Dynamic phototuning of 3D hydrogel stiffness

Significance Extracellular matrix (ECM) stiffness is an influential factor in many biological processes. Temporal changes in ECM stiffness are observed in cancer, cardiovascular disease, and wound healing, and are likely involved in disease progression. However, no cell culture systems exist to appropriately model temporal changes in ECM stiffness to determine the biological relevance and mechanisms involved. Here, we present a 3D hydrogel cell culture system in which the matrix stiffness can be tuned by light. Our approach offers both spatial and temporal control of stiffness, is compatible with cell culture, and can be used transdermally for in vivo applications. This system is amenable to many applications to investigate the influence of matrix stiffness on cell behavior and fate. Hydrogels are widely used as in vitro culture models to mimic 3D cellular microenvironments. The stiffness of the extracellular matrix is known to influence cell phenotype, inspiring work toward unraveling the role of stiffness on cell behavior using hydrogels. However, in many biological processes such as embryonic development, wound healing, and tumorigenesis, the microenvironment is highly dynamic, leading to changes in matrix stiffness over a broad range of timescales. To recapitulate dynamic microenvironments, a hydrogel with temporally tunable stiffness is needed. Here, we present a system in which alginate gel stiffness can be temporally modulated by light-triggered release of calcium or a chelator from liposomes. Others have shown softening via photodegradation or stiffening via secondary cross-linking; however, our system is capable of both dynamic stiffening and softening. Dynamic modulation of stiffness can be induced at least 14 d after gelation and can be spatially controlled to produce gradients and patterns. We use this system to investigate the regulation of fibroblast morphology by stiffness in both nondegradable gels and gels with degradable elements. Interestingly, stiffening inhibits fibroblast spreading through either mesenchymal or amoeboid migration modes. We demonstrate this technology can be translated in vivo by using deeply penetrating near-infrared light for transdermal stiffness modulation, enabling external control of gel stiffness. Temporal modulation of hydrogel stiffness is a powerful tool that will enable investigation of the role that dynamic microenvironments play in biological processes both in vitro and in well-controlled in vivo experimen