An integrated design strategy for structurally stable full-thickness human skin models with regulatory-qualified barrier assessment

Three-dimensional full-thickness (FT) human skin models provide advantages over reconstructed human epidermis (RhE) by incorporating dermal-epidermal interactions but face persistent challenges with scaffold detachment, contraction, and OECD-compatible barrier validation. In this study, we establish design strategy for in vitro full-thickness (FT) human skin by defining and implementing coordinated dermal and epidermal design principles that stabilize construct geometry and support regulatory-relevant barrier function. Quantitative analysis reveals that fibroblast-mediated detachment and contraction in dermal equivalents are governed by a critical fibroblast-per-collagen threshold, defining conditions that maintain structural integrity. In epidermal equivalents, keratinocytes alone can generate sufficient mechanical stress to detach and deform COL1 scaffolds, and poly(dopamine) (PDA) coating reinforces tissue-insert anchorage to suppress detachment and geometric deformation without altering keratinocyte activation. Incorporating a Matrigel/COL1 dermal matrix that provides a basement-membrane-derived matrix environment improved epidermal stratification and spatial separation of proliferative (Ki67-positive) and terminally differentiated (loricrin-positive) keratinocytes, while bulk RNA-seq and qPCR analyses show Matrigel-dependent upregulation of keratin, junctional, and barrier-associated genes. These structural and transcriptional changes translate into enhanced barrier function, as quantified by a direct gravimetric transepithelial water-loss (TEWL) and transepithelial electrical resistance (TEER) measurements. Under Triton X-100 exposure, the optimized FT-skin achieves an ET 50 of 4.5 h, satisfying OECD Test Guideline 431 performance criteria (>2.0 h) and closely matching the ET 50 of 5.0 h obtained with an RhE model, while OECD Test Guideline 439-compatible irritation testing correctly classifies non-irritant and irritant chemicals. This structurally stable, regulatory-aligned FT-skin integrates dermal and epidermal contributions to barrier function and embodies an integrated design strategy that provides practical, experimentally grounded design principles for in vitro assessment of skin corrosion, irritation, and future multi-component skin equivalents.