Generation of Polyacrylamide and Silicone Extracellular Matrix Substrates with Defined Stiffness for Cell Biology...

The extracellular matrix (ECM) is a critical regulator of cell behavior, with ECM stiffness serving as a key mechanical cue that governs cellular signaling, morphology, and fate. However, experimental investigation of stiffness-dependent cellular responses is often limited by the availability of reproducible, cost-effective, and accessible culture platforms with precisely tunable mechanical properties. This protocol describes polyacrylamide (PA)- and silicone-based methods for fabricating ECM substrates with tunable stiffness. The procedures outline substrate preparation, surface functionalization, and ECM protein conjugation to ensure consistent cell adhesion across a physiologically relevant stiffness range. PA-based substrates are compatible with downstream biochemical assays, including protein and RNA extraction, as well as high-resolution fluorescence imaging. Silicone-based substrates are optimized for total internal reflection fluorescence (TIRF) microscopy, enabling visualization of cell-ECM interactions at the basal membrane. In addition, soft substrates can be adapted to support spheroid cultures positioned within a consistent imaging plane, facilitating consistent image acquisition. These methods provide robust, cost-effective, and reproducible platforms for systematically probing how ECM stiffness regulates cellular processes, advancing mechanistic insight into cell-ECM crosstalk in both physiological and disease contexts.