Multiphoton techniques for dynamic manipulation of cellular microenvironments

A multitude of biophysical signals, including chemical, mechanical, and contact guidance cues, are embedded within the extracellular matrix (ECM) to dictate cell behavior and determine cell fate. To understand the complexity of the cell-matrix interaction and how changes to the ECM contribute to the development of tissues or diseases, three-dimensional (3D), culture systems that can decouple the effects of these cues on cell behavior are required. This dissertation describes the development and characterization of approaches based on multiphoton excitation (MPE) to control the chemical, mechanical, and topographical presentation of micro-3D-printed (μ-3DP) protein hydrogels independently. Protein hydrogels were chemically functionalized via the MPE-induced conjugation of benzophenone-biotin without altering the physical properties of the matrix. Complex, immobilized patterns and chemical gradients were generated within protein hydrogels with a high degree of spatial resolution in all axes. Hydrogel surfaces were also labeled with adhesive moieties to promote localized Schwann cell adhesion and polarization. Laser shrinking, a method based on MPE to manipulate the topographical and mechanical presentation of protein hydrogels after fabrication, is also presented. Topographical features on an originally flat substrate are created with depths approaching 6 μm. The Young’s modulus of protein hydrogels can also be increased by 6-fold (~15 – ~90 kPa) using laser shrinking, and parameters can be adjusted to create continuous gradient profiles for studying durotaxis. At determined scan conditions, the two properties can be adjusted independently of each other. Most importantly, the physical properties of the hydrogels can be manipulated in situ to study the effects of dynamic changes to the substrates on cells. As a potential tool to monitor cellular responses to presented cues, fluorescent probes that detect nitric oxide are characterized. Collectively, these technologies represent a key advance in hydrogel tunability, as the platforms presented offer independent, dynamic, and spatiotemporal control of the chemical, mechanical, and topographical features of protein hydrogels. The introduced technologies expand the possibilities of protein hydrogels to clarify underlying factors of cell-matrix interactions that drive morphogenesis and pathogenesis, and are broadly applicable to a multitude of physiological systems.