Chong, S. W.; Sardharwalla, J.; Masonsong, G. S. P.; Cosgrove, J.; Katselas, A.; Gresham, I. J.; Bilek, M. M.; Shen, Y.; Neto, C.; Vigolo, D.

Engineered stiffness gradient hydrogels offer exciting opportunities to probe fundamental mechanobiological processes in vitro. However, the need to spatially manipulate the properties of soft hydrogels at the micron scale poses challenges in developing fabrication platforms that can reliably modulate the gradient gel characteristics according to user needs. This study describes a modular approach leveraging thermophoresis in microfluidics to create high-fidelity stiffness gradients with linear and complex profiles, including periodic and anisotropic gradients. This study describes the platform\'s design and optimization, demonstrating achievable stiffness ranges and gradient slopes that correlate with many physiologic and diseased tissue types. This platform is also compatible with different hydrogel crosslinking chemistries, providing a versatile tool to engineer microenvironments with increased complexity. Directionally biased fibroblast cell proliferation and migration on fabricated stiffness gradient gelatin methacryloyl (GelMA) hydrogels indicate the effectiveness of this platform in modulating the mechanical microenvironment of cells. The results indicate that both the absolute stiffness range and the pattern of stiffness variation jointly affect cell behaviors. Considering its remarkable flexibility, the fabrication platform can significantly advance the development of biophysical gradient hydrogels that better replicate the intricacies of native tissues and help realize the next breakthroughs in mechanobiology.