Real-time monitoring of HT29 epithelial cells as an in vitro model for assessing functional differences among intestinal microbiotas from different human population groups

Several in vitro screening tests have been used for selecting probiotic strains; however they often show low predictive value and only a limited number of strains have demonstrated functionality in vivo. The most used in vitro tests represent a very simplified version of the gut environment, especially since they do not consider the accompanying microbiota. Therefore, there is a need to develop sensitive and discriminating in vitro models including the microbiota. Here we developed an in vitro model to discriminate among microbiotas/fecal waters from different population groups. To this end samples were obtained from seven healthy adults, five IBD-patients, ten full-term and ten preterm newborns. Fecal microbiotas were purified and their impact, as well as that of the fecal waters, on HT29 cells was continuously monitored for 22 h using a real-time cell analyzer (RTCA). The composition of the purified microbiotas was assessed by 16S rRNA gene profiling and qPCR and the levels of short chain fatty acids (SCFA) determined by gas chromatography. The microbiota fractions and SCFA concentrations obtained from IBD-patients, full-term and preterm babies, showed clear differences with regard to those of the control group (healthy adults). Moreover, the purified intestinal microbiotas and fecal waters also differed from the control group in the response induced on the HT29 cells assay developed. In short, we have developed a real-time, impedance-based in vitro model for assessing the functional response induced by purified microbiotas and fecal waters upon intestinal epithelial cells. The capability of the assay for discriminating the functional responses induced, by microbiotas or fecal waters from different human groups, promises to be of help on the search for compounds/strains to restore the functionality of the microbiota-host's interaction.This work was financed by projects AGL2013-43770-R and AGL2015-64901-R from Plan Estatal de I + D + I (Spanish Ministry of Economy and Competitiveness, MINECO) and by Grant GRUPIN14-043 from Plan Regional de Investigación del Principado de Asturias, Spain. Both national and regional grants received cofounding from the European Union FEDER funds. AMN is the recipient of a predoctoral contract and NS benefits from a Juan de la Cierva postdoctoral contract, both granted by MINECOPeer reviewe