Thesis Advisor:

Andrés J. García, Ph.D., George W. Woodruff School of Mechanical Engineering, Georgia

Institute of Technology

Thesis Committee Members:

Hang Lu, Ph.D., School of Chemical and Biomolecular Engineering, Georgia Institute of

Technology

Ankur Singh, Ph.D., George W. Woodruff School of Mechanical Engineering, Georgia Institute

of Technology

Synthetic PEG-Maleimide Hydrogel For In vitro Culture of Primary Human

Intestinal Enteroids

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.

https://gatech.zoom.us/j/96045411587?pwd=hZJ6E6XuOEtCbbwiNNXlmgDUBwSDPa.1

Meeting ID: 960 4541 1587

Passcode: 109969