Synergistic impact of 3D multicellular architecture and capillary-like flow on intestinal drug permeability.

Understanding intestinal drug permeability requires in vitro systems capable of reproducing both the structural complexity and the dynamic fluidic environment of the human gut. Traditional two‑dimensional (2D) models, although widely used in Absorption, Distribution, Metabolism and Excretion (ADME) screening, offer limited physiological relevance and often fail to predict in vivo absorption. Three‑dimensional (3D) models and microfluidic technologies have emerged as promising tools to address these limitations by incorporating multiple cell types, extracellular matrix components, and biomechanical cues. In this study, we compared the permeability of three model drugs, metoprolol (high permeability), atenolol (moderate permeability), and colchicine (low permeability), across a 2D Caco‑2/HT29‑MTX coculture and an advanced 3D intestinal model composed of the same epithelial cells, stromal fibroblasts embedded in a collagen-alginate matrix, and a basolateral endothelial layer mimicking the vascular compartment. Both systems were evaluated under static conditions and under dynamic basolateral flow using the PDMS‑free MIVO® millifluidic platform designed to reproduce capillary‑like fluid dynamics. Permeability and TEER measurements revealed consistently higher transport across the 3D model than the 2D system, confirming its enhanced physiological relevance. Dynamic flow further increased permeability in both models, with the most pronounced effect observed for metoprolol, whose permeation was highest under dynamic 3D conditions. Flow also accelerated TEER decline in the 3D setup, suggesting increased barrier stress associated with elevated transport. Overall, integrating 3D multicellular architecture with physiologically relevant flow substantially improves the predictive capacity of intestinal in vitro models. These findings support dynamic 3D systems as advanced tools for drug permeability assessment and early‑stage drug development.