Improving the compatibility of INFOGEST digesta with intestinal epithelial models through post-digestion storage conditions

: The INFOGEST protocol is widely used for standardized in vitro digestion studies, but its application to cell models remains challenging due to residual enzyme activity, bile salts, and matrix effects that impair cell metabolism. This study investigated a post-digestion processing strategy to enable direct testing of INFOGEST digesta on 2D Caco-2 monolayers and intestine-on-chip systems without chemical inhibitors. Five post-digestion storage methodologies were compared: A, no storage; B, liquid nitrogen at - 80 °C; C, liquid nitrogen/freeze-drying; D, - 20 °C/freeze-drying; and E, - 20 °C. Residual enzymatic activities and effects on epithelial cell morphology and viability were evaluated, and the optimized workflow was validated in a microfluidic intestine-on-chip, including plant matrices. The condition D significantly reduced residual enzymatic activity, especially trypsin, identified as a key mediator of epithelial cell disruption. This storage method preserved Caco-2 monolayer integrity at a 25% dilution, with no cytotoxicity or cell detachment. When applied to digested Brassica oleracea and Malus domestica matrices, condition D did not compromise cell viability as evidenced by the results obtained with INFOGEST digesta in the absence of plant food matrices. In intestine-on-chip experiments, treated digesta produced stable TEER values and continuous ZO-1 localization, confirming barrier integrity. This simple freeze-then-lyophilize workflow enables a scalable and biocompatible integration of INFOGEST digestion with advanced cellular models for studying food bioaccessibility and intestinal absorption.