Engineering the extracellular matrix environment to investigate dynamic cellular responses

Cellular behavior is precisely regulated by a variety of cues within the extracellular environment that vary across time and space. In the following studies, we examine cellular responses to dynamic stimuli using micropatterning and microfabrication approaches that enable greater spatiotemporal control over the stimulus. Specifically, we investigate how the controlled presentation of new extracellular matrix and growth factors regulates multi-cellular behaviors such as cell migration and tubulogenesis in a variety of contexts.;We first developed a novel method to investigate mechanisms underlying epithelial sheet migration on 2D surfaces. We applied a micro-contact printing approach to pattern gold surfaces with carboxylic acid-terminated self assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that can be rendered permanently non-adhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically switched from non-adhesive to adhesive, thereby enabling cell migration from a pre-specified pattern onto a new pattern. We used these substrates to investigate the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to examine whether lead cells influence the direction of movement of followers. We observed that on average 5 cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets.;We then developed an approach to culture cells within micro-scale collagen gels (microgels) in order to eliminate the reduced rate of soluble factor penetration through traditional 3D cultures (macrogels). Comparing responses in 2D culture, microgels, and macrogels demonstrated that ERK signaling associated with HGF-mediated kidney epithelial cell scattering and tubulogenesis was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment. In contrast, HGF induced opposite effects on myosin activation in microgel versus 2D cultures, demonstrating also the existence of responses regulated primarily by matrix dimensionality. These studies highlight the importance of dissecting diffusional effects that lead to altered growth factor stimulation dynamics from the dimensional effects of 3D environments on cellular signaling and function.;Lastly, we applied the microgel culture approach to spatially pattern cells within a collagen gel in order to investigate endothelial tubulogenesis. Endothelial cells cultured within micro-scale channels filled with collagen gel organized into tubes with lumens within 24 - 48 hours of seeding, and exhibited cell-cell junction formation characteristic of capillaries. Tube diameter could be expanded by culturing cells at higher collagen concentrations or by increasing channel width. The microfabricated template was used to guide branched tube formation, allowing for the generation of more complex capillary architectures. This platform may be of use to create vascular networks for tissue engineering applications as well as to better understand the process of vasculogenesis.;Together, these studies introduce several new techniques to control the spatiotemporal presentation of extracellular cues to cells, and generate novel insights into how these cues guide multi-cellular behaviors such as cell migration and tubulogenesis.