4.2. Cell Culture

Mel-Ab cell line is derived from spontaneously immortalized mouse melanocytes and synthesizes large quantities of melanin. Mel-Ab cells were maintained in DMEM supplemented with 10% FBS, 100 nM TPA, 1 nM CT, and 1% antibiotic/antimycotic solution at 37 °C in 5% CO₂. Human neonatal epidermal melanocytes with moderate pigmentation were cultured in medium 254 supplemented with human melanocyte growth supplement (Cascade Biologics, CA, USA). Melanocytes were used between passages 3 and 7. Human neonatal keratinocytes were cultured in Epilife^® medium supplemented with human keratinocyte growth supplement (HKGS; Cascade Biologics, CA, USA). Keratinocytes were used between passages 2 and 5. Both melanocytes and keratinocytes were incubated at 37 °C in 5% CO₂.

For our coculture model, melanocytes were plated on 6-well plates at a density of 6 × 10⁴ cells per well. After 24 h, keratinocytes were added to each well at a density of 3 × 10⁵ cells for cocultures, with an initial seeding ratio of 5:1. Cocultures were then maintained in keratinocyte media. Twenty-four hours later, wells were treated two times with PDRN and Placentex^®, and melanin content was measured 5 days later.