Ostrowski, Z.; Price, T.; Zhang, J.; Kaufmann, T.; Judka, N.; Gardner, T.; Thatcher, M.; Miller, E.; Mesyk, L.; Koep, A.; Melvin, A. T.; Ren, J.; Schneider, I. C.

Mechanobiology or the response of cells to forces or mechanical properties of their environment drives many physiological and pathological processes including development, wound healing, fibrosis and cancer. A variety of cell biological behaviors are driven by local mechanical properties including stem cell differentiation and drug resistance. Furthermore, cells can sense stiffness gradients and migrate up the gradient in a process called durotaxis. The development of 3D hydrogel systems with tunable mechanical gradient patterns affords the ability to run multiple experiments at different stiffness. This is critical as some cell behavior is not monotonically dependent upon stiffness. Additionally, the creation of mechanical property gradients within 3D hydrogels may be able to guide cells to particular targets forming complex cellular structures within the hydrogel or enhancing wound healing through directed migration. In this paper, we developed an approach to spatially imprint within alginate hydrogels, gradients in mechanical properties that can be used to probe mechanobiology. Stencils were easily designed and fabricated using a common craft cutter to control the presentation of a calcium crosslinking solution to alginate gels. Different stencil shapes result in different gradients in opacity that can be imprinted into both thick and thin alginate gels of arbitrary shape. The steepness of the opacity gradient as well as the maximum opacity can be controlled based on reproducible crosslinking kinetics regulated through calcium concentration and gradient developing time. Calcium crosslinking results in both opacity changes as well as increases in elastic modulus in the bulk hydrogel. Opacity correlates with elastic modulus, allowing it to be used as a proxy for local elastic modulus. Consequently, spatial gradients in elastic modulus can also be imprinted into alginate gels using this stenciling approach. This stenciling approach represents a facile way to control stiffness gradients in alginate gels.