Stable glutamine

Thirty ml of peripheral blood was withdrawn from three groups of CAD patients. Peripheral blood mononuclear cells (PBMCs) were isolated by collecting the buffy coat generated after Lymphoprep™ (Axis Shield, Oslo, Norway) density centrifugation. Mojosort™ magnetic cell separation system (Biolegend, San Diego, California) was employed to further isolate monocytes from the pooled PBMCs. Next, monocytes were re-suspended (1 × 10⁶/ml) in RPMI1640, with stable glutamine (Capricorn Scientific, Germany) and seeded into 25 cm² tissue culture flask (SPL Life Sciences, Korea). Monocytes were allowed to adhere at 37 °C, 5% CO₂ for 3 h. Non-adherent cells were washed off using RPMI 1640 with stable glutamine media. The adherent monocytes were cultured for 5 days in RPMI 1640 with stable glutamine media supplemented with 10% heat-inactivated fetal bovine serum (Capricorn Scientific, Germany), 1% penicillin-streptomycin (Nacalai Tesque, Japan), and 20 ng/ml recombinant GMCSF (Miltenyi Biotec, Germany) for M1 macrophage or 10 ng/ml recombinant MCSF (Gold Biotechnology, Missouri) to generate M2 macrophages. After day 5, M1 macrophages were polarized with 10 ng/ml LPS (Nacalai Tesque, Japan) and 20 ng/ml IFN-γ (Miltenyi Biotec, Germany) for 2 days. Meanwhile, 20 ng/ml IL-4 and 20 ng/ml IL-13 (Stemcell Technologies, Canada) were added into culture media to polarize M2 macrophages.

Both lipoma-derived stem cells (LDSCs) and adipose-derived stem cells (ADSCs) were isolated by enzymatic digestion of tissue samples, respectively, as we previously described [26 (link)]. Stromal vascular fraction (SVF) of cells, obtained from tissue homogenates after collagenase I digestion, was seeded in 25 cm² cell culture flask (Greiner Bio One, Kremsmünster, Austria) in standard cell culture medium that contained Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 2 mM stable glutamine and 1% antibiotic-antimycotic solution (all purchased from Capricorn Scientific, Ebsdorfergrund, Germany). Media were changed 16–18 h after isolation to remove non-attached cells. After reaching confluency, the first cell passage was performed (P1), which enabled purification of mesenchymal stem cells. Cells were cultured in standard cell culture conditions, meaning temperature of 37 °C and humidified atmosphere with the presence of 5% CO₂. Medium was changed every three days. Conditioned media (CM) of LDSCs (LDSC-CM) and ADSCs (ADSC-CM) were collected as a three-day medium just before passage 2 (P2) and stored at –80 °C until further analyses.

The human melanoma (Mewo) cell line (kindly given by Prof. Marc Mareel, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, Belgium) [38 (link)] was maintained in DMEM culture medium with stable glutamine (Capricorn Scientific, Ebsdorfergrund, Germany), supplemented with 10% fetal bovine serum (FBS, GIBCO, Invitrogen, UK), 1x penicillin/streptomycin (Biowest, Nuaillé, France), and 1.25 μg/mL amphotericin B (Corning, NY, USA). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂. Cell lines were authenticated following genotyping at i3S Genomics Core Facility (Porto, Portugal) using the PowerPlex^® 16 HS System (Promega, Madison, WI, USA) and confirmation with the DNA profiles available at ATCC and ECACC STR profiles database.