A Scalable Method to Fabricate 2D Hydrogel Substrates for Mechanobiology Studies with Independent Tuning of Adhesiveness and Stiffness

Mechanical signals from the extracellular matrix are crucial in guiding cellular behavior. Two-dimensional hydrogel substrates for cell cultures serve as exceptional tools for mechanobiology studies because they mimic the biomechanical and adhesive characteristics of natural environments. However, the interdisciplinary knowledge required to synthetize and manipulate these biomaterials typically restricts their widespread use in biological laboratories, which may not have the material science expertise or specialized instrumentation. To address this, we propose a scalable method that requires minimal setup to produce 2D hydrogel substrates with independent modulation of the rigidity and adhesiveness within the range typical of natural tissues. In this method, norbornene-terminated 8-arm polyethylene glycol is stoichiometrically functionalized with RGD peptides and crosslinked with a di-cysteine terminated peptide via a thiol-ene click reaction. Since the synthesis process significantly influences the final properties of the hydrogels, we provide a detailed description of the chemical procedure to ensure reproducibility and high throughput results. We demonstrate examples of cell mechanosignaling by monitoring the activation state of the mechanoeffector proteins YAP/TAZ. This method effectively dissects the influence of biophysical and adhesive cues on cell behavior. We believe that our procedure will be easily adopted by other cell biology laboratories, improving its accessibility and practical application.