Dulbecco vogt modified eagle medium

Dermis was fragmented using scissors and digested using a stirring solution of 2 mg/mL of Type I collagenase (Gibco^TM) in Dulbecco–Vogt modified Eagle medium (Gibco^TM) for 20 h at 37 °C. The digested tissue was filtered using a Cell Strainer of 100 µm (Fisherbrand^®), neutralized using fibroblast medium and centrifuged (20 min/24 °C/450× g) [11 (link)]. Finally, dermal cells were counted and seeded at 115,000 cells/cm² in fibroblast medium. This primary culture was named passage 0 (P0). The estimated time and cost (considering only the price of the enzymes) for the dermal cell isolation were 22 h and USD 11.00, respectively.

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)).