Isolation and Characterization of Adipose-Derived Stem Cells

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.