A tunable in vitro Caco-2/HT-29 cell co-culture mimicking the intestinal barrier aging: a morphofunctional study

The intestinal epithelial barrier (IEB) of elderlies undergoes a myriad of changes due to the microbiota and intestinal cell alterations, which drive the feed-forward cycle of leaky gut, inflammaging, dampened cell renewal, and defective mucus production 1. Despite different in vivo and ex vivo studies 2 have shed some light on the age-associated impairment of IEB integrity and cytokine production, the involved molecular mechanisms are not completely understood. Thus, there is 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 Caco2 and HT-29 cells, differentiated in absorptive and mucus-secreting phenotypes respectively 3, and sub-cultivated both in standard condition and for a longer time so that cell aging is gradually induced in a “physiological” way, avoiding the administration of exogenous stimuli. Preliminary results evidenced in the aged co-culture i) a diminished epithelial electrical resistance (TEER); ii) an increased paracellular permeability; iii) a slight decrease in cell proliferation; 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. Taken together, these preliminary results suggest the presence of impaired barrier integrity associated with a modulation of the morphological features mimicking the leaky/aged gut as reported in clinics. Future experiments could ascertain the use of the aged in vitro Caco-2 and HT-29 cell co-culture as a useful model for both studying the molecular process and testing potential drug/nutraceutical treatments to ameliorate gut aging.