LAUSR

BACKGROUND AND AIMS The goal of this project was to validate the functional relevance and utility of mucus produced by an in-vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. METHODS Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, non-inflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle tracking microrheology (PTMR), multi-angle laser light scattering (MALLS), refractometry, and immunohistochemical imaging. RESULTS An air-liquid-interface (ALI) culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight % of solids over one week (1.3±0.5% at 2 days vs. 2.4±0.3% at 7 days). The full range of mucus concentrations (0.9-3.3%) observed during culture was comparable to that displayed by ex-vivo mucus (1.3-1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in-vitro and ex-vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. PTMR showed higher complex viscosities for ex-vivo mucus samples than in-vitro mucus which was explained by a greater fraction of water present in in-vitro mucus than ex-vivo, i.e. in-vitro mucus is more heterogeneous than ex-vivo. Refractometry, MALLS, and immunostaining showed increased mucus complex size in ex-vivo mucus compared to in-vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex-vivo mucus collection. CONCLUSIONS The ALI culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.