A multi-layered polydimethylsiloxane microfluidic device with an integrated suspended membrane has been fabricated that allows dynamic and multi-axial mechanical deformation and simultaneous live-cell microscopy imaging. The transparent membrane’s strain field can be controlled independently along two orthogonal directions. Human foreskin fibroblasts were immobilized on the membrane’s surface and stretched along two orthogonal directions sequentially while performing live-cell imaging. Cyclic deformation of the cells induced a reversible reorientation perpendicular to the direction of the applied strain. Cells remained viable in the microdevice for several days. As opposed to existing microfluidic or macroscale stretching devices, this device can impose changing, anisotropic and time-varying strain fields in order to more closely mimic the complexities of strains occurring in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-013-1381-5) contains supplementary material, which is available to authorized users.

The strain-induced reorientation response of cyclically stretched cells has been well characterized in uniform strain fields. In the present study, we comprehensively analyse the behaviour of human fibroblasts subjected to a highly non-uniform strain field within a polymethylsiloxane microdevice. Our results indicate that the strain gradient amplitude and direction regulate cell reorientation through a coordinated gradient avoidance response. We provide critical evidence that strain gradient is a key physical cue that can guide cell organization. Specifically, our work suggests that cells are able to pinpoint the location under the cell of multiple physical cues and integrate this information (strain and strain gradient amplitudes and directions), resulting in a coordinated response. To gain insight into the underlying mechanosensing processes, we studied focal adhesion reorganization and the effect of modulating myosin-II contractility. The extracted focal adhesion orientation distributions are similar to those obtained for the cell bodies, and their density is increased by the presence of stretching forces. Moreover, it was found that the myosin-II activity promoter calyculin-A has little effect on the cellular response, while the inhibitor blebbistatin suppresses cell and focal adhesion alignment and reduces focal adhesion density. These results confirm that similar internal structures involved in sensing and responding to strain direction and amplitude are also key players in strain gradient mechanosensing and avoidance.

On-chip microvalves regulate electrical and fluidic access to an array of nanopores integrated within microfluidic networks. This configuration allows for on-chip sequestration of biomolecular samples in various flow channels and analysis by independent nanopores.

Cyclically stretched cells are known to exhibit a strain-induced reorientation response. In this study, we comprehensively analyse this behaviour for human fibroblasts subjected to a highly non-uniform strain field within a polymethylsiloxane microdevice. We demonstrate a strong correlation between the strain amplitude and the degree of cell alignment perpendicular to the principal strain direction (stretching avoidance). Analogously, our results indicate that the strain gradient amplitude and direction also regulate this reorientation through a coordinated gradient avoidance response. We stipulate that strain gradients are thus biologically relevant mechanical cues sensed by cells. To gain insight into the underlying mechanosensing processes, we also studied focal adhesion reorganization and the effect of modulating myosin-II contractility. The extracted focal adhesion orientation distributions are similar to those obtained for the cell bodies, and their density is increased by the presence of stretching forces. Moreover, it was found that the myosin-II activity promoter calyculin-A has little effect on the cellular response, while the inhibitor blebbistatin suppresses cell and focal adhesion alignment and reduces focal adhesion density.These results confirm that similar internal structures involved in sensing and responding to strain direction and amplitude are also key players in strain gradient mechanosensing and avoidance.

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Carcinoma, the most common type of cancer, develops in the sheets of cells forming the epithelium and lining our organs and cavities. It usually begins with the transformation of a single cell via the activation of oncogenes such as Ras. The capacity of epithelia to eliminate newly transformed cells via apical extrusion is believed to be a critical defense mechanism to eradicate initial stages of carcinoma. Our organs and tissues are in constant motion, exposing epithelial cells to mechanical stretch. How these external forces impact the onset and progression of tumor growth is thus of primary interest, but little is known currently. Here we show that mechanical strains jeopardize the epithelial defense mechanisms against Ras V12 -transformed MDCK cells by impeding their apical extrusion. Concurrently, they prevent the formation of strong circumferential belts of actin in Ras V12 cells, previously established as a primary step of apical extrusion under static conditions. Cyclic stretching also changes the metastatic phenotype of newly transformed cells by greatly promoting the formation of Ras V12 protrusions. We show that Ras V12 and wild type MDCK cells possess distinct sensitivity to strain. External forces remodel their actin cytoskeletons and adhesion complexes differently, resulting in a more invasive system dynamic. Our work also shows that the Rho-ROCK mechanotransduction pathway is involved in regulating the mechanically-induced switch to a more aggressive phenotype. Such insight may lead to the targeting of mechanotransduction pathways in innovative future therapies. Significance statement:Cancer progression is increasingly viewed as a complex journey in which the mechanical properties of the microenvironment play a key role. The entire human body is in constant motion, yet the initial stage of cancer development at the cellular level is commonly studied in static petri dishes. Here we demonstrate that in a mechanically dynamic microenvironment, oncogenic Ras-transformed cells exhibit drastically different cellular dynamics and movements when compared to static conditions. They grow larger invasive protrusions, and they are much less likely to be eliminated from the healthy tissues. A deeper understanding of how external physical cues regulate early stage microtumor growth can reveal potentially new and unexplored avenues for cancer therapies.