Changes in extracellular matrix stiffness impact a variety of biological processes including cancer progression. However, cells also actively remodel the matrices they interact with, dynamically altering the matrix mechanics they respond to. Further, cells not only react to matrix stiffness, but also have a distinct reaction to matrix viscoelasticity. The impact of cell-driven matrix remodeling on matrix stiffness and viscoelasticity at the microscale remains unclear, as existing methods to measure mechanics are largely at the bulk scale or probe only the surface of matrices, and focus on stiffness. Yet, establishing the impact of the matrix remodeling at the microscale is crucial to obtaining an understanding of mechanotransduction in biological matrices, and biological matrices are not just elastic, but are viscoelastic. Here, we advanced magnetic probe-based microrheology to overcome its previous limitations in measuring viscoelasticity at the cell-size-scale spatial resolution within 3D cell cultures that have tissue-relevant stiffness levels up to a Young’s modulus of 0.5 kPa. Our magnetic microrheometers exert controlled magnetic forces on magnetic microprobes within reconstituted extracellular matrices and detect microprobe displacement responses to measure matrix viscoelasticity and determine the frequency-dependent shear modulus (stiffness), the loss tangent, and spatial heterogeneity. We applied these tools to investigate how microscale viscoelasticity of collagen matrices is altered by fibroblast cells as they contract collagen gels, a process studied extensively at the macroscale. Interestingly, we found that fibroblasts first soften the matrix locally over the first 32 hours of culture, and then progressively stiffen the matrix thereafter. Fibroblast activity also progressively increased the matrix loss tangent. We confirmed that the softening is caused by matrix-metalloproteinase-mediated collagen degradation, whereas stiffening is associated with local alignment and densification of collagen fibers around the fibroblasts. This work paves the way for the use of measurement systems that quantify microscale viscoelasticity within 3D cell cultures for studies of cell–matrix interactions in cancer progression and other areas.

细胞外基质硬度的变化会影响多种生物过程，包括癌症进展。然而，细胞也会积极地重塑它们与之交互的矩阵，动态地改变它们响应的矩阵机制。此外，细胞不仅对基质刚度有反应，而且对基质粘弹性也有明显的反应。细胞驱动的矩阵重构对微观尺度上的矩阵刚度和粘弹性的影响尚不清楚，因为现有的力学测量方法主要是在体尺度上或仅探测矩阵的表面，并专注于刚度。然而，在微观尺度上确定基质重塑的影响对于了解生物基质中的机械转导至关重要，生物基质不仅具有弹性，而且具有粘弹性。这里，我们改进了基于磁探针的微流变学，以克服其先前在 3D 细胞培养中以细胞大小尺度空间分辨率测量粘弹性的局限性，该 3D 细胞培养具有高达 0.5 kPa 的杨氏模量的组织相关刚度水平。我们的磁性微流变仪对重组细胞外基质内的磁性微探针施加受控磁力，并检测微探针位移响应以测量基质粘弹性并确定频率相关的剪切模量（刚度）、损耗角正切和空间异质性。我们应用这些工具来研究成纤维细胞在收缩胶原凝胶时如何改变胶原基质的微尺度粘弹性，这是一个在宏观尺度上广泛研究的过程。有趣的是，我们发现，在培养的前 32 小时内，成纤维细胞首先局部软化基质，然后逐渐使基质变硬。成纤维细胞活性也逐渐增加基质损失正切。我们证实软化是由基质金属蛋白酶介导的胶原降解引起的，而硬化与成纤维细胞周围胶原纤维的局部排列和致密化有关。这项工作为使用测量系统在 3D 细胞培养物中量化微尺度粘弹性以研究癌症进展和其他领域中的细胞-基质相互作用铺平了道路。我们证实软化是由基质金属蛋白酶介导的胶原降解引起的，而硬化与成纤维细胞周围胶原纤维的局部排列和致密化有关。这项工作为使用测量系统量化 3D 细胞培养物中的微尺度粘弹性以研究癌症进展和其他领域中的细胞-基质相互作用铺平了道路。我们证实软化是由基质金属蛋白酶介导的胶原降解引起的，而硬化与成纤维细胞周围胶原纤维的局部排列和致密化有关。这项工作为使用测量系统量化 3D 细胞培养物中的微尺度粘弹性以研究癌症进展和其他领域中的细胞-基质相互作用铺平了道路。