The cells were grown in serum containing media until reaching 80% confluence, at which time they were serum starved overnight in endothelial basal medium (EBM; Lonza) or smooth muscle basal medium (SMBM; Lonza) with 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO).
Human coronary artery ECs and human coronary artery smooth muscle cells (SMC) were purchased from Lonza (Cologne, Germany) and cultured in endothelial basal medium and smooth muscle basal medium, respectively, at 37°C, 5% CO2, air humidity until the third passage as previously described.
The proliferation medium consisted of Skeletal Muscle Cell Basal Medium (Cat. No. C-23260, PromoCell), Skeletal Muscle Cell Growth Medium Supplement Pack (Cat. No. C-39360, PromoCell), 10% FBS and 1.5% L-alanyl-L-glutamine (Invitrogen).
Human coronary artery smooth muscle cells (Lonza CC-2583) were grown in smooth muscle cell basal medium (SmBM, Lonza) containing growth factors, cytokines and supplements (SingleQuots Kit, Lonza).
Cells treated with the DMEM β-adrenergic agonist media had a greater amino acid uptake than cells in control DMEM (P < 0.05), whereas there were no differences when placed in skeletal muscle cell basal medium.
The 7304-1 cells were cultured in Skeletal Muscle Cell Basal Medium supplemented with 15% FBS, 5 μg hEGF, 0.5 μg hFGF, 25 mg Fetuin, 5 mg Insulin and 200 μg Dexamethasone (PromoCell GmbH, Germany).
In order to induce differentiation of myoblasts into myotubes, cultures with a confluence of up to 80% were not split and proliferation medium was replaced by differentiation medium composed of Skeletal Muscle Cell Basal Medium, 5% horse serum (Invitrogen) and 5 mg Insulin (PromoCell).
Immortalised VSMCs were cultured in Smooth Muscle Cell Basal Medium (PromoCell, Heidelberg, Germany) supplemented with decomplemented FCS (5%), insulin (5 μg/mL), basic fibroblast growth factor (bFGF) (2 ng/mL), epidermal growth factor (EGF) (0.5 ng/ml), streptomycin/penicillin (1%) and ciprofloxacin (1%) at 37°C in 5% CO2.
Vascular smooth muscle cells were grown in Vascular Cell Basal Medium supplemented with Vascular Smooth Muscle Growth Kit components (ATCC) to a confluency of 80 90 %.
Both strains were cultivated in synthetic growth medium (Bold's Basal Medium) to enhance biomass production.
HASMC (human aortic smooth muscle cell) were grown in SmBM Basal Medium supplemented with SmGM-2 SingleQuot (Lonza).
