Dynamic biaxial loading of vascular smooth muscle cell seeded tissue equivalents.

An intricate reciprocal relationship exists between adherent synthetic cells and their extracellular matrix (ECM). These cells deposit, organize, and degrade the ECM, which in turn influences cell phenotype via responses that include sensitivity to changes in the mechanical state that arises from changes in external loading. Collagen-based tissue equivalents are commonly used as simple but revealing model systems to study cell-matrix interactions. Nevertheless, few quantitative studies report changes in the forces that the cells establish and maintain in such gels under dynamic loading. Moreover, most prior studies have been limited to uniaxial experiments despite many soft tissues, including arteries, experiencing multiaxial loading in vivo. To begin to close this gap, we use a custom biaxial bioreactor to subject collagen gels seeded with primary aortic smooth muscle cells to different biaxial loading conditions. These conditions include cyclic loading with different amplitudes as well as different mechanical constraints at the boundaries of a cruciform sample. Irrespective of loading amplitude and boundary condition, similar mean steady-state biaxial forces emerged across all tests. Additionally, stiffness-force relationships assessed via intermittent equibiaxial force-extension tests showed remarkable similarity for ranges of forces to which the cells adapted during periods of cyclic loading. Taken together, these findings are consistent with a load-mediated homeostatic response by vascular smooth muscle cells.

A 3D micro-printed single cell micro-niche with asymmetric niche signals - An in vitro model for asymmetric cell division study.

Intricate microenvironment signals orchestrate to affect cell behavior and fate during tissue morphogenesis. However, the underlying mechanisms on how specific local niche signals influence cell behavior and fate are not fully understood, owing to the lack of in vitro platform able to precisely, quantitatively, spatially, and independently manipulate individual niche signals. Here, microarrays of protein-based 3D single cell micro-niche (3D-SCµN), with precisely engineered biophysical and biochemical niche signals, are micro-printed by a multiphoton microfabrication and micropatterning technology. Mouse embryonic stem cell (mESC) is used as the model cell to study how local niche signals affect stem cell behavior and fate. By precisely engineering the internal microstructures of the 3D SCµNs, we demonstrate that the cell division direction can be controlled by the biophysical niche signals, in a cell shape-independent manner. After confining the cell division direction to a dominating axis, single mESCs are exposed to asymmetric biochemical niche signals, specifically, cell-cell adhesion molecule on one side and extracellular matrix on the other side. We demonstrate that, symmetry-breaking (asymmetric) niche signals successfully trigger cell polarity formation and bias the orientation of asymmetric cell division, the mitosis process resulting in two daughter cells with differential fates, in mESCs.

Generation of 3D Fibroblast-Derived Extracellular Matrix and Analysis of Tumor Cell-Matrix Interactions and Signaling.

Fibroblasts are the major producers of the extracellular matrix and regulate its organization. Aberrant signaling in diseases such as fibrosis and cancer can impact the deposition of the matrix proteins, which can in turn act as an adhesion scaffold and signaling reservoir promoting disease progression. To study the composition and organization of the extracellular matrix as well as its interactions with (tumor) cells, this protocol describes the generation and analysis of 3D fibroblast-derived matrices and the investigation of (tumor) cells seeded onto the 3D scaffolds by immunofluorescent imaging and cell adhesion, colony formation, migration, and invasion/transmigration assays.

Unexpected softening of a fibrous matrix by contracting inclusions.

Cells respond to the stiffness of their surrounding environment, but quantifying the stiffness of a fibrous matrix at the scale of a cell is complicated, due to the effects of nonlinearity and complex force transmission pathways resulting from randomness in fiber density and connections. While it is known that forces produced by individual contractile cells can stiffen the matrix, it remains unclear how simultaneous contraction of multiple cells in a fibrous matrix alters the stiffness at the scale of a cell. Here, we used computational modeling and experiments to quantify the stiffness of a random fibrous matrix embedded with multiple contracting inclusions, which mimicked the contractile forces of a cell. The results showed that when the matrix was free to contract as a result of the forces produced by the inclusions, the matrix softened rather than stiffened, which was surprising given that the contracting inclusions applied tensile forces to the matrix. Using the computational model, we identified that the underlying cause of the softening was that the majority of the fibers were under a local state of axial compression, causing buckling. We verified that this buckling-induced matrix softening was sufficient for cells to sense and respond by altering their morphology and force generation. Our findings reveal that the localized forces induced by cells do not always stiffen the matrix; rather, softening can occur in instances wherein the matrix can contract in response to the cell-generated forces. This study opens up new possibilities to investigate whether cell-induced softening contributes to maintenance of homeostatic conditions or progression of disease. STATEMENT OF SIGNIFICANCE: Mechanical interactions between cells and the surrounding matrix strongly influence cellular functions. Cell-induced forces can alter matrix properties, and much prior literature in this area focused on the influence of individual contracting cells. Cells in tissues are rarely solitary; rather, they are interspersed with neighboring cells throughout the matrix. As a result, the mechanics are complicated, leaving it unclear how the multiple contracting cells affect matrix stiffness. Here, we show that multiple contracting inclusions within a fibrous matrix can cause softening that in turn affects cell sensing and response. Our findings provide new directions to determine impacts of cell-induced softening on maintenance of tissue or progression of disease.