Development of Novel 3D Co-Culture Models of the Human Intestinal Mucosa and their Applications to Study Epithelial Transport and Inflammatory Bowel Diseases

Abstract

Investigations into intestinal epithelial function frequently require the use of animal models or ex vivo human tissue samples. However, human tissue has limited availability while animal models exhibit species differences to the human intestinal structure and function. Additionally, in accordance with the principles established by the National Centre for the 3Rs (NC3Rs), there is a drive to move away from animal-based models. As such, several in vitro cell culture-based models have been developed to investigate epithelial function, the gold standard of these models is the Caco-2 monolayer, frequently employed throughout industry for the purposes of assessing absorption in the development of new chemical entities. Numerous limitations exist regarding this gold standard culture system: epithelial monolayers have differences in protein expression and activity, there is a lack communication between other intestinal cell lineages, and the in vivo microenvironment which provides structural support and signalling molecules to the epithelium is not considered using this reductionist approach to modelling the intestine structure and function.
This project aimed to address two key issues surrounding the Caco-2 epithelium: the lack of cell-cell communication, and absence of cell-extracellular matrix (ECM) communication. It was hypothesised that the culture of Caco-2 epithelial cells in combination with other cell lineages including, epithelial, stromal, and immune cells would enhance the structure and function of the model system to recreate the characteristics of the intestinal mucosa more accurately.
Through the use of 3D tissue culture technologies, it was demonstrated that fibroblast lineages produced de novo ECM representative of the intestinal lamina propria. Furthermore, differentiation of a Caco-2 monolayer on the surface of the lamina propria highlighted an altered epithelial polarisation, with greater apical-basolateral polarity as evidenced by the highly elongated, columnar morphology of the cells when compared to the gold-standard culture system. Moreover, this co-culture was shown to have functional properties of the epithelial barrier more like that of in vivo tissue. The complexity of this 3D co-culture system could be further enhanced by the addition of a mucus-producing cell lineage to represent the goblet cell component of the intestinal epithelium. Additionally, a constituent of the intestinal immune system was successfully incorporated into the lamina propria compartment of the model – specifically, macrophages differentiated from a monocytic cell line. Macrophages were selected for this purpose as they have principal roles in maintaining a state of anergy within the intestine, however perturbations of macrophage function can result in chronic inflammation and a subset of intestinal diseases collectively known as inflammatory bowel disease (IBD). The final immune-competent co-culture was demonstrated to instigate a proinflammatory response that altered the epithelial barrier integrity of the model when exposed to stimulatory effector molecules, recapitulating aspects of IBD.
Overall, this model has the potential to be utilised as a platform for further investigations regarding normal and inflammatory transport processes within the intestinal mucosa and a proof-of-concept study has demonstrated that the model has further applications in understanding the mechanisms underpinning IBD.