10 research outputs found

    Combining dynamic stretch and tunable stiffness to probe cell mechanobiology in vitro.

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    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 1 Hz 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

    Schematic of polyacrylamide gel on flexible silicone membrane under static (A) and stretched (B) conditions.

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    <p>Top view of a 22 mm diameter collagen-coated gel (∼70 µm thickness) is cast into a 35 mm diameter flexible-bottomed Flexcell™ well (C) and STREX well (C, insert). Image of Flexcell™ well (D) stretched above an Arctangle™ loading post and labeled with retroreflective beads for strain field analysis. Rectangle shows region analyzed in HDM software, arrows point to edge of gel. Scale bars = 10 mm in all panels.</p

    Average strain (± SD) within central region used for analysis of cell morphology for equibiaxial stretch (round loading post) and uniaxial stretch (Arctangle™ loading post).

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    <p>Average strain (± SD) within central region used for analysis of cell morphology for equibiaxial stretch (round loading post) and uniaxial stretch (Arctangle™ loading post).</p

    hMSC response to stretch is unclear due to spreading on static soft gels.

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    <p>Micrographs of hMSCs cultured statically (left column) and following ∼10% cyclic equibiaxial strain (right column) for 6 hours on soft gels (0.3 kPa, top row) and stiff gels (50 kPa, bottom row). Staining for f-actin (green) and nuclei (blue) shows that hMSCs on soft gels (static and stretched) have unorganized actin fibers whereas cells on stiff gels have more organized actin fibers. Unlike VICs, hMSCs spread well on soft gels and stretch appears to increase the spread area of the cells slightly on stiff gels. Scale bar = 100 µm.</p

    VICs on soft (0.3 kPa) and stiff (50 kPa) gels cultured under static and pure uniaxial stretch conditions (1 Hz, 10% stretch, 6 hrs).

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    <p>Cells cultured on soft substrates appear to have less realignment with stretch compared to the classic realignment perpendicular to the direction of stretch on the stiff substrates. Scale bar = 100 µm.</p

    When cyclically stretched, cells on stiff substrates reduce spread area whereas cells on soft substrates increase spread area: Area (A) and perimeter (B) of VICs cultured on low (0.3 kPa) and high (50 kPa) stiffness gels subjected to 10% cyclic stretch at 1 Hz for 6 hours (grey bars) or static (black bars) culture.

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    <p>Shape factor (C) quantifies how rounded a cell is (a shape factor of 1 is perfectly circular, whereas a shape factor of 0 is extremely spread with many extensions). Cells of low and high shape factor are shown in C. Brackets above bars show significance between individual groups (two-way ANOVA, p<0.05).</p

    Strain field in region of interest is roughly uniform for pure uniaxial stretch.

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    <p>Strain maps for a soft gel (0.3 kPa) undergoing pure uniaxial strain in the X (A), Y (B), and XY (shear, C) directions demonstrating relatively homogenous strain and minimal shear within the area of analysis of cell morphology (dashed box). (D) CAD representation of the Arctangle™ loading platen over which the silicone membrane is stretched by vacuum pressure. Scale bars = 5 mm.</p

    Strain field in region of interest is roughly uniform for equibiaxial stretch.

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    <p>Strain maps for a soft gel (0.3 kPa) undergoing equibiaxial strain in the X (A), Y (B), and XY (shear, C) directions demonstrating relatively homogenous strain and minimal shear within the area of analysis of cell morphology (dashed box). (D) CAD representation of the circular loading platen over which the silicone membrane is stretched by vacuum pressure. Scale bars = 5 mm.</p
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