM2; Lonza) and incubated at 37°C in a humidified atmosphere with 5% CO₂. Nonadherent cells were removed after 72 hours by washing with phosphate-buffered saline (PBS). Culture media was changed every 3 days until testing. When cells were near confluence, they were expanded in vitro for at least 14 passages. Before the isolation, a small piece of each vascular segment as well as the remaining digested tissue was fixed, hematoxylin and eosin stained and analyzed to verify the efficiency of the isolation method.

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)].