Nhdf adult

Commercial human dermal fibroblasts (NHDF adult, Promocell, Heidelberg, Germany) were used for all cell experiments. NHDF fibroblasts were cultured in medium containing 72 % Dulbecco’s modified Eagle medium (ATCC, Manassas, USA), 18 % Medium M199 (Sigma-Aldrich, Steinheim, Germany), 9 % fetal calf serum (PAA Laboratories, Pasching, Austria) and 1 % Penicillin–Streptomycin (Invitrogen, Karlsruhe, Germany). Cells were incubated at 37 °C in humidified air with 5 % CO₂ atmosphere. Culture medium was changed twice a week. Cells were subcultured by detachment with Accutase (PAA Laboratories, Pasching, Austria) at approximately 90 % confluence. Fibroblasts from the 2nd to 6th passage were used for all experiments.

Gene transfer of eGFP into NDHF-p adult fibroblasts was achieved by lentiviral transduction according to a published protocol [19 (link)]. The eGFP encoding lentiviral vector pHR′-SEW was used to prepare vector supernatants by transfection of 293T cells as previously described [19 (link)]. For gene transfer, 15.000 fibroblasts (NHDF adult, Promocell, Heidelberg, Germany) were seeded into 24-well tissue culture plates (Greiner, Frickenhausen, Germany). Two rounds of transduction on day 1 and 3 were performed at a cumulative multiplicity of infection (MOI) of ~100 to achieve >98 % gene marking. Transduction efficiency was confirmed by fluorescence microscopy (Wilovert AFL30, Hundt GmbH, Wetzlar, Germany) and flow cytometry FACSCalibur (BD Biosciences, San Jose, USA) using the CellQuestPro Software (BD Biosciences, San Jose, USA).