Insulin transferrin sodium selenite solution

Approximately 0.5–1 cm² of normal skin was collected from 6 healthy donors with their signed consent after clinical surgery. After removing the fat and blood on the skin, the skin was rinsed three times with PBS. The skin tissues were minced into 0.5- to 1.0-mm³ fragments and digested with type II collagenase (2 mg/ml; Sigma-Aldrich) at 37°C for 4–6 hours. When SGs were released from skin tissues, they were collected with a fine needle and transferred to culture plates containing basic SG medium containing DMEM supplemented with 10% FBS (Gibco/Thermo Fisher Scientific), 100 U/ml penicillin (Sigma-Aldrich), 100 mg/ml streptomycin (Sigma-Aldrich), 2 mM l-glutamine (Sigma-Aldrich), insulin-transferrin-sodium selenite solution (1 ml/100 ml; Sigma-Aldrich), 2 nM/ml triiodothyronine (T3; Sigma-Aldrich), 0.4 mg/ml hemisuccinate hydrocortisone (Sigma-Aldrich), and 10 ng/ml human recombinant EGF (Invitrogen/Thermo Fisher Scientific) [10 (link), 12 (link)]. The SGs from skin tissues were cultured for approximately 1–2 weeks, and the medium was changed every 2–3 days. The SGs were maintained at a density of 1 × 10⁴ cells/cm² as positive controls in this study [10 (link), 12 (link)].