Exploring the usefulness of the complex in vitro intestinal epithelial model Caco-2/HT29/Raji-B in nanotoxicology

The use of in vitro barrier models is gaining relevance as an alternative to animal studies in risk assessment, pharmacokinetic, and toxicological studies in general. These models permit an easier evaluation of the underlying mechanisms taking place at the molecular and cellular levels on the barrier site. Here, we report several methodological modifications of the three-dimensional in vitro intestinal epithelial model Caco-2/HT29/Raji-B for its successful application in the Nanotoxicology field. In addition, new insights in the study of specific molecular markers and new confocal microscope approaches have also been incorporated. Due to the multiple variables and parameters playing a part when the model's complexity is increased, we have monitored the barrier's formation and cell differentiation over time. Finally, the practical usability of the proposed model was tested by evaluating the action of the food additives titanium dioxide and silica dioxide nanoparticles (TiO2NPs and SiO2NPs). The NPs-associated effects were evaluated by confocal microscopy. We have demonstrated the essential role of the mucus layer in the decrease of cellular uptake, avoiding potential NPs-cell nuclei interactions.