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Building a Thick Mucus Hydrogel Layer to Improve the Physiological Relevance of In Vitro Primary Colonic Epithelial Models

Human colonic mucosa is covered with a mucus layer several hundred microns in thickness, which serves as a barrier physically separating the underlying epithelium from bacteria, and hindering the movement of molecules derived from food and microbiota. Despite its importance, a thick, continuous mucus layer that mimics in vivo functions has not been achieved in in vitro colonic epithelial models constructed from primary intestinal epithelial cells. The organoid “mini-gut” model possesses goblet cells, but mucus is secreted and accumulated within the organoids’ inaccessible lumen making the mucus properties difficult to quantify. Prior monolayer models exposed the tissue luminal surface by culturing primary cells on scaffolds, but the mucus layer was very thin and diffuse and unable to act as a physiologic barrier. In this study, we report strategies to generate a thick mucus layer and demonstrate its function act as a physiological barrier to bacteria and toxins. An air-liquid interface (ALI) similar to that used for respiratory cell culture was tested based on the hypothesis that overlying aqueous medium with convective mixing might dilute the mucus as it formed. Removal of the fluid might then enable accumulation of a thick, condensed mucus. Human colonic epithelial monolayers were differentiated for 5 days as either a submerged or as an ALI culture (Supplementary Figure 1A). A contiguous mucus layer with irregular thickness (71–381 mm) was observed in the ALI culture by immunofluorescence (Supplementary

Human colonic mucosa is covered with a mucus layer several hundred microns in thickness, which serves as a barrier physically separating the underlying epithelium from bacteria, and hindering the movement of molecules derived from food and microbiota. [1][2][3] Despite its importance, a thick, continuous mucus layer that mimics in vivo functions has not been achieved in in vitro colonic epithelial models constructed from primary intestinal epithelial cells. The organoid "mini-gut" model possesses goblet cells, but mucus is secreted and accumulated within the organoids' inaccessible lumen making the mucus properties difficult to quantify. 4,5 Prior monolayer models exposed the tissue luminal surface by culturing primary cells on scaffolds, but the mucus layer was very thin and diffuse and unable to act as a physiologic barrier. 6,7 In this study, we report strategies to generate a thick mucus layer and demonstrate its function act as a physiological barrier to bacteria and toxins.
An air-liquid interface (ALI) similar to that used for respiratory cell culture 8 was tested based on the hypothesis that overlying aqueous medium with convective mixing might dilute the mucus as it formed. Removal of the fluid might then enable accumulation of a thick, condensed mucus. Human colonic epithelial monolayers were differentiated for 5 days as either a submerged or as an ALI culture (Supplementary Figure 1A). A contiguous mucus layer with irregular thickness (71-381 mm) was observed in the ALI culture by immunofluorescence (Supplementary   Figure 1C). Additionally, the mucus layer served as a barrier to 1-mm red fluorescent beads and green fluorescent protein-expressing Escherichia coli separating them from the epithelium (Supplemental Figure 1D).
The ALI culture was modified by adding an endogenous intestinal hormone, vasoactive intestinal peptide (VIP), 9,10 to mediate the luminal water homeostasis with a goal of supporting hydrated mucus layer of uniform thickness ( Figure 1A). VIP stimulated the apical water secretion in a dosedependent manner with an effective dose 50 of 210 pg/mL ( Figure 1B). When the monolayers were differentiated under ALI culture with VIP, a layer of liquid (42 mg/cm 2 ) accumulated on the apical side within 24 hours and was maintained during 5 days of differentiation. The ALI culture with VIP significantly increased the number of goblet cells ( Figure 1C; Supplementary   Figure 2A, B). The apical mucus volume increased in quantity over time and by day 5 a slippery hydrogel could be lifted off the epithelium ( Figure 1D). The thickness of the bacteria-separating mucus layer was readily adjusted by the duration of ALI culture ( Figure 1E, F; Supplemental Figure 2C). The thickness of the mucus was even across the surface (302 ± 28 mm), likely due to its ability to flow and redistribute as a result of its water content unlike that in the ALI culture without VIP. The mucus effectively separated 1-mm red fluorescent beads from the underlying epithelium ( Figure  2G, Supplemental Figure 2E). Scanning electron microscopy images additionally demonstrated a confluent layer of uninterrupted mucus upon which microbes could be visualized (Supplemental Figure 2D). The mucus layer contained other expected constituents including Muc5AC, TFF3, and AGR2 ( Figure 1H; Supplementary Figure 2G). As the single mucus layer acted as barrier to microbes, its properties resembled that of the compact or microbe-impermeant layer observed in vivo. However, vigorous washing removed the mucus layer entirely, a property most closely associated with the diffuse mucus layer bordering the luminal contents in vivo (Supplementary Figure 2E). Overlaying the mucus layer with a mixture of 0.02and 5-mm beads suggested the presence of a single layer (Supplementary Figure 2F).
To demonstrate the physiological barrier function of the mucus, intestinal epithelial monolayers were incubated with Clostridium difficile toxin A (Figure 2A  served as a physical barrier to segregate E coli from the epithelium and underlying white blood cells for up to 24 hours with elimination of cytokine production (tumor necrosis factor alpha, IL-6, and IL-1b) in the presence of the mucus layer ( Figure 2E-G). These results demonstrate that the mucus layer successfully slowed toxin movement, delaying intestinal damage, and acted as a physical barrier for bacteria, delaying or eliminating the immune response and emulating in vivo conditions.

