Development of a novel bioengineered tissue model for inflammatory bowel disease research and drug testing applications

Abstract

Inflammatory bowel disease (IBD), which includes Crohn’s Disease (CD) and Ulcerative Colitis (UC), are chronic
inflammatory disorders of the gastrointestinal tract. The exact aetiology of disease is unknown, but it is likely
to arise from a combination of factors, encompassing genetic factors, environmental influences, and lifestyle
contributions. Whilst CD and UC differ from each other in terms of genetic predisposition, affected tissues,
and treatment regimes, they share many key disease hallmarks. For example, both CD and UC patients exhibit
excessive immune cell activation and infiltration into the intestinal mucosal tissue, epithelial barrier
disruption and pathological stromal remodelling events.
Current therapeutic intervention in IBD is insufficient and remission rates remain low. Current approaches
primarily target activated leukocytes to dampen the immune response, neglecting other critical mediators of
inflammation, such as fibroblasts. There now exists irrefutable evidence that fibroblasts are not just
bystanders in mucosal inflammation but are pivotal participants in orchestrating the immune response in IBD.
Although more recent therapies such as biologic agents have shown more success in treating IBD than
previous immunosuppressive drugs, 40% of patients elicit no response. Moreover, approximately 90% of
clinical trials for investigational new drugs (IND) fail, which is likely attributable to the fact that most IND
research is performed using animal models, and outdated in vitro culture methods. The scarcity, diseaseseverity
and short-lived nature of human IBD explant tissue limits the scope and application of such cultures
in disease research, so alternative model systems are required. However, the species differences between
animal models and human tissue, and the lack of physiologically relevant in vitro models impedes research
progression and subsequent drug development.
This project aimed to address the unmet clinical requirement for more advanced in vitro models of IBD tissue.
Thus, it was hypothesised that the development of novel in vitro models incorporating stromal, immune, and
epithelial components could faithfully replicate significant aspects of the pathogenesis of IBD, while also
addressing certain constraints inherent in current models. To do this, the models were compared directly to
IBD patient tissue alongside existing literature.
3D tissue culture technologies were utilised to construct immune competent lamina propria tissue
compartments with an overlying epithelial layer. Within the models, subepithelial fibroblasts secrete
abundant endogenous extracellular matrix (ECM) which supports the growth and migration of a co-cultured
immune component that simulates the mass immune-cell infiltration observed in IBD. An inflammatory
phenotype is induced through addition of appropriate inflammatory stimuli. Inflammatory parameters
characteristic of IBD are observed, including endogenous inflammatory cytokine and chemokine secretion,
barrier disruption, and ECM remodelling. The potent MMP response elicited by inflamed models represents
a novel opportunity to study the mechanisms underpinning MMP activity in IBD, including MMP-9. The
inflamed models provided novel insight into ultrastructural epithelial aberrations relevant to IBD, as well as
basement membrane remodelling mechanisms. Moreover, this in vitro system allows for investigation into
the complex interplay between epithelial, mesenchymal and immune cells and the resultant effects on barrier
function. The versatility of the inflammatory response generated by IBD mucosal models was further evidenced through diverse stimulation methods. These data suggested that models could be guided towards
a specific disease phenotype based on the chosen stimulation approach.
Through treatment with four test drug compounds including standard-of-care treatments for IBD, we
demonstrated that the in vitro models can be applied for use in drug screening assays. Using prophylactic and
therapeutic approaches over 24-72 hour treatment windows, we were able to generate datasets that would
otherwise be impossible to obtain using explant tissue cultures due to their short-lived viability window.
These datasets demonstrated that the drug compounds exhibited differential anti-inflammatory effects
within inflamed IBD mucosal models and revealed novel insights into drug-mechanisms in epithelial repair
and stromal remodelling events.
Taken together, the data herein demonstrates the prospective utility of this innovative bioengineered system
for pre-clinical research and drug testing, while also highlighting its potential for further advancement.