LGG was chosen as a positive control, because human in vivo studies showed that the beneficial effects of LGG are, in part, attributed to a strong colonization of the colonic mucus layer upon oral administration [41]. This strong adhesion capacity of LGG has recently been attributed to a SpaC pilin, which is located on the top of the pili and exerts a strong mucus-binding activity [42]. After 1.5 h of incubation in the upper compartment of the HMI module, LGG showed an adhesion percentage of 15.7 ± 3.2%, as compared to the original concentration Hydroxychloroquine molecular weight dosed to the model. This value

is in line with what described by Van den Abbeele et al. [21], who tested the adhesive properties of LGG in presence of a complex gut microbiota in a M-SHIME. The colonization capacity of mucus by LGG was thus confirmed in the HMI module. Finally, the HMI module containing enterocytes in the lower compartment was challenged for the first time with a complex microbiota originated from the simulated ascending colon of the SHIME. In parallel the enterocytes were also directly exposed to the same complex microbiota. A MTT test showed that the viability of Copanlisib molecular weight Caco-2 cells directly exposed to the complex microbial community decreased by 80% after 2 hours of co-culture. In contrast, when the interaction occurred within an HMI module, the cells’ viability

after 48 h of incubation was not significantly different as compared to a control system in which only sterile SHIME medium was dosed (Figure 2). Although the use of cell cultures, such as Caco-2 cells, is not novel for mechanistic studies [29, 43, 44], the output of these reductionist studies is limited by the fact that they are normally

conducted using pure bacterial cultures, a mix of few bacterial strains or filtered growth media. This is mainly related to the fact that mixed microbial slurries are too cytotoxic (Figure 2), thus limiting the experimental only time (a few hours at most) and the adaptation of the host to the microbial metabolism. On the contrary, the HMI module allows to indirectly expose the Caco-2 cells to the gut microbiota for up to 48 h, the average in vivo exposure time of an enterocyte to the content of the gut lumen when migrating from the crypts to the top of the villi [45]. Figure 2 MTT values (expressed as Optical Density – OD) of Caco-2 cells directly exposed for 2 h to the complex microbial community of the ascending colon of a SHIME reactor (direct contact), exposed to the same microbial community within a HMI module (HMI 1 and 2) or to sterile SHIME medium (control) for 48 h. Values are averages ± standard deviation (n = 2). * = statistically different from the control condition according to a Student’s two-tailed t-test (p < 0.05).