(J-361) Development of an In Vitro Colon Crypt Model to Study the Impact of Underlying Fibroblasts on Intestinal Epithelial Cells

Introduction:: The colonic epithelium is situated above a layer of fibroblasts that supply growth factors to support proliferative stem cells at the crypt base and differentiation factors to promote cell maturation at the luminal intestinal surface. Disruptions in this epithelium-fibroblast relationship play a role in many diseases including colorectal cancer and inflammatory bowel disease. A model system to recapitulate the interplay between the fibroblasts and epithelial cells within the context of the crypt is needed to understand fibroblast roles in health and disease.

Materials and Methods:: With cross-linked collagen we have created a planar and molded three-dimensional crypt scaffold that can be suspended within a Transwell insert to permit control of environmental conditions on either side of the tissue. With this scaffold, we have created a 3D, fully polarized in vitro tissue model with an array of crypts comprised of primary human colonic epithelial cells located above a layer of human primary pericryptic fibroblasts. Transepithelial electrical resistance (TEER), permeability studies, and immunofluorescence confocal imaging have allowed us to evaluate the barrier integrity, stem cell proliferation, and differentiation behavior in these tissues.


Results, Conclusions, and Discussions:: When a gradient of Wnt, R-spondin, and Noggin was applied across the crypt long axis, a stem cell niche with proliferative cells formed at the crypt base as well as a differentiated cell zone at the luminal crypt end. The underlying layer of fibroblasts enhanced epithelial cell barrier function as evidenced by a faster increase in the transepithelial electrical resistance (TEER). It has also been observed that coculture with fibroblasts enhanced epithelial cell survival, though intriguingly it has also been observed that fibroblasts may modulate epithelial cell proliferation. Additional studies focusing on cell phenotype and physiology in the presence and absence of healthy or perturbed fibroblasts are underway. These early data suggest that colonic crypts can be used to study the bidirectional relationship between the epithelium and fibroblasts enabling an improved understanding of the role of dysfunctional fibroblasts, for example, senescent or cancer-associated fibroblasts (CAFs), in disease initiation and development.

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