A Vascularized, Adipose-Containing Human Skin Equivalent Enables Long-Term Culture and Models Orthopoxvirus-Mediated Immune Suppression

Human skin is a complex organ whose functions depend on coordinated interactions between the epidermal and stromal layers. Reproducing this architecture in vitro remains challenging, as existing human models reproduce a restricted subset of the structural and functional features inherent to native tissue. Here, we describe a fibrin-based, multicellular human skin equivalent (HSE) composed of primary human keratinocytes, fibroblasts, preadipocytes, and endothelial cells organized into epidermal and stromal compartments. The resulting constructs achieved mature epidermal stratification with appropriate phenotypic markers, developed robust barrier properties, underwent spontaneous endothelial network assembly, and exhibited transcriptional profiles consistent with native skin. Optimized culture conditions supported long-term structural and functional stability through 42 days, maintaining a proliferative basal cell layer and intact epidermal architecture. Non-destructive optical coherence tomography enabled longitudinal monitoring of epidermal growth, providing a practical method for real-time quality assessment. To evaluate their utility for disease modeling, HSEs were challenged with cowpox virus to model infection by a classic dermotropic virus. Infected HSEs reproduced classic epithelial pathologies of human orthopoxvirus infection and exhibited dose-dependent suppression of host interferon signaling pathways, recapitulating known viral immune-evasion mechanisms. Together, these findings establish the vascularized, adipose-integrated HSE as a platform for long-term studies of human skin biology, host–pathogen dynamics, and therapeutic development.