Dynamic micro-3D-printed substrates for characterizing cellular responses to topography
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Cell cultures provide researchers the opportunity to observe cell behavior in response to specific, well-defined environmental cues, leading to insights that enable better engineering design for tissue culture and other biomedical applications. Chemical and electrical stimuli have been successfully applied to cultured cells to approximate aspects of the dynamic conditions experienced in vivo. However, in vitro topographical cues have mostly been limited to static substrates that do not subject cells to the dynamic conditions they experience in vivo when tissue remodels during development and wound healing. Delivering dynamic topographical cues to cultured cells can answer long-standing questions about mechanisms of cell morphology changes. Such capabilities could also facilitate engineering of wound-healing matrices and nerve guidance conduits by promoting migration of cells and providing directional guidance to cellular processes. This dissertation describes the development of approaches for introducing in situ topographical cues to cell cultures and inducing responses such as neurite guidance and cell alignment. Both strategies undertaken in this work make use of multiphoton-promoted photochemistry to print and manipulate three-dimensional microscopic protein hydrogel structures. In one approach, a technique referred to as micro-3D printing, topographical guidance cues are printed in the proximity of cultured cells to guide the growth of cellular processes. By translating a tightly-focused pulsed laser beam through a printing reagent solution flooding cultured cells, features are printed that provide physical guidance to extending neurites from NG108-15 cells, a neuronal model cell type. In another approach, an innovative technique known as micro-3D imprinting is developed for producing micrometer-scale depressions on the surfaces of photoresponsive protein hydrogels. The impact of various experimental parameters on topographical feature dimensions is characterized. Micro-3D imprinting is used to introduce dynamic topographical changes on a cell culture substrate, demonstrating that NIH-3T3 cells, a fibroblast cell model, alter their morphology and alignment in response to the introduction of a grooved surface topography. This set of approaches introduces new tools to the repertoire of cell biologists for exploring the behavior of cells growing in a spatio-temporally dynamic environment, opening possibilities for studies of cellular behavior in conditions that may better reflect environments cells experience in vivo.