Live-cell subcellular measurement of cell stiffness using a microengineered stretchable micropost array membrane

Abstract

Forces are increasingly recognized as major regulators of cell structure and function, and the mechanical properties of cells, such as cell stiffness, are essential to the mechanisms by which cells sense forces, transmit them to the cell interior or to other cells, and transduce them into chemical signals that impact a spectrum of cellular responses. Here we reported a new whole-cell cell stiffness measurement technique with a subcellular spatial resolution. This technique was based on a novel cell stretching device that allowed for quantitative control and real-time measurements of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. Using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached on the tops of the microposts. The micropost top positions before and after mPAM stretches were recorded using fluorescence microscopy and further utilized to quantify local cell stretching forces and cell area increments. A robust computation scheme was developed and implemented for subcellular quantifications of cell stiffness using the data of local cell stretching forces and cell area increments generated from mPAM cell stretch assays. Our cell stiffness studies using the mPAM revealed strong positive correlations among cell stiffness, cellular traction force, and cell spread area, and illustrated the important functional roles of actin polymerization and myosin II-mediated cytoskeleton contractility in regulating cell stiffness. Collectively, our work reported a new approach for whole-cell stiffness measurements with a subcellular spatial resolution, which would help likely explain the complex biomechanical functions and force-sensing mechanisms of cells and design better materials for cell and tissue engineering and other applications in vivo.

Figure 1

Live-cell subcellular measurement of cell stiffness using a microengineered stretchable micropost array membrane (mPAM) and a cell stretching device (CSD). (a) Schematic of the CSD and its implementation for stretching the mPAM and thus single cells adhered on the tops of the PDMS microposts. Only one-half of the CSD is shown for visualization of its internal structure. A vacuum was applied to the CSD evacuation chamber to draw the periphery of the mPAM, causing the central region of the mPAM holding the PDMS micropost array and pre-seeded with adherent cells to stretch equibiaxially. Inset: Scanning electron microscopy image of a single cell seeded on the PDMS micropost array. Scale bar: 20 μm. (b) Cartoon of an adherent cell on the mPAM under a static equibiaxial stretch. Left inset shows a triangular subcellular region formed between three adjacent microposts deflected by cellular traction forces. Right inset shows the same triangular region under a static equibiaxial stretch with an increased cell area and heightened traction forces. Mechanical forces exerted on the cell by the PDMS microposts were labelled by blue and light blue arrows before and after the mPAM stretch, respectively. (c) Grid arrangement for theoretical computation of subcellular cell stiffness. Hexagonally arranged microposts formed the nodes in a regular triangular grid surface. Inset: Relative displacements of the micropost tops before and after static equibiaxial mPAM stretches used to calculate stretching forces and deformations of grid elements.

Figure 2

(a) Equibiaxial stretch of the mPAM as a function of the level of vacuum pressure applied to the CSD evacuation chamber. Error bars represent ± standard deviations of data obtained from three independent measurements. Data trend in a was plotted using linear least square fitting (dark line), with the square of the correlation coefficient R² indicated. (b) Instantaneous changes of cell spread area (top) and cellular traction force (bottom) of single live vascular smooth muscle cells (VSMCs) in response to 10% static equibiaxial cell stretches. Datawere recorded within 30 sec after the onset of mPAM stretches. Data were normalized to the average values of cellular traction force and cell spread area prior to mPAM stretches. Error bars represent ± standard errors of data collected from 10 individual VSMCs (n = 10). (c) Increment rate of normalized cellular traction force (min⁻¹) after different periods of 10% static equibiaxial mPAM stretches. Asterisk indicates a statistically significant change (p < 0.05).

Figure 3

Subcellular measurement of cell stiffness for single fixed vascular smooth muscle cells (VSMCs). (a) Representative single fixed and stained VSMCs adhered on the PDMS micropost array before (left) and after (right) a 9% static equibiaxial mPAM stretch. The cell was stained for nucleus (blue) and actin microfilaments (green), and the PDMS microposts were labelled with DiI (red). Scale bar, 20 μm. (b) Spatial maps of subcellular traction forces before (left) and 30 sec after (right) a 9% static equibiaxial mPAM stretch. (c) Spatial maps of stretching force (top), cell area increment (lower left), and computed subcellular cell stiffness (lower right).

Figure 4

Live-cell subcellular measurement of cell stiffness for vascular smooth muscle cells (VSMCs) and its correlation with cellular traction force and cell spread area. (a) Cell stiffness measurement for a representative live single VSMC. Spatial maps were shown for stretching force (top), cell area increment (middle), and computed subcellular cell stiffness (bottom). (b) Whole-cell elastic modulus plotted against cellular traction force (top) or initial cell spread area before mPAM stretches (middle). The bottom figure plotted cellular traction force against cell spread area before mPAM stretches.

Figure 5

Effects of drug treatments to inhibit myosin II and ROCK activities and actin polymerization on cell stiffness of live single vascular smooth muscle cells (VSMCs). (a) Representative immunofluorescence images showing untreated (control) and drug-treated single VSMCs plated on the PDMS micropost array. For drug-treated VSMCs, blebbistatin, Y27632, and cytochalastin D were applied to block myosin II and ROCK activities and actin polymerization, respectively. Cells were stained for nucleus (blue), myosin IIA (white), and actin microfilaments (green). The PDMS microposts were labelled with DiI (red). Scale bar, 20 μm. (b) Temporal evolutions of cellular traction force (normalized to the initial value prior to drug treatments) for individual VSMCs (thin dashed lines) and population means (heavy lines with symbols) under different drug treatment conditions as indicated. Error bars represent ± standard errors of data collected from 3-5 individual VSMCs. (c) Whole-cell elastic modulus of VSMCs that were either untreated (control) or treated with different drugs for 1 hr as indicated. Each p-value calculated using the Student’s t-test was included to indicate statistical significance. (d) Correlations between whole-cell elastic modulus, cellular traction force, and cell spread area for untreated (control) and drug-treated VSMCs as indicated. Data trends in d were plotted using linear least square fitting, with the square of the correlation coefficient R² indicated.