We describe a detailed procedure to create photolabile, polyethylene glycol (PEG)-based hydrogels and manipulate material properties in situ. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in two-dimensional (2D) or 3D cell culture. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. In this study, we provide coupling procedures to PEG-bis-amine to form a photodegradable cross-linker or to the fibronectin-derived peptide RGDS to form a photoreleasable tether. Hydrogels are synthesized with the photodegradable cross-linker in the presence or absence of cells, allowing direct encapsulation or seeding on surfaces. 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. Degradation is readily achieved with cytocompatible wavelengths of low-intensity flood irradiation (365–420 nm) in minutes or with high-intensity laser irradiation (405 nm) in seconds. In this protocol, synthesis and purification of photodegradable monomers take approximately 2 weeks, but the process can be substantially shortened by purchasing the o-nitrobenzylether precursor. Preparation of sterile solutions for hydrogel fabrication takes hours, whereas the reaction to form the final hydrogel is complete in minutes. Hydrogel degradation occurs on demand, in seconds to minutes, with user-directed light exposure. This comprehensive protocol is useful for controlling peptide presentation and substrate modulus during cell culture on or within an elastic matrix. 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. Although 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.
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We acknowledge the National Institutes of Health (NIH) (DEO12998 and DEO16523) and HHMI for funding this work. M.W.T. acknowledges the US Department of Education's Graduate Assistantships in Areas of National Need program and the NIH (T32 GM-065103) for fellowship assistance. A.M.K. acknowledges the NASA Graduate Student Researchers Program and the US Department of Education's Graduate Assistantships in Areas of National Need program for fellowship assistance. We thank A.M. Kasko for initial guidance and contributions toward the photolabile molecule synthesis, A.A. Aimetti for discussions and training related to peptide synthesis and J.A. Benton for assistance with VIC culture. We also thank the MediaLab in the Department of Biochemistry at the University of Wisconsin at Madison for the use of an Adobe Illustrator template.
The authors declare no competing financial interests.
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Kloxin, A., Tibbitt, M. & Anseth, K. Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms. Nat Protoc 5, 1867–1887 (2010). https://doi.org/10.1038/nprot.2010.139
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