Epilife medium

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.

Human epidermal keratinocytes (Thermo Fisher Scientific, Waltham, MA, USA) were cultured in EpiLife medium (Thermo Fisher Scientific) with human keratinocyte growth supplement (HKGS, Thermo Fisher Scientific). Before treatment with the reagents, the cells were cultured in EpiLife medium (Thermo Fisher Scientific) for starvation overnight. The cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C, and the medium was replaced every 2 days. The keratinocytes were stimulated with 10 MOI HKSA to induce inflammatory cytokines and TLR2 signaling.

Reconstructed human epidermis (RHE) samples were prepared as previously described [40 (link)]. Briefly, suspensions of primary human keratinocytes from surgical samples of pediatric foreskins were cultured on 0.5 cm² polycarbonate culture inserts (Millipore, Molsheim, France) in Epilife medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with Epilife supplements and then transferred to the air–medium interface for 10 days and grown in Epilife medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 1.5 mmol calcium chloride and 50 µg/mL ascorbic acid. The 10-day-old RHE samples were then placed in a culture medium containing or not (control) the test compounds (LE and GAGs; systemic application), alone or in combination, at 0.02 mg/mL each, and preincubated for 24 h at 37 °C and 5% CO₂. Then, the RHE samples were stimulated with a cytokine mix of interleukin 4 (IL-4; R&D systems, Minneapolis, MN, USA), IL-13 (R&D systems, Minneapolis, MN, USA), IL-22 (R&D systems, Minneapolis, MN, USA) and TNF- α (R&D systems, Minneapolis, MN, USA) at 3 ng/mL each, and the treatment with the test compounds was renewed or not (stimulated control). The RHE samples were further incubated for 48 h at 37 °C and 5% CO₂. A non-stimulated and non-treated control condition was performed in parallel. All experimental conditions were performed in triplicate.