Isolation and Culture of Primary Skin Cells

Human primary keratinocytes (HEK) were derived from prepuce tissue discarded after surgery in children 3–14 years of age. The study was approved by the ethics committee of the Affiliated Hospital of Guizhou Medical University. All subjects’ parents or guardians provided written informed consent. Purified HEK was obtained through a two-step enzyme digestion method and was grown in EpiLife® medium (MEPI500CA, Invitrogen Cascade Biologics, USA) with human keratinocyte growth supplement (HKGS, S0015, Invitrogen Cascade Biologics, USA), 2 mM L-glutamine (1051024, Gibco, USA), and a 1% penicillin and streptomycin solution (2114092, Biological Industries, Israel). HEK in the second passage was used in this study. Human immortalized keratinocytes (HaCaT) were purchased from Kunming Cell Bank of Type Culture Collection, Chinese Academy of Science (KCB200442YJ, Kunming, China). HaCaT was cultured in Dulbecco’s modified Eagle’s medium (DMEM, 0030034DJ, Gibco, USA) containing 10% fetal bovine serum (FBS, FBSSA500-S, AUS GeneX) and a 1% penicillin and streptomycin solution and cultured in an incubator with 5% CO₂ at 37 °C. HaCaT in passages 10–25 was used in this study.
Human primary melanocytes (MC) were obtained from human children’s foreskin with a two-step enzyme digestion method, and MC was immediately established in Medium 254(M254500, Gibco, USA) supplemented with a human melanocyte growth supplement (S0165, Gibco, USA), 2 mM L-glutamine, and 1% penicillin and streptomycin solution.