Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Pathology and Laboratory Medicine

Supervisor

Asfaha, Samuel.

2nd Supervisor

Flynn, Lauren E.

Co-Supervisor

Abstract

The generation of a tissue-specific intestinal hydrogel using small intestinal extracellular matrix (ECM) has the potential to support and promote the growth of intestinal organoids. In this study, we aimed to develop hydrogels derived exclusively from intestinal ECM or composites comprised of intestinal ECM combined with alginate, which may allow greater tuning of the hydrogel properties. A novel mouse intestinal decellularization protocol was developed and the ECM was characterized. Our analysis demonstrates that cellular and nuclear content was removed effectively, while preserving key ECM components. When decellularized ECM was used to generate hydrogels, the resulting ECM displayed bioactivity as demonstrated by metabolic and pro-proliferative effects on NIH 3T3 murine fibroblasts. More importantly, our novel ECM hydrogel also supported intestinal organoid growth. These studies demonstrate that tissue-specific ECM-derived hydrogels can indeed support and promote the growth of intestinal organoids in vitro.

Summary for Lay Audience

The small intestine is the organ in the body where most food digestion and nutrient absorption takes place. It has been shown previously that stem cells give rise to all of the cell types in the intestine, which is important for normal tissue turnover and healing. Interestingly, these stem cells can be isolated from tissues and used to form “mini-intestines” in a petri dish, called organoids. These organoids may allow researchers to develop models of intestinal diseases for testing the effects of different drugs, or potentially could be used to develop cell-based therapies for regenerative medicine applications. Currently, the only way to form organoids is by encapsulating and culturing them within a jello-like material called Matrigel, which contains essential proteins needed for the stem cells. However, Matrigel is produced by mouse cancer cells, which means that the cells generated using this approach cannot be used for clinical applications. The purpose of this study was to develop new biomaterials to replace Matrigel for the growth of organoids, using proteins sourced from intestinal tissues. There is evidence to support that such intestinal-derived materials could support the survival and growth of stem cells, and help them to give rise to the other cell types in the intestine. This thesis developed a new method for isolating intestinal-specific proteins from mouse tissues. Further, these proteins were further processed to enable the formation of gels that could be used to encapsulate cells. Cell culture studies confirmed that the intestinal protein gels supported cell viability and the growth of mouse intestinal organoids, similar to Matrigel. In addition, the effects of combining the intestinal proteins with alginate, a natural gel that comes from seaweed, were explored to develop composite materials that had more tunable mechanical properties. While the organoids were successfully encapsulated and cultured within these composites, further studies are needed to refine the conditions to promote organoid growth. Overall, this thesis contributed to the development of promising new biomaterials that hold the potential to replace Matrigel as a more clinically translational tissue-specific platform for studies of intestinal organoids.

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