Abstract
Artificial skin models have emerged as valuable tools for evaluating cosmetic ingredients and developing treatments for skin regeneration. Among them, 3D skin equivalent models (SKEs) using human primary skin cells are widely utilized and supported by standardized testing guidelines. However, primary cells face limitations such as restricted donor availability and challenges in conducting genotype-specific studies. To overcome these issues, recent approaches have focused on differentiating skin cells from human-induced pluripotent stem cells (hiPSCs). In this study, we developed a protocol to differentiate high-purity skin cells, such as fibroblasts (hFIBROs) and keratinocytes (hKERAs), from hiPSCs. To construct the hiPSC-derived SKE (hiPSC-SKE), a dermis was first formed by culturing a collagen and hFIBROs mixture within an insert. Subsequently, hKERAs were seeded onto the dermis, and keratinization was induced under air-liquid culture conditions to establish an epidermis. Histological analysis with hematoxylin and eosin staining confirmed that the hiPSC-SKE recapitulated the layered architecture of native human skin and expressed appropriate epidermal and dermal markers. Moreover, exposure to Triton X-100, a known skin irritant, led to marked epidermal damage and significantly reduced cell viability, validating the model's functional responsiveness. These findings indicate that the hiPSC-SKE model represents a promising alternative for various skin-related applications, including the replacement of animal testing.