Methods to Characterize Granular Hydrogel Rheological Properties, Porosity, and Cell Invasion

Granular hydrogels are formed through the packing of hydrogel microparticles and are emerging for various biomedicalapplications, including as inks for 3D printing, substrates to studycell-matrix interactions, and injectable scaffolds for tissue repair.Granular hydrogels are suited for these applications because of theirunique properties including inherent porosity, shear-thinning andself-healing behavior, and tunable design. The characterization of their material properties and biological response involves technicalconsiderations that are unique to modular systems like granularhydrogels. Here, we describe detailed methods that can be used toquantitatively characterize the rheological behavior and porosity ofgranular hydrogels using reagents, tools, and equipment that aretypically available in biomedical engineering laboratories. Inaddition, we detail methods for 3D cell invasion assays usingmulticellular spheroids embedded within granular hydrogels and describe steps to quantify features of cell outgrowth (e.g.,endothelial cell sprouting) using standard image processing software. To illustrate these methods, we provide examples wherefeatures of granular hydrogels such as the size of hydrogel microparticles and their extent of packing during granular hydrogelformation are modulated. Our intent with this resource is to increase accessibility to granular hydrogel technology and to facilitate the investigation of granular hydrogels for biomedical applications