Hydrodynamically generated multilayer skin spheroids enable in vitro screening of biologically active ingredients and toxicity tests

Human tissues often have a multilayer structure, with each layer performing a distinct physiological task. Reconstructing layered tissue structures with their respective functions is crucial for disease modeling, screening biologically active ingredients, and performing toxicology tests; however, multicellular spheroids used for these purposes generally lack a well-defined multilayer architecture. Here, to recapitulate a multilayer structure of the skin, we developed a hydrodynamically mediated approach to the generation of large arrays of fibroblast spheroids (a dermal core) that were engulfed with an epidermal layer of keratinocytes. These spheroids expressed biomarkers of the epidermis, epidermal-dermal junction, and dermis, and exhibited skin-like barrier properties. Screening of the synergistic effect of vitamins and peptides on protein synthesis by the spheroids and evaluation of skin toxicity with chemical agents showed a correlation with clinical results or existing standards. This approach offers enhanced control over spatial cell distribution in spheroids for advanced in vitro models of multilayer tissues.