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 • Steps for formulating extracellular vesicle (EV)-crosslinked hydrogels • Rheological methods to assess stiffness, self-healing, and injectability • Guidance for interpreting rheological data to reveal EV-polymer interactions • Framework for linking polymer chemistry, EV concentration, and hydrogel mechanics Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.