Methods for drug delivery and compatibility studies in an ex vivo mucus-covered Caco-2 cell monolayer model

Oral drug delivery studies are increasingly done using the Caco-2 cell monolayer model overlaid with simple or complex in vitro mucus matrices as the model then becomes more robust towards otherwise cytotoxic biorelevant conditions. However, in vitro mucus does not accurately mimic native mucus properties. Here, we present a more biorelevant model using hyperosmotic and isosmotic ex vivo porcine intestinal mucus (HYP-PIM and ISO-PIM, respectively) overlaying the Caco-2 cell monolayer, which more closely mimics the native intestinal barrier. Biocompatibility of the mucus preparations were evaluated using i: a basolateral viability assay designed to prevent damage to the monolayer integrity by apical PIM removal and to ensure accurate metabolic activity assessment, ii: transepithelial electrical resistance measurements and iii: nuclear morphology assessment using advanced live cell microscopy and nuclear segmentation. These orthogonal methods demonstrated that all mucus preparations were well tolerated by the Caco-2 cell monolayer. Lastly, HYP-PIM and ISO-PIM on the Caco-2 cell monolayer were shown to constitute a significant barrier to the permeation of the peptide cyclosporin A compared to the commonly used porcine gastric mucin suspension (PGMII), highlighting the biorelevance of this model over in vitro mucus alternatives.