Supplemental Information
Methods Cell culture and generation of a mucus layer. The human-derived colonic epithelial stem cells were expanded in stem medium (SM) (formulation is listed in Supplementary Table 1) using a previously published protocol. 1-3 Twelve-well Transwell inserts possessing a porous membrane (0.4-mm pore size) (#3460; Corning, Corning, NY) were coated with 1 vol% Matrigel in ice cold phosphate-buffered saline at 37 C overnight and rinsed with phosphate-buffered saline. Cells were plated as described previously in EM. 3 The medium was switched to differentiation medium (DM) after 5 days. 3 In the submerged culture, 1-mL DM was added in the apical reservoir and 2 mL was added in the basal reservoir. In the air-liquid interface culture, the medium in the apical reservoir was completely aspirated, 1-mL DM or DM vasoactive intestinal peptide (VIP) (DM containing 330-ng/mL VIP [#AS-22872; AnaSpec, Fremont, CA]) was added to the basal reservoir. DM or DM-VIP was changed daily. Cells were cultured for 10 days before assay.
To demonstrate the barrier capability, the cells were stained with Hoechst 33342 (2 mg/mL) from basal side and then green fluorescent protein-expressing Escherichia coli (EC) (0.5 mL) (#25922GFP; ATCC, Manassas, VA) at 200 million colonyforming units (CFU)/mL, or red fluorescent beads (1-mm diameter) (#F13083; ThermoFisher) at a density of 10 8 beads/mL was added to the apical reservoir. Twenty minutes after seeding, the Transwell insert were imaged by confocal microscopy.
Co-culture with EC and peripheral blood mononuclear cells. Fresh human peripheral blood mononuclear cells (Physician's Plasma Alliance, Johnson City, TN) were suspended in RPMI containing 10% FBS and 100mg/mL gentamicin at 2.86 million cells/mL. GFP-expressing EC (200 million CFU/mL) were washed and suspended in 0.2-mL RPMI with FBS and gentamicin (100 mg/mL). 7 GFPexpressing EC (20 million CFU, 20 mL) was added to the apical epithelial side. Peripheral blood mononuclear cells (1.43 million, 500 mL) were added to the basal reservoir. After 24 hours, the basal reservoir media reservoir was collected, centrifuged (5000 rpm, 6 minutes), aliquoted, and stored at -20 C.