2.9.1. Culture of Human Skin Biopsies and Treatment

Skin punch biopsies (12 mm) were collected from elective abdominoplasties purchased from HKB Surgery Hospital in Huntersville, NC. After trimming the subcutaneous fat from the skin, the 12 mm biopsies were washed in PBS and placed in 6-well plates containing 1 mL of fresh DMEM High Glucose Media containing 10% FBS, 100 U/mL Penicillin, 100 mg/mL Streptomycin, and 0.25 mg/mL Amphotericin B (Gibco, ThermoFisher Scientific, Waltham, MA, USA). Skin explants were left overnight in an incubator at 37 °C and 5% CO₂ to recover; these were then treated with 20 µL of the ethanol solution (Sol) and different LBNs that were unloaded (Sol, TB1, TP1, MAD) or FA-loaded (Sol-FA, TB1-FA, TP1-FA, MAD-FA) by applying them onto the skin using a glass rod. Twenty-four hours after the treatment, skin biopsies were exposed to 0.4 ppm of ozone (O₃) for 4 h by using an ozone generator, as previously described [34]. Samples were collected 6 and 24 h after the end of the O₃ exposure and processed for the subsequent analysis. The experiment was conducted in three different donors.