A Dynamic Double‐Flow Gut‐On‐Chip Model for Predictive Absorption Studies In Vitro

Abstract Human gut is crucial for digestion, drug absorption, and overall health; however traditional in vitro and animal models struggle to accurately replicate its complex mechanisms. This study introduces an innovative gut‐on‐chip based on the MIVO millifluidic device, designed to faithfully replicate the human intestinal environment. CaCo‐2 and HT‐29 cells were co‐cultured under different ratio under dynamic flow conditions, resembling the bloodstream. Intestinal tissue differentiation was assessed through Trans‐epithelial electrical resistance (TEER) measurements, Zonula Occludens‐1, and Alcian blue staining. After model establishment, a second dynamic flow was applied on the apical side recapitulating the intestinal lumen niche. The dynamic culture conditions significantly reduced cell maturation time, obtaining a differentiated intestinal layer within 7‐10 days, compared to 21 days of static culture. In addition, CaCo‐2:HT‐29 co‐cultures enables to finely tune the mucus thicknesses and barrier function, essential for studying specific conditions. Furthermore, the introduction of a double apical‐basal flow system recapitulated intestinal permeability characteristics more closely resembling those observed in vivo. The Double‐Flow millifluidic Gut‐on‐Chip described and successfully validated enables to cross‐correlate the barrier function of the epithelial layer with the CaCo‐2:HT‐29 cells ratios, finally providing a predictive model useful for drug development and disease modelling.