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.

细胞形状在调节细胞行为中起重要作用。然而，在三个维度上独立控制细胞形状是一项艰巨的任务，并且细胞形状如何影响细胞对三个方面对机械和生化线索的反应仍不清楚。在这里，我们提出了一种基于水凝胶的平台，通过使用由胶原蛋白和藻酸盐组成的顺序形成的混合水凝胶来控制三维空间形状。通过调节藻酸盐的交联时间，我们将NIH 3T3成纤维细胞的形状固定在不同的扩散状态。然后，我们通过使用心脏成纤维细胞（CF）作为模型细胞，探索了细胞形状对细胞对微环境提示反应的影响。我们发现CF的扩散状态会影响其对机械（即基质刚度）和生化（即 转化生长因子-β1（TGF-β1））的三个维度提示。其他实验表明，粘着斑中的整合素β1和Smad2 / 3分别参与介导CF对基质硬度和TGF-β1信号的细胞形状依赖性反应。这项工作代表了了解细胞形状如何在三个维度上影响细胞对机械和生化线索的反应的第一步，并且可以为开发靶向细胞形状调节的新方法提供指导，以治疗纤维化和其他疾病。