A Standardized Protocol for Inducing Stress-Induced Premature Senescence in Primary Human Melanocytes Using Tert-butyl Hydroperoxide.

Environmental stressors, including UV radiation, air pollution, or cigarette smoke, drive extrinsic skin aging. Exposure to these extrinsic aging factors promotes the accumulation of senescent cells, which contributes to the formation of wrinkles and pigmentation disorders. Melanocytes, which provide primary UV protection, exhibit reduced activity with age. Senescent melanocytes accumulate in aged skin and contribute to its aging appearance; However, there are few models to study melanocyte senescence, and this work aims to develop a reproducible model for further research. In this study, we present a senescence model of human neonatal melanocytes (HNM) using tert-butyl hydroperoxide (tBHP), a well-characterized oxidative stress inducer. tBHP disrupts redox homeostasis by depleting cellular antioxidant defenses and promoting radical overproduction, resulting in DNA and protein damage. We characterize the senescence phenotype of tBHP-treated melanocytes through the analysis of established senescence markers, including restricted growth potential, morphological changes, and SA-β-Galactosidase activity. This model provides a valuable tool for investigating stress-induced premature senescence in melanocytes, offering insight into their role in extrinsic skin aging and pigmentation disorders.