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

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.