Stepwise Generation of Vascularized Multilayered 3D Organotypic Skin Models

Skin models play critical roles in understanding disease mechanisms and advancing therapeutic development. However, conventional systems based on 2D cell cultures, in vivo animal models, and ex vivo tissue explants are limited by insufficient physiological complexity, interspecies differences, and restricted accessibility, respectively. Advances in biofabrication technologies have enabled the engineering of 3D skin equivalents that better balance biological complexity and experimental scalability. Here, we present a biofabrication protocol inspired by the regenerative processes of wound healing to construct vascularized 3D organotypic skin models in a stepwise manner. The approach integrates bioprinting for precise spatial organization of cellular compartments with guided cell self-organization to achieve native-like tissue complexity and heterogeneity. Through a programmable culture strategy, tissue maturation proceeds sequentially through keratinocyte proliferation and collective migration, microchannel endothelialization, basal-to-suprabasal differentiation, and progressive extracellular matrix remodeling within a fibrin-based scaffold. The resulting tissue constructs comprise stratified epidermal layers positioned atop a vascularized, fibroblast-remodeled dermal matrix. Beyond reproducing key structural features of human skin, this protocol recapitulates cellular processes associated with tissue regeneration, providing a dynamic platform for investigating disease pathogenesis, progression, and therapeutic responses. Key features • This protocol provides detailed procedures for preparing cells and biomaterials used in bioink formulation. • This protocol outlines a stepwise biofabrication process for integrating keratinocytes, fibroblasts, and endothelial cells into a multicellular skin model. • This protocol employs PolyJet 3D printing for tissue culture chamber fabrication and extrusion-based 3D bioprinting for spatial placement of cell-laden compartments. • This protocol leverages a dynamic, wound healing-inspired cell self-organization process under a programmable culture strategy to promote tissue maturation and architectural development.