Generation of Human Epidermal Equivalents

Human N/TERT keratinocyte cell line N/TERT-2G, purchased from J. Rheinwald laboratory (Harvard Medical School, Boston, MA, USA), was cultured in Epilife medium (MEPI500CA, ThermoFisher Scientific, Waltham, MA, USA), complemented with human keratinocyte growth supplement (S0015, ThermoFisher Scientific) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich, Saint-Louis, MO, USA). Human epidermal equivalents (HEEs) were generated as previously described^{62 (link)}, with minor adjustments. Briefly, inert Nunc cell culture inserts (141002, ThermoFisher Scientific) were coated with rat tail collagen (100 μg/mL, BD Biosciences, Bedford, USA) at 4°C for 1 hour. 1.5×10⁵ N/TERT-2G keratinocytes (either wildtype, ΔAHR, or ΔTFAP2A keratinocytes) were seeded on the transwells in 150 μL Epilife medium (ThermoFisher Scientific) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) in a 24 wells format. After 48 h, cultures were switched to a mixture of CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) and DMEM medium (60:40 (v/v)) without penicillin/streptomycin for 24 h and then cultured at the air-liquid interface for an additional ten days. Culture medium was refreshed every other day until harvesting at day ten of the air-exposed phase.