Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms
- 4 November 2010
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Protocols
- Vol. 5 (12), 1867-1887
- https://doi.org/10.1038/nprot.2010.139
Abstract
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.Keywords
This publication has 55 references indexed in Scilit:
- In situ elasticity modulation with dynamic substrates to direct cell phenotypeBiomaterials, 2010
- Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cellsNature Materials, 2009
- Control of Stem Cell Fate by Physical Interactions with the Extracellular MatrixCell Stem Cell, 2009
- Sequential click reactions for synthesizing and patterning three-dimensional cell microenvironmentsNature Materials, 2009
- Hydrogels as extracellular matrix mimics for 3D cell cultureBiotechnology & Bioengineering, 2009
- A thermoresponsive, microtextured substrate for cell sheet engineering with defined structural organizationBiomaterials, 2008
- The enhancement of chondrogenic differentiation of human mesenchymal stem cells by enzymatically regulated RGD functionalitiesBiomaterials, 2008
- Copper-free click chemistry for the in situ crosslinking of photodegradable star polymersChemical Communications, 2008
- Controlled differentiation of stem cellsAdvanced Drug Delivery Reviews, 2008
- Electroactive Self-Assembled Monolayers that Permit Orthogonal Control over the Adhesion of Cells to Patterned SubstratesLangmuir, 2006