Structural and mechanical remodeling of the cytoskeleton studied in 3D microtissues under acute dynamic stretch

Abstract When mechanically stretched, cells cultured on 2D substrates share a universal softening and fluidization response that arises from poorly understood remodeling of well-conserved cytoskeletal elements. It is known, however, that the structure and distribution of these cytoskeletal elements are profoundly influenced by the dimensionality of a cell’s environment (ie. on a 2D surface vs. within a 3D matrix). Therefore, in this study we aimed to determine whether cells cultured in a 3D extracellular matrix also follow the same softening response and to link this mechanical change to direct evidence of cytoskeletal remodeling. To achieve this, we developed a new high-throughput approach to measure the dynamic mechanical properties of cells and allow for sub-cellular imaging of physiologically relevant 3D microtissue cultures. We found that fibroblast, smooth muscle and skeletal muscle microtissues strain softened but did not fluidize, and upon loading cessation, they fully regained their initial mechanical properties. These responses required a filamentous actin cytoskeleton, and were mirrored by changes to actin remodeling rates, and direct visual evidence of actin depolymerization during stretching and repolymerization after stretch cessation. On the other hand, the response could not be attributed to either remodeling of microtubules or myosin motor activity. Our new approach for assessing cell mechanics has linked behaviors seen in 2D cultures to a soft 3D extracellular matrix, and connected visual remodeling of the cytoskeleton to changes in mechanical properties at the tissue-level. Significance StatementWith every breath and movement, cells in our body are subjected to mechanical forces. These forces are key regulators of normal development and function, as well as disease progression. To understand how cells “feel” mechanical cues in their microenvironment, we have previously relied on two-dimensional experimental approaches and often assessed single cells in isolation. Here, we present a novel lab-on-chip device, which enables simultaneous mechanical stimulation and sub-cellular imaging of three-dimensional multi-cellular microtissues. In this article with this device, we quantitatively linked force-induced mechanical changes in microtissues to specific molecular remodelling pathways in the cytoskeleton. The approaches and insights presented in this study will deepen our understanding of the mechanobiological pathways governing tissue development and function in health and disease.