Isolation and Culture of Human Keratinocytes and Fibroblasts

Human keratinocytes and fibroblasts were isolated from the foreskin of a healthy individual (14 days old) as previously described [27 (link)]. Primary fibroblasts and HeLa cells (ATCC CCL-2) were cultured in fibroblast medium (Dulbecco-Vogt modified Eagle medium (Gibco™, Waltham, MA, USA) supplemented with 10% Avantor Seradigm FB Essence serum (Avantor^®, Radnor, PA, USA), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 25 μg/mL gentamicin (Gemini Bio, West Sacramento, CA, USA)). Human keratinocytes were cultured on a feeder layer composed of irradiated human fibroblasts cultured in keratinocyte medium (Dulbecco-Vogt modified Eagle medium (Gibco™): Ham’s F12 (Gibco™), ratio 3:1, supplemented with 24.25 μg/mL adenine (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 0.4 μg/mL hydrocortisone (Galenova, Saint-Hyacinthe, QC, Canada), 0.212 μg/mL isoproterenol hydrochloride (Sigma-Aldrich), 5% bovine HyClone FetalClone II serum (GE Healthcare, Chicago, IL, USA), 10 ng/mL human epidermal growth factor (Austral Biologicals, San Ramon, CA, USA), 100 U/mL penicillin (Sigma-Aldrich) and 25 μg/mL gentamicin (Gemini Bio)).