The oxygen permeability was measured in a HMI module (with a mucus layer of 200 μm) maintaining a completely

anaerobic upper chamber (water previously gassed with 95% N2-5% CO2) and an aerobic lower chamber (liquid constantly gassed with an air pump). Measurements were carried out at 37°C by following the increasing oxygen concentration in the upper chamber by means of a luminescent LDO oxygen probe (Hach Lange, Mechelen, Belgium) placed on the outlet connection of the luminal side of the module. Data of the increasing oxygen concentration in the upper chamber, collected in the first 30 minutes, were used to calculate the relative permeability (PmO2) using the following equation, as shown by Autophagy inhibitor cost Saldena et al. [40]: where MO2 is the mass of oxygen transferred in the time t; (cO2)A and (cO2)B are the concentrations of oxygen in the upper and lower chamber of the HMI module with a mucus layer with a surface S and a thickness x. The quotient DO2/x corresponds to the oxygen permeability (PmO2). Characterization of the biological parameters Lactobacillus rhamnosus GG (LMG 18243, BCCM/LMG, Ghent, Belgium) was used as a positive control to assess the capacity of bacteria to colonize

the double functional layer [55]. LGG was grown in MRS medium, quantified by plate count (LGG t0). The fully grown OICR-9429 liquid culture was then circulated through the upper chamber of an HMI module at a flow correspondent to a shear stress of 3 dynes cm−2 (6.5 mL min−1). After 1.5 h, the simulation was Temsirolimus stopped Cytidine deaminase and the luminal suspension removed. The functional layer was rinsed twice with phosphate buffer solution to remove the non-adhered bacteria. Subsequently, the luminal side of the functional layer was rinsed with Triton X-100 to remove the adhering bacteria. The obtained bacterial suspension was analyzed for microbial concentration measurements using the plate count technique on MRS (LGG t1.5). Percent of adhering bacteria was calculated

as LGG t1.5/LGG t0. In a second set of experiments, it was evaluated the capacity of Caco-2 cells to survive in the HMI module in presence of a complex microbial community (derived from a SHIME reactor). An HMI module was set up as described in the first paragraph of the Methods section and the complex microbial community was introduced in the upper chamber of the HMI module. In a parallel experiment, the enterocytes were directly exposed to the same microbiota (i.e. viability after direct contact) in a microtiter plate. The cell viability in the 2 setups was compared by means of the MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric test [56] after 48 h of incubation in the HMI module and after 2 h of direct contact.