Keratinocyte and Fibroblast Cell Culture

Primary cultures of keratinocytes and fibroblasts were isolated according to standard protocols from skin biopsies taken from the thigh of individuals P5 and P6 of family three, both of whom carried the splice site mutation. Keratinocytes were cultured in defined, serum-free keratinocyte growth medium containing human keratinocyte growth supplement (SFM and Keratinocyte Supplements, Life Technologies). Fibroblasts were cultured in serum-free medium (FibroLife Serum-Free Cell Culture Medium, Cellsystems). For control experiments, we used the neonatal normal human epidermal keratinocyte cell line (NHEK-Neo, LONZA). Upon confluency, epidermal differentiation of keratinocytes was induced by maintaining the keratinocytes in medium supplemented with 1.1 mM Ca²⁺, 30 μM palmitic acid, and 15 μM linoleic acid.^{10, 25} Fatty acids were complexed to fatty acid-free BSA (both Sigma-Aldrich) in a ratio of 3:1. All cells were kept at 37°C in a humidified atmosphere with 5% CO₂. Media were changed every second day.