Microfabricated Biomimetic Hydrogel Platforms to Investigate the Role of Physical Cues on Cell Behavior

Historically, cell behavior has been investigated by removing cells from their native environment and culturing them in two-dimensional plastic or glass substrates. Cell fate and function were believed to be influenced only by biochemical cues such as growth factors and signaling molecules, whereas the extracellular matrix (ECM) was considered a passive structural component. However, multiple landmark discoveries in the 1990s have led to the widespread appreciation of ECM physical properties like stiffness and geometry as functional and dynamic regulators of diverse cellular processes involved in development, homeostasis, and disease progression. This recognition of physical cues has led to the development of in vitro cell culture models that recapitulate mechanical and geometric features of the extracellular niche. Although these models have expanded our understanding, they frequently decouple the mechanical and geometric cues due to challenges in producing in vitro tissue models featuring tunability in shape and stiffness simultaneously in high a throughput and cost-effective manner. Such substrates lead to disparate results which do not correlate to cell behavior in vivo. Thus, the focus of this dissertation is to develop a facile fabrication strategy for in vitro tissue culture substrates that integrate the tissue's mechanical and geometric features and investigate the influence of such integrated cues on cell behavior. The first part describes the development of a high throughput and cost-effective fabrication strategy to create three-dimensional hierarchically curved mechanically tunable hydrogel platforms that mimic the stiffness, micro-curvature, and meso-curvature of cellular microenvironments. Photolithographic patterning and strain engineering were used to create soft anatomically mimetic gelatin cell culture models. The second part utilizes this mechanically tunable micropatterned platform to investigate the role of mechanical and geometric combinatorial cues in multicellular self-organization. The study reports the discovery of a new mode of endothelial cell self-organization guided by the mechano-geometric combinatorial cue, independent of protein or chemical patterning. This new mode of self-organization can be used to program reproducible cell patterning in customized shapes simply by providing appropriate topological and mechanical cues. Experimental insights gained using quantitative epifluorescence/confocal microscopy and pharmacological studies combined with mathematical modeling revealed that self-organization results from the balance of tangential cytoskeletal tension, cell-cell, and cell-substrate adhesion forces. These findings highlight the importance of the combinatorial effects of geometry and stiffness in complex cellular organization and open a new frontier in cell biophysics and biology