Synthesis of photodegradable hydrogels as dynamically tunable cell culture platforms

Abstract

We describe a detailed procedure to create photolabile, poly(ethylene glycol)-based (PEG) hydrogels and manipulate material properties in situ. The cytocompatible chemistry and degradation process enable dynamic, tunable changes for applications in 2D or 3D cell culture. The materials are created by synthesizing an o-nitrobenzylether-based photodegradable monomer that can be coupled to primary amines. 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. Hydrogels are synthesized with the photodegradable crosslinker 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 to 420 nm) in minutes or with highintensity laser irradiation (405 nm) in seconds. 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. Preparation of the sterile solutions for hydrogel fabrication takes hours, while 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. 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.

Design and Characterization of a Synthetically Accessible, Photodegradable Hydrogel for User-Directed Formation of Neural Networks

“…(15) While the above advances have demonstrated the power of controlled biological signaling to cells through regulation of material properties in space and time, complementary materials and simplified systems provide opportunities to expand the accessibility of the chemistry to a broader community of biomaterial researchers. (26,27) Here, recent advances in molecular building blocks are exploited to synthesize a step-growth poly(ethylene glycol) (PEG) hydrogel cross-linked through copper-free click chemistry capable of undergoing degradation upon irradiation by either single-(334 and 365 nm) or two-photon light (740 nm). A SPAAC reaction between dibenzylcyclooctyne (DBCO) and an aliphatic azide is employed for cross-linking and a nitrobenzyl ether moiety is engineered into the cross-linker to enable degradation.…”

Photochemical Patterning of Ionically Cross-Linked Hydrogels

“…Photoisomerizable groups, including the azobenzene group [29], stilbenes [30], and spiropyrans [31,32] have been used to induce disassembly or dissolution of biomaterials. The most commonly used approach to control this material involves photolysis of covalent cross-links, such as 2-nitrobenzyl derivatives [27,28,[33][34][35]. These materials were suggested for applications involving controlled drug release from micelles [36] and liposomes [37] as well as gene delivery using photoisomerizable cationic surfactants [38].…”

“…Conventional photolithographic methods that use short wave UV light and organic solvents are not suitable for sensitive macromolecules and cells. However, by using visible and to a lesser degree long wave UV light, it is possible to fabricate materials and dynamically change properties of biomaterials with already attached or embedded living cell cultures [27,28]. Photoisomerizable groups, including the azobenzene group [29], stilbenes [30], and spiropyrans [31,32] have been used to induce disassembly or dissolution of biomaterials.…”

Stiffening hydrogels to probe short- and long-term cellular responses to dynamic mechanics

“…More recently, Anseth and colleagues 16 reported on photocleavable polyethylene glycol-based hydrogels, where crosslinking can be decreased both temporally and spatially with light exposure. However, matrix stiffening is more relevant for many biological events, including tissue development, wound healing and disease progression, such as fibrosis and tumour formation.…”