Creation of a spatially complex mucus bilayer on an in vitro colon model.

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X-Y-Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air-liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 ± 180 µm thick) possessing an inner (149 ± 50 µm) and outer (435 ± 111 µm) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8-10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X-Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease.

In vitro co-culture of Clostridium scindens with primary human colonic epithelium protects the epithelium against Staphylococcus aureus.

A complex and dynamic network of interactions exists between human gastrointestinal epithelium and intestinal microbiota. Therefore, comprehending intestinal microbe-epithelial cell interactions is critical for the understanding and treatment of intestinal diseases. Primary human colonic epithelial cells derived from a healthy human donor were co-cultured with Clostridium scindens (C. scindens), a probiotic obligate anaerobe; Staphylococcus aureus (S. aureus), a facultative anaerobe and intestinal pathogen; or both bacterial species in tandem. The co-culture hanging basket platform used for these experiments possessed walls of controlled oxygen (O2) permeability to support the formation of an O2 gradient across the intestinal epithelium using cellular O2 consumption, resulting in an anaerobic luminal and aerobic basal compartment. Both the colonic epithelial cells and C. scindens remained viable over 48 h during co-culture. In contrast, co-culture with S. aureus elicited significant damage to colonic epithelial cells within 24 h. To explore the influence of the intestinal pathogen on the epithelium in the presence of the probiotic bacteria, colonic epithelial cells were inoculated sequentially with the two bacterial species. Under these conditions, C. scindens was capable of repressing the production of S. aureus enterotoxin. Surprisingly, although C. scindens converted cholic acid to secondary bile acids in the luminal medium, the growth of S. aureus was not significantly inhibited. Nevertheless, this combination of probiotic and pathogenic bacteria was found to benefit the survival of the colonic epithelial cells compared with co-culture of the epithelial cells with S. aureus alone. This platform thus provides an easy-to-use and low-cost tool to study the interaction between intestinal bacteria and colonic cells in vitro to better understand the interplay of intestinal microbiota with human colonic epithelium.

Mucus-coated, magnetically-propelled fecal surrogate to mimic fecal shear forces on colonic epithelium.

The relationship between the mechanical forces associated with bowel movement and colonic mucosal physiology is understudied. This is partly due to the limited availability of physiologically relevant fecal models that can exert these mechanical stimuli in in vitro colon models in a simple-to-implement manner. In this report, we created a mucus-coated fecal surrogate that was magnetically propelled to produce a controllable sweeping mechanical stimulation on primary intestinal epithelial cell monolayers. The mucus layer was derived from purified porcine stomach mucins, which were first modified with reactive vinyl sulfone (VS) groups followed by reaction with a thiol crosslinker (PEG-4SH) via a Michael addition click reaction. Formation of mucus hydrogel network was achieved at the optimal mixing ratio at 2.5 % w/v mucin-VS and 0.5 % w/v PEG-4SH. The artificial mucus layer possessed similar properties as the native mucus in terms of its storage modulus (66 Pa) and barrier function (resistance to penetration by 1-µm microbeads). This soft, but mechanically resilient mucus layer was covalently linked to a stiff fecal hydrogel surrogate (based on agarose and magnetic particles, with a storage modulus of 4600 Pa). The covalent bonding between the mucus and agarose ensured its stability in the subsequent fecal sliding movement when tested at travel distances as long as 203 m. The mucus layer served as a lubricant and protected epithelial cells from the moving fecal surrogate over a 1 h time without cell damage. To demonstrate its utility, this mucus-coated fecal surrogate was used to mechanically stimulate a fully differentiated, in vitro primary colon epithelium, and the physiological stimulated response of mucin-2 (MUC2), interleukin-8 (IL-8) and serotonin (5HT) secretion was quantified. Compared with a static control, mechanical stimulation caused a significant increase in MUC2 secretion into luminal compartment (6.4 × ), a small but significant increase in IL-8 secretion (2.5 × and 3.5 × , at both luminal and basal compartments, respectively), and no detectable alteration in 5HT secretion. This mucus-coated fecal surrogate is expected to be useful in in vitro colon organ-on-chips and microphysiological systems to facilitate the investigation of feces-induced mechanical stimulation on intestinal physiology and pathology.