Penicillin streptomycin amphotericin

Primary rat aortic smooth muscle cells (SMCs) were isolated from the aorta via careful removal of the adventitia and endothelial cell lining, followed by a 1 h incubation with trypsin (0.25%) and collagenase (5 units ml⁻¹). Only passage numbers under ten were used. All cells were cultured in monolayer cultures at 37 ºC and 5% of CO₂ until spheroid assembly. SMCs were cultured using Dulbeco’s modified Eagle medium: F-12 (ATCC, 1:1, DMEM:F-12) supplemented with 10% fetal bovine serum (Atlanta Biologics) and 1% penicillin–streptomycin–amphotericin (MediaTech, Inc.).

VICs were harvested from aortic valve leaflets of fresh porcine hearts obtained from a local commercial abattoir (Fisher Ham and Meats, Spring, TX) according to a validated protocol.³¹ To loosen endothelial cells, dissected leaflets were digested in a collagenase 2 solution (500 U/mL, Worthington Biochemical, Lakewood, NJ) for 30 min at 37 °C. After the endothelial cells were scraped from both surfaces of the leaflets, the residual leaflet tissue was minced. The minced tissue was digested in a collagenase 3 solution (300 U/mL, Worthington Biochemical) for 4 h at 37 °C. The solution was passed through a 70 µm cell strainer and then VICs were plated in tissue culture polystyrene (TCPS) flasks. VICs were cultured in a standard humidified incubator (37 °C, 5% CO₂) in 50:50 DMEM/F12 media (Corning, Tewksbury, MA) with 10% bovine growth serum (BGS, Lonza, Walkerville, MD), 1% 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Thermo Fisher Scientific, Waltham, MA), and 1% penicillin/streptomycin/ amphotericin (Corning). For consistency, all VICs were frozen after passage 1 and used in passages 2–3. VICs were used from three separate harvests with cells pooled from all aortic valve leaflets of six porcine hearts during each harvest.