Collagenase blend f

Skin cell isolation and the following cultivation was performed as previously described in Böttcher-Haberzeth et al. (Böttcher-Haberzeth et al., 2013 (link)). Briefly, skin samples were reduced to small pieces (about 3 mm³) and digested in 12 U/mL dispase (BD Biosciences, Switzerland) combined with Hank’s balanced salt solution containing 5 mg/ml gentamicin (all from Invitrogen, Switzerland) at 4°C overnight. Thereafter, forceps where used to separate the epidermis and dermis for subsequent cell isolation. Epidermal cells were extracted from the epidermis by further digestion in 1% trypsin and 5 mM EDTA (both from Invitrogen, Switzerland) for 10 min at 37°C and afterwards cultured in serum-free keratinocyte medium containing 25 mg/ml bovine pituitary extract, 0.2 ng/ml epidermal growth factor, and 5 mg/ml gentamicin (all from Invitrogen, Switzerland). Further digestion of the dermal tissue in 2 mg/ml collagenase blend F (Sigma, Switzerland) for 4 h at 37°C maintained to the extraction of fibroblasts, which were further cultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 4 mM L-alanyl-l- glutamine, 1 mM sodium pyruvate, and 5 mg/ml gentamicin (all from Invitrogen, Switzerland).

Human fetal fibroblasts were extracted from fetal skin samples, as described previously in^{37 (link)}. Human postnatal dermal fibroblasts and keratinocytes were isolated and expanded from foreskin samples, as described previously^{21 (link),38 (link)}. Briefly, skin samples were cut into small pieces and digested overnight at 4 °C in 12 U ml⁻¹ dispase (BD Biosciences, Allschwil, Switzerland) in Hank's balanced salt solution containing 5 μg ml⁻¹ gentamycin (all from Invitrogen, Basel, Switzerland). Subsequently, epidermis was mechanically separated from dermis using forceps. Epidermis was used for the isolation of keratinocytes while dermis for extraction of fibroblasts. For keratinocytes isolation, epidermal pieces were further digested in 0.5% Trypsin–EDTA (Thermo Fisher Scientific, Basel, Switzerland) at 37 °C for 3 min. Keratinocytes were cultivated in serum free keratinocyte medium (CnT-57, CellnTec, Bern, Switzerland) containing 5 μg/ml gentamycin (Thermo Fisher Scientific, Basel, Switzerland). For fibroblasts isolation, dermis was digested in 2 mg ml⁻¹ collagenase blend F (Sigma, Buchs, Switzerland) at 37 °C for ∼ 60 min. Dermal fibroblasts were grown in DMEM supplemented with 10% fetal calf serum (FCS), 4 mM l-alanyl-l-glutamine, 1 mM sodium pyruvate, and 5 μg ml⁻¹ gentamycin (all from Thermo Fisher Scientific, Basel, Switzerland).