Control of fibroblast shape in sequentially formed 3D hybrid hydrogels regulates cellular responses to microenvironmental cues

Cell shape plays important roles in regulating cell behavior; however, independently controlling cell shape in three dimensions is a challenging undertaking, and how cell shape affects cellular responses to mechanical and biochemical cues in three dimensions remains unclear. Here, we present a hydrogel-based platform to control cell shape in three dimensions by using sequentially formed hybrid hydrogels consisting of collagen and alginate. By adjusting the cross-linking time of the alginate, we fixed the shape of NIH 3T3 fibroblasts at different spreading states. Then, we explored the influence of cell shape on the cell responses to microenvironmental cues by using cardiac fibroblasts (CFs) as model cells. We found that the spreading state of the CFs influences their responses to both mechanical (i.e., matrix stiffness) and biochemical (i.e., transforming growth factor-β1 (TGF-β1)) cues in three dimensions. Additional experiments revealed that integrin β1 in focal adhesions and Smad2/3 are involved in mediating the cell shape-dependent responses of CFs to matrix stiffness and TGF-β1 cues, respectively. This work represents the first step in understanding how cell shape influences cell responses to mechanical and biochemical cues in three dimensions and can be instructive for developing novel approaches to target cell shape regulation for treating fibrosis and other diseases.