Building the colon epithelial microenvironment in vitro for investigation of intestinal disease mechanisms

Abstract

Colonic epithelium is situated directly above the lamina propria which houses fibroblasts and resident immune cells that provide support and protection for proliferative stem cells and terminally differentiated epithelial cells. In health, fibroblasts maintain the extracellular matrix (ECM) and provide signals to help enforce spatial organization in the epithelial layer. In the case of injury or inflammation, resident immune cells initiate the immune response while fibroblasts help to rebuild a healthy tissue, though in some cases persistent, pathological fibroblast activity may lead to tissue thickening and stiffening inducing fibrosis. Disruptions in the epithelium-fibroblast relationship play a role in many diseases including colorectal cancer (CRC) while epithelium-immune cell interactions are critical for maintaining intestinal homeostasis, yet the complexity of the intestinal microenvironment makes it challenging to study these relationships in vivo. To better understand intestinal disease initiation and progression, in vitro model systems are needed to recapitulate the direct contact between fibroblasts and epithelial cells and to study the immune cell response within a controlled environment.This dissertation describes the development and testing of in vitro model systems to mimic interplay of multiple cell types present in the colonic mucosa. In Chapter 2, we describe 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 pericryptal fibroblasts. Model crypts form a stem cell niche in their base and a differentiated cell zone at the luminal end. In this in vitro context underlying fibroblasts also support epithelial survival and barrier function while modulating proliferation. This model will enable work towards an improved understanding of the role fibroblasts play in healthy colon tissue and in disease initiation and development. Chapter 3 describes a new model of colonic fibrosis wherein fibroblasts and epithelial cells are grown on two scaffolds that exhibit healthy or fibrotic biophysical characteristics. Though fibrosis is often the result of a persistent inflammatory response, fibrinogenesis is a self-perpetuating process and so we sought to model excessive ECM deposition within a tissue by altering the underlying substrate and then culturing primary fibroblasts and epithelial cells together. The hydrogel scaffolds on which cells were cultured possess diffusivity and stiffness characteristics similar to healthy and fibrotic tissues. Cells exhibited altered morphology and phenotype when cultured on stiff scaffolds, demonstrating the usefulness of this model for future fibrosis related investigations. In chapter 4 a different in vitro model system was used to observe the interaction between another pair of cell types present in the colonic mucosa: epithelium and resident immune cells. In this case, a soft, highly permeable hydrogel was used as a model of the interstitial space and the migration of macrophages towards a normal or damaged epithelial layer was measured. The epithelial cell barrier function and phenotype along with immune cell migration in response to stimuli were measured demonstrating the usefulness of this system for modeling acute inflammation. The model systems described in this dissertation will enable further discovery regarding colonic tissue repair and CRC initiation and progression.