In Vitro Expansion of Dermal Fibroblasts

Adult NHDF were obtained from Coriell (Camden, NJ, USA). Dermal fibroblasts from patients with PXE were provided according to the authors’ description in [28 (link)], who also listed the clinical characteristics of the PXE patients. The study was approved by the Ethics Committee of the HDZ NRW, Department of Medicine, Ruhr University of Bochum (registry no. 32/2008, approval date is 3 November 2008). Primary cells were maintained under standardized conditions (37 °C, 5% CO₂) as a monolayer culture in tissue culture dishes (100 × 20 mm, Greiner bio-one, Frickenhausen, Germany) with Dulbecco’s modified Eagle’s medium without phenol-red addition (DMEM; Thermo Fisher Scientific, Waltham, MA, USA). DMEM was supplemented with either 10% (v/v) fetal calf serum (FCS; Biowest, Nuaillé, France) or 10% (v/v) lipoprotein-deficient FCS (LPDS) and 4 mM L-glutamine (PAN Biotech, Aidenbach, Germany), 1% (v/v) Penicillin-Streptomycin-Amphotericin B solution (100×; PAN Biotech, Aidenbach, Germany), as described previously [34 (link)]. The LPDS was prepared according to our previous work [28 (link)]. Medium changes were performed twice a week. The subculturing of near confluent primary NHDF was performed with an expansion ratio of 1:3 utilizing 0.05% (v/v) trypsin (PAN Biotech, Aidenbach, Germany) in Dulbecco’s phosphate buffered saline (PBS, 1×; Thermo Fisher Scientific, Waltham, MA, USA).