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. 2003 Feb 18;100(4):1484-9.
doi: 10.1073/pnas.0235407100. Epub 2003 Jan 27.

Cells lying on a bed of microneedles: an approach to isolate mechanical force

John L Tan  1 , Joe Tien, Dana M Pirone, Darren S Gray, Kiran Bhadriraju, Christopher S Chen
Affiliations

Cells lying on a bed of microneedles: an approach to isolate mechanical force

John L Tan et al. Proc Natl Acad Sci U S A. .

Abstract

We describe an approach to manipulate and measure mechanical interactions between cells and their underlying substrates by using microfabricated arrays of elastomeric, microneedle-like posts. By controlling the geometry of the posts, we varied the compliance of the substrate while holding other surface properties constant. Cells attached to, spread across, and deflected multiple posts. The deflections of the posts occurred independently of neighboring posts and, therefore, directly reported the subcellular distribution of traction forces. We report two classes of force-supporting adhesions that exhibit distinct force-size relationships. Force increased with size of adhesions for adhesions larger than 1 microm(2), whereas no such correlation existed for smaller adhesions. By controlling cell adhesion on these micromechanical sensors, we showed that cell morphology regulates the magnitude of traction force generated by cells. Cells that were prevented from spreading and flattening against the substrate did not contract in response to stimulation by serum or lysophosphatidic acid, whereas spread cells did. Contractility in the unspread cells was rescued by expression of constitutively active RhoA. Together, these findings demonstrate a coordination of biochemical and mechanical signals to regulate cell adhesion and mechanics, and they introduce the use of arrays of mechanically isolated sensors to manipulate and measure the mechanical interactions of cells.

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Figures

Figure 1
Figure 1
Fabrication of arrays of posts. (A) With the appropriate surface density of vertical posts positioned on a substrate, a cell should spread across multiple posts as depicted. Under the proper geometric constraints of post height and width, cells exerting traction forces would deflect the elastomeric posts. (B) Schematic drawing of the method used to fabricate posts. (C–G) Scanning electron micrographs of fabricated arrays (C, D, and F) and schematic drawings indicating the compliance of posts (E and G). (C) A uniform array of posts. (D and E) An array of posts whose tips all lie in one plane, but the bases of certain posts are raised with respect to surrounding posts to generate spatially controlled step-increases in substrate stiffness. (F and G) An array of posts with oval cross sections to introduce anisotropic stiffness. Lengths of arrows in E and G indicate the relative magnitude of the deflection with the application of a constant force in the direction of the arrow. (Scale bars indicate 10 μm.)
Figure 2
Figure 2
Cell culture on arrays of posts. (A) Scanning electron micrograph of a representative smooth muscle cell attached to an array of posts that was uniformly coated with fibronectin. Cells attached at multiple points along the posts as well as the base of the substrates. (B) Schematic of microcontact printing of protein (red), precoated on a PDMS stamp, onto the tips of the posts (gray). (C) Differential interference contrast (Upper) and immunofluorescence (Lower) micrographs of the same region of posts where a 2 × 2 array of posts has been printed with fibronectin. (D and E) Scanning electron micrograph (D) and phase-contrast micrograph (E) of representative smooth muscle cells attached to posts where only the tips of the posts have been printed with fibronectin by using a flat PDMS stamp. Cells deflected posts maximally during the 1- to 2-h period after plating, were fully spread after 2 h, and were fixed and critical point dried 4 h after plating. (Scale bars indicate 10 μm.)
Figure 3
Figure 3
Measurement of contractile forces in cells. (A–D) Differential interference contrast micrographs of a smooth muscle cell (outlined in blue) cultured for 2 h on an array of posts in 10% serum (A and B), 20 min after 20 mM 2,3-butanedione monoxime (BDM) was added to the culture to inhibit myosin contractility (C), and after 2 μg/ml cytochalasin D (cyto D) was added to the same culture for an additional 10 min to disrupt the actin cytoskeleton (D). In each case, longer treatments did not result in additional loss of contractility. (E–G) Confocal images of immunofluorescence staining of a smooth muscle cell on posts. Position of fibronectin (E, red) on the tips of the posts was used to calculate force exerted by cells (white arrows). The force map was spatially correlated to immunofluorescence localization of the focal adhesion protein vinculin (F, white; G, green). A similar correlation in the orientation and the quantity of focal adhesion with the traction forces was observed in all cells examined (n > 10). The lengths of arrows indicate the magnitude of the calculated force (top right arrow indicates 50 nN); white circles on undeflected posts depict the background error in the force measurement, where the diameter of the circle (same length scale as the arrows) indicates the magnitude of calculated force on each post not attached to a cell. (Scale bars indicate 10 μm.) (H) Plot of the force generated on each post as a function of total area of focal adhesion staining per post. Each point represents the force and area of vinculin staining associated with each post; focal adhesions from five cells were analyzed. The shaded region (blue) indicates the adhesions smaller than 1 μm2. (Inset) Image of a typical small adhesion (<1 μm2) formed by a cell (green) generating substantial force on a post (red).
Figure 4
Figure 4
Role of cell spreading in regulating contractile forces. (A–F) Confocal micrographs of smooth muscle cells attached onto different-sized sets of fibronectin-coated posts. Cells attached to 2 × 2 (A and B), 3 × 3 (C and D), or 4 × 4 (E and F) sets of posts were costained for fibronectin (A, C, and E) and filamentous actin (B, D, and F). (Scale bars indicate 10 μm.) (G) Plot of average force generated per post for cells spread to different degrees (140, 440, 900, or 1,520 μm2) on arrays of posts (2 × 2, 3 × 3, 4 × 4, or 5 × 5, respectively) for 20 h in standard culture media. (H) Plot of average force generated per post over time for a cell cultured on either a 5 × 5 (“spread”) or 3 × 3 (“unspread”) set of posts. Cells were serum-starved for 12 h on the posts and were exposed to LPA (10 μg/ml) at time 0. (I) Plot of force exerted on each of the 25 posts over time for the spread cell plotted in H and shown as the Inset. The forces exerted on the posts colored in the Inset are indicated by line plots of the same color. (J) Plot of average force exerted per post for cells cultured on 5 × 5 (spread) or 3 × 3 (unspread) posts and were serum-starved, exposed to LPA for 12 min, or transfected with constitutively active RhoA. A total of 18 cells in G and 25 cells in J were analyzed across three independent experiments; error bars indicate standard error of the mean.
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