Combining Dynamic Stretch and Tunable Stiffness to Probe Cell Mechanobiology In Vitro

Abstract

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G′ = 0.3 kPa) to stiff (G′ = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.

A Nonhuman Primate Model of Lung Regeneration: Detergent-Mediated Decellularization and Initial In Vitro Recellularization with Mesenchymal Stem Cells

“…This surface has a substantially higher rigidity than natural lung matrix does; therefore, cells may have different attachment properties when compared to 3D matrix cultures. 36,37 To validate the data herein showing the relative positioning of rhesus BMSC and ASC within the acellular lung matrix, further studies will be necessary to elucidate the mechanisms by which MSC bind to and interact with the lung-derived scaffolds to reconstitute the native tissue. Ode and colleagues have provided an excellent profile of human MSC integrin expression and a morphological characterization of the functional behavior of MSC on various ECM proteins.…”

Magneto-Active Substrates for Local Mechanical Stimulation of Living Cells

“…The magneto-active substrates developed here complement the current cell stretching technologies used to establish that cells change contractility, spreading phenotype, fibre formation and proliferation differently for different mechanical stimulation patterns 8,[17][18][19] . Indeed, the present work demonstrates that magneto-active substrates made of soft magnetic micro-pillars embedded in a soft elastomer uniquely combine advantages that were not associated so far.…”

“…Although local enough to address the subcellular mechanisms of mechanotransduction, these methods involve intrinsic perturbations of the cell structure through mechanical interactions with a stiff object of fixed geometry. Cell stretchers were developed to induce mechanical stimulation via substrates of tunable substrate rigidity 8,17 . Despite being more physiological and less invasive, such approaches only enable global deformation at the cellular scale.…”

Prediction of matrix-to-cell stress transfer in heart valve tissues

“…Using triads of VIC nuclei as fiducials, local strains calculated at a uniaxially applied tissue-level strain of 30% only reached~10% and~20% under circumferential and radial stretches, respectively. Nevertheless, while studies have begun to address the influence of mechanical stretch on in vitro cultured VICs [24,26,[52][53][54][55] and explanted leaflet tissues [19,20], comparatively few studies have investigated the micromechanical interactions between the VICs and ECM in situ [13,56], in order to quantify regional stress distributions associated with the heterogeneous ECM or the stress-strain state of an individual VIC.…”