Caco-2/HT-29 cell co-culture mimicking the intestinal barrier is a tunable model for gut aging

Considering the physiological role played by the intestinal epithelial barrier (IEB), the research related to modifications due to the microbiota and intestinal cell alterations with aging is attracting more and more attention. The necessity to standardize the appropriate experimental models is still unmet and is accompanied by a critical need to develop an in vitro study model of the IEB reproducing the interactions between the absorptive and the secreting cells related to aging. The present study aimed at characterizing the morphology and the physiology of the aged IEB through an in vitro model constituted by a co-culture of the two cell lines Caco-2 and HT-29 that we previously differentiated and characterized in absorptive and mucus-secreting cells respectively1,2. The co-culture was set up by plating a 70/30 ratio mixture of differentiated Caco2 cells from the 24th to 50th passage and HT-29 cells from the 8th to 35th passages for inducing “physiological” aging, in the absence of any exogenous stimulus. In the aged co-culture set up by plating Caco-cells which had reached at least 40th sub cultivation passage and HT-29 the 21st passage, we observed relevant morphofunctional impairments as i) a diminished epithelial electrical resistance (TEER); ii) an increased paracellular permeability; iii) a slight decrease in cell proliferation; and iv) a less homogeneous distribution of the membrane-associated claudin-1 immunostaining. Transmission electron microscopy (TEM) analysis revealed that the intracellular mucus and desmosomes were less represented in the aged co-culture, together with underdeveloped apical microvilli. These results suggest that this experimental setting can reproduce some of the main morphofunctional modifications of IEB reported in clinics in the leaky/aged gut. Future experiments could ascertain the use of the aged in vitro Caco-2 and HT-29 cell co-culture as a useful model for studying the molecular process and testing potential drug/nutraceutical treatments to ameliorate gut aging.