Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article


Master of Science


Pathology and Laboratory Medicine


Asfaha, Samuel


Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and Ulcerative Colitis (UC), is characterized by chronic inflammation of the gut. A strategy to study IBD in-vitro includes epithelial organoids, but this approach does not permit studying epithelial-stromal interactions. In contrast, Air-Liquid Interface (ALI) organoids—whole tissue explants grown in collagen gel exposed to air—potentially contain all intestinal layers. I thus generated colonic ALI organoids from healthy neonatal and adult mice. These organoids encapsulated epithelial and stromal components; however, organoid formation from adult tissue was less efficient than neonatal tissue. Addition of exogenous growth factors to the culture medium increased organoid efficiency from adult tissue while retaining stromal elements. Finally, colonic ALI organoids generated from mice recovering from Dextran Sodium Sulfate (DSS)-induced colitis appeared to contain epithelium and stroma, including immune cells in short-term culture. Thus, the ALI technique holds promise for studying epithelial-stromal interactions in health and disease.

Summary for Lay Audience

Inflammatory bowel disease (IBD) is an umbrella term for various lifelong intestinal diseases—primarily Crohn’s Disease (CD) and Ulcerative Colitis (UC). IBD affects approximately 1 in 150 people in Canada. Currently, we lack a model of IBD to study the disease and its treatment. Organoids, miniature versions of organs grown in 3D in a petri dish, are best suited to provide such a model. However, intestinal organoids (“mini-guts”) typically only contain one layer of the intestine and colon and therefore lack many cell types that are important in IBD, and for that reason, these organoids cannot fully represent this disease. However, another organoid method, called Air-Liquid Interface (ALI), contains all layers of the intestine and thus holds greater promise for modeling both the healthy gut and IBD. ALI organoids have not been generated from either healthy or IBD tissue.

Thus, we began by generating these organoids from healthy mice, ranging in age from a few days to several weeks old. These organoids appeared to contain several layers of the gut, but tissue from older mice formed fewer organoids than tissue from younger mice. So, we conducted several experiments adding various growth factors (special proteins to make cells grow and divide faster) to our organoids derived from adult tissue. These changes led to more organoids forming from adult tissue while still containing many layers of the gut. Then, we used an animal model of IBD where mice are given a chemical to damage their colonic tissue, similar to UC in human beings. ALI organoids generated from this tissue again appeared to contain several layers of the gut, but, importantly, these organoids also had more immune cells than healthy organoids. This suggests we maintained some parts of the disease in our organoids.

In sum, we have shown that ALI organoids can be generated from the healthy and diseased mouse colon. Therefore, this model holds promise for studying how IBD causes disease of the colon.

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