Unravelling Inflammatory Bowel Disease: Gut Mucosal in vitro Models and the Effects of Food Bioactives on its Cellular Components

Inflammatory Bowel Disease (IBD), including Crohn’s Disease and Ulcerative Colitis, represents chronic inflammatory conditions often marked by intestinal epithelial impairment triggered by environmental factors, such as diet. The molecular mechanisms underlying barrier damage, diet and IBD remain unclear. Despite the lack of tissue complexity, two-dimensional (2D) cell culture systems provide valuable insights into the cellular responses to dietary and inflammatory stimuli. Recently, a more advanced perspective has been provided by three dimensional (3D) in vitro co-culture models. However, they mostly rely on the use of colon epithelial cancer cell lines, such as Caco-2, traditionally used to study gut leakiness in 2D. This project aims to (i) generate transcriptomics and secretory inflammatory profiles following 2D exposure of each cell line to fructose, lipopolysaccharide (LPS) and resveratrol at 1, 24 and 48 hours; (ii) build a 3D co-culture model of the healthy intestinal epithelium using non-neoplastic colon epithelial cells (HCEC-SV40) and dermal fibroblasts (HDF/tert164) supported by the Alvetex® Scaffold; and (iii) label each cell line employed in the model by inserting fluorescent genes via CRISPR/Cas9 homology-directed repair (HDR) within the ROSA26 locus. 2D studies revealed that resveratrol and LPS activated antiviral and interferon-related genes in HCEC-SV40 and HDF/tert164, promoting cell proliferation. Over time, these cell lines revealed a significant increase in IL-17A, MUC16 and N-Cadherin upon exposure to fructose, LPS and resveratrol. Co-culturing HDF/tert164 and Caco-2 on collagen showed the potential to recapitulate the intestinal mucosa, while further optimisation is required for HCECSV40. CRISPR/Cas9-mediated HDR successfully incorporated fluorescent transgenes into the ROSA26 locus, demonstrating the feasibility of exploiting fluorescence to study individual cells within the 3D co-cultures. The work presented in this thesis allows to dissect the molecular differences between cancer and non-neoplastic cells and lays the foundation for optimising 3D co-culture systems to investigate the cell types profiled in the 2D monocultures.