To realize this need for dynamically tunable culture systems, we recently developed an approach for in situ hydrogel property manipulation with light, allowing intimate control of a cell's microenvironment in both time and space 8. Such materials systems would have the potential to significantly improve our understanding of how cells receive information from their microenvironment and the role that these dynamic processes may play in biological questions ranging from directing stem cell differentiation 4, 5 to understanding cancer metastasis 6, 7. However, as advances in real-time tracking of dynamic cellular functions have emerged 4- 6, complementary approaches to alter the surrounding extracellular environment in a user-defined and highly-controlled fashion are extremely limited. In particular, hydrogel systems based on both protein components (e.g., collagen and Matrigel) and highly-tunable synthetic chemistries (e.g., PEG) have evolved to address some of these needs 1- 3. Physiological processes are guided by interactions between cells and their extracellular matrix, and this understanding has led to a growing interest in the development of material systems for improved 3D cell culture. While other protocols are available for controlling peptide presentation or modulus, few allow manipulation of material properties in situ and in the presence of cells down to the micrometer scale. These PEG-based materials are useful for probing the dynamic influence of cell-cell and cell-material interactions on cell function in 2D or 3D. This comprehensive protocol is useful for controlling peptide presentation and substrate modulus during cell culture on or within an elastic matrix. Hydrogel degradation occurs on-demand, in seconds to minutes, with user-directed light exposure. Preparation of the sterile solutions for hydrogel fabrication takes hours, while the reaction to form the final hydrogel is complete in minutes. In this protocol, synthesis and purification of the photodegradable monomers take approximately 2 weeks, but can be substantially shortened by purchasing the o-nitrobenzylether precursor. Degradation is readily achieved with cytocompatible wavelengths of low intensity flood irradiation (365 to 420 nm) in minutes or with highintensity laser irradiation (405 nm) in seconds. Cell-material interactions can be probed in 2D or 3D by spatiotemporally controlling the gel microenvironment, which allows unique experiments to be performed to monitor cell response to changes in their niche. Hydrogels are synthesized with the photodegradable crosslinker in the presence or absence of cells, allowing direct encapsulation or seeding on surfaces. Here, we provide coupling procedures to PEG- bis-amine to form a photodegradable crosslinker or to the fibronectin-derived peptide RGDS to form a photoreleasable tether. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in 2D or 3D cell culture. We describe a detailed procedure to create photolabile, poly(ethylene glycol)-based (PEG) hydrogels and manipulate material properties in situ.

Hdyroxy photolinker