Anin vitromodel of stiffened colonic mucosa exhibits altered epithelial behavior

Stiffening of the extracellular matrix underlying the epithelial cells of the large intestine is associated with aging as well as many diseases. Yet the impact of the stiffened matrix on epithelial physiology remains poorly understood. A 2D and 3D microphysiological model of the large intestine was developed using a collagen scaffold with a physiologic or excessive stiffness (Young's moduli of 2.84 ± 0.85 kPa and 15.9 ± 0.73 kPa) by altering the collagen concentration within the substrate. Diffusion of a 10 and 40 kDa fluorescent dextran was significantly different between the physiologic and stiff scaffold (97.8 vs 79.8µm²s^-1[10 kDa] and 68.2 vs 56.8µm²s^-1[40 kDa], respectively). When primary human epithelial cells of the large intestine were grown as a 2D monolayer, cultures on the physiologic scaffold grew to a significantly higher density with more proliferative and fewer differentiated cells than cultures on the stiffened scaffold. Three-dimensional crypt arrays were also fabricated with the physiologic and stiff substrates, populated with cells, and a growth factor gradient applied. The cell density, proliferation, and height-to-width ratio was significantly greater for cells on the physiologic scaffold relative to that of cells on the stiffened scaffolds. Placement of a layer of intestinal fibroblasts below the epithelium on the crypt arrays did not mitigate the impact of the stiffened substrate. Bulk-RNA sequencing revealed 378 genes that were significantly upregulated and 385 genes significantly downregulated in the stiffened vs physiologic scaffolds. This work demonstrates that a molded collagen hydrogel can be used to mimic the biophysical characteristics of a stiffened intestinal stroma, recapitulating physiology observedin vivo. Thisin vitromodel of polarized crypts with a tunable underlying substrate will enable an improved understanding of intestinal epithelial cell morphology, stem cell maintenance and lineage allocation within a stiffened environment.