Cnt 57

Human primary keratinocytes and fibroblasts were isolated from normal human adult female skin obtained from surgical waste skin remnants (abdominoplasty), with written, informed patient consent and full local Institutional Review Board ethics approval. The use of the primary cells was also approved by the Skin Cell Bank IMB‐IRB, Singapore. Fibroblasts were cultured in high glucose DMEM, supplemented with 10% fetal bovine serum and 1% penicillin‐streptomycin. Keratinocytes were expanded in serum‐free keratinocyte media (CnT‐57; Cell‐N‐Tec). All cell culture reagents were purchased from Life Technologies (Carlsbad, CA).

Normal human epidermal keratinocytes (NHEKs) and normal human epidermal melanocytes (NHEMs) were isolated from human neonatal foreskins with modifications as described previously (Yoshida et al., 2007 (link)). NHEKs were preliminarily incubated either in EpiLife medium (Life Technologies, Carlsbad, CA, USA) or in supplemented-PCT epidermal keratinocyte medium (CnT-57; CELLnTEC Advanced Cell Systems Inc., Bern, Switzerland), both of which were supplemented as described previously (Murase et al., 2013 (link)). NHEMs were maintained in Medium 254 (Life Technologies) or in DermaLife^® M Melanocyte Culture Medium (Lifeline Cell Technology, Walkersville, MD, USA) as previously described (Murase et al., 2009 (link)). MNT-1 cells were preliminarily incubated in supplemented-RPMI-1640 medium as described previously (Murase et al., 2013 (link)). In co-culture experiments, NHEKs and NHEMs were seeded in 4-well culture slides pre-coated with Coating Matrix Kit (Life Technologies) at a density of 0.5×10⁵ cells/well and 0.1×10⁵ cells/well, respectively, in culture medium mixed with supplemented-PCT epidermal keratinocyte medium and supplemented-DermaLife^® M Melanocyte Culture Medium at a 3:1 ratio as described elsewhere (Murase et al., 2013 (link)).

Human periodontal ligament fibroblasts (HPLF) were purchased from ScienCell (Cat. 2630) and cultured in DMEM/F12 ((Gibco, 31331-028) containing 20% Fetal bovine serum (FBS) (Sigma, F7524), 1% penicillin-streptomycin (Hyclone, SV30079.01). Human gingival epithelial cells (HGEPp, CELLnTEC, Cat. HGEPp) were cultured in CnT-57 (CELLnTEC, CnT-57). For both cell lines, passage 3–6 cells were used.

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).