The prediction of nanomaterial (NM) intestinal absorption is a key step in the evaluation of risks after oral exposure. The Caco-2 monolayer model is widely used to predict the permeability of substances across the intestinal barrier. The addition of M-cells should improve the relevance of the model for NMs, since transcytosis through M-cells is considered to play a major role in the intestinal absorption of particles. A Caco-2/Raji B co-culture was established to induce the transformation of enterocytes into M-cells. The model was characterized by monitoring the transepithelial electrical resistance (TEER) values and by scanning electron microscope (SEM) imaging. The translocation of TiO2 nanoparticles (NPs) was evaluated in the Caco-2/M-cell system and compared to that in a Caco-2 monolayer model. In addition, two different co-incubation periods (co-culture day 4 vs. co-culture day 7) and two different NP dispersion protocols (probe sonication for different periods and amplitude) were used to evaluate whether these experimental variables could influence translocation. The exposure to TiO2 NPs did not cause changes in the morphology or TEER values of the cell monolayers. A three-way ANOVA for the titanium levels in the basal compartment showed no interactions between the variables tested, but a significant contribution to the overall variance of the data by the cell model (p < 0.05). The levels of titanium recorded in the basal compartment were significantly higher (p < 0.05) than the background titanium levels only in the presence of M-cells. This work supports a relevant role for M-cells in nanoparticle absorption. However, a standardized protocol for the Caco-2/M-cell co-culture model should be established and well characterized before this can be used to compare translocation data among NMs, particularly if tested in different laboratories.

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