Human mesofluidic intestinal model for studying transport of drug carriers and bacteria through a live mucosal barrier

Abstract

The intestinal mucosal barrier forms a critical interface between lumen contents such as bacteria, drugs, and drug carriers and the underlying tissue. Current in vitro intestinal models, while recapitulating certain aspects of this barrier, generally present challenges with respect to imaging transport across mucus and uptake into enterocytes. A human mesofluidic small intestinal chip was designed to enable facile visualization of a mucosal interface created by growing primary human intestinal cells on a vertical hydrogel wall separating channels representing the intestinal lumen and circulatory flow. Type I collagen, fortified via cross-linking to prevent deformation and leaking during culture, was identified as a suitable gel wall material for supporting primary organoid-derived human duodenal epithelial cell attachment and monolayer formation. Addition of DAPT and PGE2 to culture medium paired with air-liquid interface culture increased the thickness of the mucus layer on epithelium grown within the device for 5 days from approximately 5 μm to 50 μm, making the model suitable for revealing intriguing features of interactions between luminal contents and the mucus barrier using live cell imaging. Time-lapse imaging of nanoparticle diffusion within mucus revealed a zone adjacent to the epithelium largely devoid of nanoparticles up to 4.5 h after introduction to the lumen channel, as well as pockets of dimly lectin-stained mucus within which particles freely diffused, and apparent clumping of particles by mucus components. Multiple particle tracking conducted on the intact mucus layer in the chip revealed significant size-dependent differences in measured diffusion coefficients. E. coli introduced to the lumen channel were freely mobile within the mucus layer and appeared to intermittently contact the epithelial surface over 30 minute periods of culture. Mucus shedding into the lumen and turnover of mucus components within cells were visualized. Taken together, this system represents a powerful tool for visualization of interactions between luminal contents and an intact live mucosal barrier.

Fig. 1. Mesofluidic gut model enabling visualization of the mucosal interface. (A) Left: Schematic of the gut chip channel pattern, showing the channel width and length dimensions. Right: Top view illustrating three channels, including the lumen channel (green) representing the intestinal lumen, the side channel (red) representing circulatory flow, and the gel wall separating them. (B) Schematic cross-sectional view. The apical surface of primary human intestinal epithelial cells cultured on the gel wall faces the lumen. Luminal stimuli (bacteria, particles, etc.) can be introduced into the lumen channel for studying their interactions with the mucosal barrier.

Fig. 2. The human mesofluidic duodenal chips differentiated using modified differentiation media (DM-RN + DAPT + PGE2) demonstrate taller cell height with columnar shape of epithelial cells. Top: human mesofluidic duodenal chip culture procedure and experimental timeline. The duodenal chips were cultured for 8 to 10 days, depending on the rate of confluent monolayer formation. Bottom: brightfield microscopic images of primary human duodenal monolayers in the mesofluidic chips differentiated for 5 days using (A) differentiation media with R-spondin and noggin (DM-RN), (B) DM-RN supplemented with 10 μM DAPT, and (C) DM-RN supplemented with 10 μM DAPT and 1.4 nM PGE2, with a higher magnification image showing the human duodenal epithelium morphology.

Fig. 3. Left: Addition of DAPT and PGE2 during duodenal epithelium differentiation moderately increased mucus production. Primary human duodenal epithelial cells in the mesofluidic gut model were differentiated for 5 days using (A) differentiation media with R-spondin and noggin (DM-RN) (B) DM-RN supplemented with 10 μM DAPT, and (C) DM-RN supplemented with 10 μM DAPT and 1.4 nM PGE2. Confocal fluorescence images are shown of the duodenal monolayers stained with fluorescently tagged lectin (WGA, red) for mucus and Hoechst 33342 (blue) for nuclei, with 200 nm fluorescent polystyrene microspheres (green) introduced to the lumen channel for visualizing their distribution relative to the edge of the mucus layer. Right: Alcian blue mucin quantification assay showing moderate enhancement in secreted mucin in the duodenal chips differentiated using DM-RN supplemented with DAPT and PGE2 relative to DM-RN alone. *Indicates α < 0.05.

Fig. 4. ALI differentiation with basolateral VIP exposure significantly increased mucus layer thickness. Top: Human mesofluidic duodenal chip culture procedure and experimental timeline. The duodenal chips were cultured for 8 to 10 days depending on the rate of confluent monolayer formation. Bottom left: The human mesofluidic duodenal chips were differentiated using 4 distinct methods: 1. +VIP at ALI, 2. +VIP submerged, 3. −VIP at ALI, 4. −VIP submerged. Human duodenal epithelium was then stained with WGA (red) for mucus and Hoechst 33342 (blue) for nuclei. 200 nm fluorescent polystyrene microspheres were introduced into the mesofluidic duodenal chips for visualizing their distribution within the lumen vs. at the edge of the mucus layer. Scale bar: 20 μm. Bottom right: Alcian blue mucin quantification assay showing significantly greater secreted mucin concentration in the duodenal chips differentiated using ALI + VIP compared to those differentiated using ALI without VIP and submerged conditions. *Indicates α < 0.05.

Fig. 5. Multiple particle tracking technique was utilized to analyze diffusion of small and large ABT-450 ANPs in ex vivo collected porcine intestinal mucus and in the mesofluidic duodenal chips. Left: 20 second videos were collected at the regions of interest (within the WGA-stained intact mucus layer, pointed by dashed red lines) and particle trajectories were tracked using the Matlab algorithm. Representative particle trajectories of small and large ABT-450 ANPs are shown here. Long and short white scale bars: 20 and 1 μm, respectively. Right: Effective diffusivities of ABT-450 ANPs calculated using mean-squared displacement (MSD) from particle trajectories at τ = 3 s indicated that larger particles have decreased effective diffusivity and particles diffuse more freely in the intact mucus layer relative to ex vivo porcine mucus. *Indicates α < 0.05.

Fig. 6. (A) Human mesofluidic duodenal chip culture procedure and experimental timeline. The duodenal chips were cultured for 8 to 10 days depending on the rate of confluent monolayer formation. (B) Distributions of ABT-450 ANPs within the mucus layer in human duodenal chips differentiated under the ALI + VIP condition 1.5 h after introduction to the lumen channel. Confocal fluorescence images of first and last frames of the time-lapse videos (30 second interval, 30 minutes duration) show changes in distribution and location of ABT-450 ANPs (green) of two different sizes (“small” = 125 nm and “large” = 230 nm) within the WGA-stained (red) mucus layer over the 30 minute of period. White circles show small ABT-450 ANPs reaching to the epithelium after 1.5 hour. Yellow arrows show increased concentration of large ABT-450 ANPs in the less WGA-stained regions over the 30 minute observation period. (C) Confocal fluorescence images collected 4 h after introduction to the lumen channel of ABT-450 ANPs (green) reveals clumping and shedding of some ANPs away from the epithelial surface over time. Dashed yellow circles highlight the same clustered ANPs shedding away from the mucus layer over 30 minute observation period. Epithelial nuclei stained with Hoechest33342: blue. Scale bar: 20 μm.