Melanocyte growth medium

Mouse melanoma B16F10 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Hyclone, Logan, UT, USA). Human melanoma MNT-1 cells were grown in Minimum Essential medium (MEM, Hyclone). All the media were supplemented with 10% fetal bovine serum (FBS, Hyclone, USA) and 1% of a penicillin–streptomycin solution. Normal human epidermal melanocytes (NHEM, PromoCell, Heidelberg, Germany) at passage number 5 or 6 were maintained in a melanocyte growth medium (PromoCell). These cells were incubated at 37 °C in a humidified atmosphere condition containing 5% CO₂.

After three weeks, 6-well- dishes were confluent. Cultured Schwann cells were transferred to a T-75 poly-l-lysin (PLL)-coated flask (TPP, Biochrome, Berlin, Germany). Then cells were detached using 0%, 25% Trypsin/EDTA (Biochrome, Germany). Cells were re-suspended in Melanocyte Growth Medium (PromoCell, Germany) and 1% Penicilin/Streptomycin +0.7 µL Forskolin (Sigma Aldrich, St. Louis, MO, USA)/mL Medium and transferred to a PLL-coated T-75 Flask. Change of medium was performed once per week. Progress in purification was observed daily in light microscopy.

Organotypic cultures were prepared using a previously established transwell system consisting of 6 well cell culture inserts containing a porous membrane (3.0 µm pore-size, BD Falcon)^{22 (link)}. Collagen type I hydrogels containing human dermal fibroblasts were used as dermal part of the skin substitutes. Briefly, 0.7 ml rat-tail collagen type I (3.2–3.4 mg/ml, BD Biosciences), was added to 0.2 ml chilled neutralization buffer containing 0.15 M NaOH and 1 × 10⁵ fibroblasts. After polymerization (10 min at room temperature and 20 min at 37 °C) these dermal equivalents were grown in DMEM supplemented with 10% FCS for 5 days. Corresponding to the physiological ratio of melanocytes to keratinocytes (~1:5), 5 × 10⁴ melanocytes or melanoma cells (M070413) were mixed with 2–2.5 × 10⁵ keratinocytes and seeded onto each dermal equivalent. To avoid dispersion, the cells were placed into siliconized polypropylene rings of 1.5 cm diameter. After 12 h the rings were removed, 1 ml keratinocyte (Keratinocyte-SFM, Invitrogen)/melanocyte medium (Melanocyte Growth Medium, PromoCell) (ratio 5:1) was added in the upper chamber, and 2 ml was added to the lower chamber. Culturing for 2 weeks with regular medium changes gave rise to the dermo-epidermal skin substitutes used for transplantation.