Protocol for the formulation and characterization of extracellular vesicle-crosslinked supramolecular hydrogels

Extracellular vesicles (EVs) can serve as supramolecular crosslinkers to form injectable, intrinsically bioactive hydrogels. This protocol details the formulation, rheological characterization, and analysis of EV-crosslinked hydrogels with tunable mechanics. We describe steps for preparing alkyl-modified hydroxypropyl methylcellulose (HPMC) polymers, mixing polymers with EVs to form supramolecular networks, and assessing properties of the resulting hydrogels. We provide guidance for linking rheological properties to polymer-EV design parameters, enabling users to tailor hydrogel mechanics for diverse biomedical applications. For complete details on the use and execution of this protocol, please refer to Margaronis et al.¹.