A Modular Microfluidic System to Generate Gradient Hydrogels with Simple‐to‐Complex Stiffness Profiles for Mechanobiology

Abstract Engineered stiffness gradient hydrogels offer exciting opportunities to probe fundamental mechanobiological processes in vitro. 2D stiffness gradients are particularly useful as a reductionist approach to study cell–material interactions in specific pathophysiological contexts. However, methods that streamline the design and fabrication of these gradient materials are needed to facilitate broad adoption and applicability. The thermophoretic technique, based on temperature‐driven gradient generation, offers a promising solution due to its compatibility with diverse hydrogel chemistries, but the prior implementations have been restricted to fixed, steep linear gradients. Therefore, this study presents a modular platform leveraging microfluidics and thermophoretic strategy to enable flexible fabrication of stiffness gradients with linear and complex spatial patterns. Key features of the platform include simplified setup and operation, rapid material optimization, and easy adaptation with various crosslinking modalities. The versatility of this platform is validated by testing a series of linear, periodic, and anisotropic stiffness gradients, demonstrating stiffness‐dependent fibroblast cell proliferation and migration. The results indicate that both the absolute stiffness range and the pattern of stiffness variation jointly affect cell behaviors. Considering its remarkable flexibility, the modular fabrication platform can significantly expand the library of biophysical gradient hydrogels and increase technology accessibility to more laboratories.