Geometrically Constrained Growth Factor Concentration Favors Enrichment of Goblet Cells and Mucus Formation

Colonic mucus forms a critical barrier to intestinal contents, providing the protection necessary for intestinal and organismal health. The mucus is composed of gel-forming mucin secreted by goblet cells residing in the epithelial layer lining the colon; yet, our knowledge of many of the attributes and functions of mucus and the goblet cells remains limited. A planar array of colonic cryptlike structures with a thick covering of goblet cell-generated mucus was developed to mimic the differentiated colonic epithelium and provide an easily accessible physiologic mucus layer for the evaluation of mucus barrier function in response to intestinal microbiota and toxins. The human microphysiological system (MPS) was created using an impermeable thin film patterned with a geometrical array of a 10 μm × 10 μm scale through holes overlaid with collagen and primary colonic stem cells. The array dimensions, collagen thickness, and growth factor concentration were optimized to assess the cell density, proliferation, migration, differentiation, and mucus thickness. A 175 μm center-to-center distance between the through holes or stem cell niches and a collagen thickness of 10 μm were found to be optimal to enable long-term culture (≥23 days) with a discrete stem/proliferative cell region and a differentiated cell zone enriched in goblet cells and supporting a 250 μm-thick adherent mucus layer. The mucus layer acted as an effective barrier to block the access of the Staphylococcus aureus α-hemolysin toxin to the epithelial cells as well as to protect the cell layer from both Staphylococcus aureus and Lactobacillus rhamnosus. The intestinal mucus MPS will be a useful tool for emulating the intestinal epithelium to study the interplay of stem cell renewal, goblet cell differentiation, mucus dynamics, and microbiota-mucus-host interactions.