Synthetic PEG-Maleimide Hydrogel For In Vitro Culture of Primary Human Intestinal Enteroids

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HEWES-THESIS-2024.pdf (41.07 MB)
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Hewes, Jacob D.
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School of Chemical and Biomolecular Engineering
School established in 1901 as the School of Chemical Engineering; in 2003, renamed School of Chemical and Biomolecular Engineering
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https://hdl.handle.net/1853/78618
Abstract
Gastrointestinal diseases are becoming increasingly prevalent in developed countries, stimulating the need for human-specific models of intestinal development and disease that can recapitulate the structure and function of the gut in vitro. In the past decade, intestinal organoid technology has advanced in vitro reproduction of intestinal organoids. Enteroids, epithelial organoids derived from human intestinal tissue, are three-dimensional (3D) structures that can model the identity, cell heterogeneity, and cell behaviors of the original tissue in vitro. This makes them a powerful tool for drug screening, disease modeling, and reconstructing damaged epithelium in conditions like ulcerative colitis.Current protocols for organoid culture require expansion of intestinal stem cells within Matrigel, a tumor-derived extracellular matrix (ECM) that exhibits considerable lot-to-lot variability, poor experimental control, and inability to decouple matrix physical and biochemical properties due to its ill-defined composition. The reliance on Matrigel for intestinal organoid culture severely limits their translational potential. This thesis project aims to reduce the requirement for biologically-derived ECMs to support intestinal organoid culture. To accomplish this aim, we developed completely synthetic hydrogels presenting ECM-derived adhesive ligands crosslinked with peptides susceptible to matrix metalloprotease (MMP) degradation to identify gel compositions supporting the culture of enteroids starting from human tissue-derived progenitor epithelial cells. The synthetic hydrogel platforms designed were based on a four-arm poly(ethylene glycol) (PEG) macromer with maleimide groups at each terminus (PEG-4MAL) and the RGD integrin-binding peptide. Hydrogel biophysical properties and crosslinker type were key parameters in engineering a synthetic ECM mimic that supported human ileum enteroids. A PEG-4MAL hydrogel platform with the protease-degradable crosslinker IPES promoted the best enteroid emergence and growth compared to Matrigel. In this synthetic matrix, human intestinal enteroids emerge from single cells and express markers of intestinal stem cells. The modular design of this synthetic matrix and its ability to support the in vitro culture of enteroids strengthens the translational potential of this platform for regenerative medicine, disease modeling, and other applications while reducing the dependency on Matrigel.
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2024-07-31
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