*2.2. Bi-Dimensional Culture of Keratinocytes*

Holoclone keratinocyte samples were thawed and amplified in bi-dimensional mass conditions one week before use for skin substitute bioengineering. Cultures were performed in a serum-containing medium, in the presence of a feeder layer of human dermal fibroblasts growth-arrested by γ irradiation (60 Gy), as described in [11]. Plastic devices coated with type I collagen were used (Biocoat, BD Biosciences, Le Pont de Claix, France). Composition of the serum-containing medium included DMEM and Ham's F12 media (Gibco, ThermoFisher, Les Ulis, France) (*v*/*v*, 3/1 mixture), 10% fetal calf serum (Hyclone, Fisher Scientific, Illkirch, France), 10 ng/mL epidermal growth factor (EGF) (Chemicon, Fisher Scientific, Illkirch, France), 5 µg/mL transferrin (Sigma, Saint-Quentin Fallavier, France), 5 µg/mL insulin (Sigma, Saint-Quentin Fallavier, France), 0.4 µg/mL hydrocortisone (Sigma, Saint-Quentin Fallavier, France), 180 µM adenine (Sigma, Saint-Quentin Fallavier, France), 2 mM tri-iodothyronine (Sigma, Saint-Quentin Fallavier, France), 2 mM L glutamine (Gibco, ThermoFisher, Les Ulis, France) and 100 U/mL penicillin/streptomycin (Gibco, ThermoFisher, Les Ulis, France).
