An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants

Abstract

The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ∼40 μm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 μm/h) and mouse (100 μm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 μm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.

Defective goblet cell exocytosis contributes to murine cystic fibrosis–associated intestinal disease

“…Since CFTR-dependent HCO 3 -secretion is often coincident with physiological mucus secretion (7), these findings indicate that the loss of CFTR-dependent HCO 3 -secretion by adjacent enterocytes and perhaps across the granule membrane (22) is an important contributor to the pathogenesis of mucoviscidosis. CFTR-dependent HCO 3 -transport can attain nearly isotonic (150 mM) NaHCO 3 concentrations in pancreatic juice (63). Although the local concentration of HCO 3 -at the luminal surface of CFTR-competent enterocytes is not known, it is conceivable that high concentrations are achieved within the Enteroid culture.…”

Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches

“…Gastrointestinal tissue was dissected, flushed with ice-cold oxygenated (95% O 2-5% CO2) Krebs transport buffer, and mounted in horizontal open Ussing-type chambers with a 4.9-mm 2 circular opening as described previously (10). To avoid disturbing the mucus gel, the apical chamber was kept unstirred with a constant volume.…”

“…Mucus measurements and video recordings were performed as described previously (9,10). Mucus was aspirated with a Gilson Pipetman P200 (Middleton, WI) set to 150 l and a yellow tip (no.…”

“…These studies were made in rats, but mice are commonly used for studies of gastrointestinal physiology and pathophysiology since these can be easily genetically manipulated. We have developed a methodology and performed studies on the mucus system of the distal colon (10,16,17), but to provide a more complete picture of gastrointestinal mucus we are now characterizing the normal mucus system of the whole GIT. Here we characterize the mucus system from stomach to distal colon, including Peyer's patches as mucus release, mucus thickness, growth over time, adhesive properties, and penetrability to fluorescent beads in C57BL/6 mice.…”

Antimicrobial peptides and the enteric mucus layer act in concert to protect the intestinal mucosa

“…6,7 The structural organization of the firmly adherent inner mucus layer was later shown to physically hinder bacterial penetration in the colon. 8,9 In contrast, the mucus in the small intestine only consists of one layer, probably to facilitate nutrient absorption. 10 In this organ, the mucus layer completely covers the crypts and lower part of the small intestinal villi and may leave the upper villus area exposed to the intestinal lumen content.…”