Fig 1.
A side view of the biaxial stretching device (A). The device consists of a linear actuator that controls the vertical motion of a traveling stage, which has a custom well (red) with a silicone membrane (light blue) on which cells are seeded. Biaxial stretch is delivered to the cells by stretching the membrane over a hollow indenter (blue), which has ball bearings to reduce friction (B). Cells can be imaged either from above, by placing a water immersion objective inside the indenter, or from below using an inverted microscope. (C) An actual image of the system under an upright microscope.
Fig 2.
(A) Nonlinear relationship between the indenter depth and the corresponding change in surface area of a demarcated region on the membrane; this calibration curve was subsequently used to prescribe strain waveforms as a function of indenter depth. Data points represent the average of n = 3 calibrations with different membranes; standard deviation bars (not shown) are smaller than symbols. (B) Representative sinusoidal waveform applied to membrane (amplitude, 20% strain; frequency, 0.0464 Hz), the input waveform (solid line) and the observed change in surface area (open circles) are shown demonstrating close agreement between input and measurement. (C) Frequency response of the device is flat for stretch frequencies between 0.01–1.0 Hz; sinusoidal waveforms with prescribed strain amplitudes of 20% (filled triangles) and 40% (open squares) are shown with reference lines, error bars represent the standard deviations (N = 3). (D) Left: characteristic image of marker beads used to track micro strain; Right: detection algorithm showing beads at baseline (green) and after applied strain (magenta), specific beads are tracked and the areas enclosed by their polygons are used to determine the corresponding micro strain. (E) Change in micro strain closely follows the prescribed macrostrain, line of identity is shown for reference; error bars represent standard deviations (n = 9).
Fig 3.
Change in surface area of sixteen triangles on the membrane shows the regional area strains at indenter depths of 0, 4, 6, 9 and 13 mm, with ΔSA ranging from 0% (blue) to 45% (red).
The average and standard deviation of ΔSA for all triangles is shown at each depth. The regional deformation is consistent for all triangles with a maximum standard deviation of 2.3% at the highest observed indenter depth.
Fig 4.
An example of a cell tracked and imaged in increments of 7% area strain (A). The outline of the cell was manually selected and the cell shape was fitted with an ellipse whose major and minor axes were measured (B). The area strain of cells was measured (n = 8) and compared to the prescribed membrane area strain. The measured cell area strain is lower than the prescribed strain, but is within one standard deviation at each measured increment. The major and minor axes of the cell increase equally, demonstrating biaxial strain.
Fig 5.
(A) A cell labeled for cytosol (green), mitochondria (red) and nucleus (blue) at 0% (left) and 14% (right) membrane area strains. (B)The top row shows the mitochondrial network of an entire cell imaged during constant strain application at increments of 0, 10, 20 and 40% change in membrane surface area. The bottom row shows a detail of an individual cluster (the green rectangle in top row) changing shape as higher strains are applied (green arrow), as well as a cluster undergoing fission and splitting into two smaller clusters (red arrow).
Fig 6.
Intracellular calcium level responses of primary bovine fibroblasts to single sinusoidal equi-biaxial strains.
Cells were loaded with Fluo4 calcium indicator dye and subjected to strains of 3 or 10% change in membrane surface area (ΔSA). (A) Following single 6 s long stretch (dotted line), cells stretched to 10% ΔSA showed large transient increases in fluorescence relative to baseline (mean of 77 cells), while cells stretched to 3% ΔSA showed little response (mean of 44 cells). Images of cells stretched to 10% ΔSA revealed differences between neighboring cells in the timing of their response to stretch, with some cells responding immediately and other requiring up to 10 s to respond. (B) The changes in fluorescence were significantly greater with 10% ΔSA than with 3% ΔSA (p<0.001).
Fig 7.
Response of intracellular calcium levels in bovine fibroblasts up to one hour of continuous sinusoidal stretching with maximum amplitude of 10% change in membrane surface area.
Average ratios of Fura2 fluorescence at 340 and 380 nm excitation wavelengths were computed in each of 11 cells. Individual cell responses varied (A), but there was an overall trend of increasing Fura2 ratios over time, with significant increases present at 20 and 60 min relative to baseline (B). **, p<0.01; ***, p<0.001