Optimizing INFOGEST Digest Conditioning for Reliable In Vitro Assessment of Nutrient Bioavailability Using Caco-2 Cell Models

<b>Background/Objectives</b>: Assessing the bioavailability of nutrients and bioactive compounds in vitro commonly relies on coupling standardized gastrointestinal digestion models with intestinal epithelial cell systems. However, digests produced using static digestion protocols such as INFOGEST often impair epithelial barrier integrity, limiting their direct application to intestinal models and reducing reproducibility across studies. <b>Methods</b>: This work systematically compared five commonly used digest conditioning strategies, including acidification, centrifugation, rapid freezing, and ultrafiltration using 10 kDa and 3 kDa molecular weight cut-off membranes, to identify the approach that best preserves intestinal epithelial viability and barrier function while enabling exposure at physiologically relevant concentrations. INFOGEST digests of yogurt were initially evaluated, followed by validation using biscuit and canned mackerel digests. Cell viability and monolayer integrity were assessed in differentiated Caco-2 cells using MTT assay and transepithelial electrical resistance (TEER) measurements. <b>Results</b>: Among the tested approaches, ultrafiltration using 3 kDa membranes consistently preserved epithelial viability and barrier integrity at a 1:10 dilution across all food matrices, whereas other conditioning methods failed to maintain TEER despite acceptable cell viability. At lower dilutions, food-dependent effects emerged, highlighting the importance of matrix-specific evaluation. <b>Conclusions</b>: These findings identify 3 kDa ultrafiltration as an effective and minimally invasive strategy to improve the compatibility of INFOGEST digests with intestinal cell models. By enabling reproducible exposure conditions that preserve epithelial integrity, this approach supports more reliable in vitro assessment of nutrient bioavailability and contributes to methodological standardization in nutrition research.