Advisor: Andrés J. García, Ph.D. (Georgia Institute of Technology)

Committee:
Thomas Barker, Ph.D. (Georgia Institute of Technology and Emory University)
David Collard, Ph.D. (Georgia Institute of Technology)
Brandon Dixon, Ph.D. (Georgia Institute of Technology)
Asma Nusrat, M.D. (Emory University)

Acute injury of major epithelial or­­gan systems (kidney, liver, lung, etc.) is collectively a principal cause of death worldwide. Regenerative medicine promises to meet these human health challenges by harnessing intrinsic cellular processes to repair or replace damaged tissues.

Epithelial morphogenesis is a hard-wired, multicellular differentiation program that dynamically integrates microenvironmental cues to coordinate cell fate processes including adhesion, migration, proliferation, and polarization. Thus, epithelial morphogenesis is an instructive mode of tissue assembly, maintenance, and repair. Three-dimensional epithelial cell cultures in natural basement membrane (BM) extracts produce hollow, spherical cyst structures and have indicated that the BM provides the critical cell adhesion ligands to facilitate cell survival, stimulate proliferation, and promote polarization and lumen formation. However, the utility of natural BMs for detailed studies is generally limited by lot-to-lot variations, uncontrolled cell adhesive interactions, or growth factor contamination.

The goal of this thesis was to engineer bioartificial extracellular matrices (ECM) that would support and modulate epithelial cyst morphogenesis. We have engineered hydrogels, based on a multi-arm maleimide-terminated poly (ethylene glycol) (PEG-4MAL), that present cell adhesive molecules and enzymatic degradation substrates and promote polarized cyst differentiation in vitro.

To investigate the influence of matrix mechanical and biochemical signals on cyst morphogenesis, we independently varied the polymer weight percentage (thus, elastic modulus), the density of a cell adhesion ligand (RGD), and crosslink degradation rates of the hydrogels. Then, we adapted immunohistochemistry protocols, confocal imaging methods, and image analysis routines to evaluate functional outcomes including cell survival, cell proliferation, cyst lumen formation, cyst polarization and morphology. We found that cell proliferation, but not cell survival, was sensitive to the polymer wt%. This result defined a working range of PEG-4MAL concentration (3.5% - 4.5%) that promotes robust proliferation. Analysis of mature cysts indicated that low wt% (3.5%) hydrogels produced cysts with higher incidence of inverted polarity and multiple lumens. Thus, we employed 4.0% PEG-4MAL hydrogels with RGD ligand density ranging over 0 – 1000 uM to discover that (1) lumen formation was eliminated in the absence of RGD, (2) extent of lumen formation increased with increasing RGD concentration, and (3) cyst polarity was inverted below a threshold RGD concentration. Together, these studies validate PEG-4MAL hydrogels as a powerful culture platform to enable detailed investigation of matrix-directed modulation of epithelial morphogenesis.