Probing cellular traction forces with magnetic nanowires and microfabricated force sensor arrays

Abstract

In this paper, the use of magnetic nanowires for the study of cellular response to force is demonstrated. High-aspect ratio Ni rods with diameter 300 nm and lengths up to 20 μm were bound to or internalized by pulmonary artery smooth muscle cells (SMCs) cultured on arrays of flexible micropost force sensors. Forces and torques were applied to the cells by driving the nanowires with AC magnetic fields in the frequency range 0.1-10 Hz, and the changes in cellular contractile forces were recorded with the microposts. These local stimulations yield global force reinforcement of the cells' traction forces, but this contractile reinforcement can be effectively suppressed upon addition of a calcium channel blocker, ruthenium red, suggesting the role of calcium channels in the mechanical response. The responsiveness of the SMCs to actuation depends on the frequency of the applied stimulation. These results show that the combination of magnetic nanoparticles and micropatterned, flexible substrates can provide new approaches to the study of cellular mechanotransduction.

Figure 1

(A) Phase contrast image of a smooth muscle cell (SMC) cultured on a PDMS micro-post array, prior to magnetic actuation. Magnetic nanowires with an average length of 20 μm are adhered on top of the cell. Scale bar = 8 μm. (B) Fluoresence image corresponding to (A), with a vector map of the forces exerted by the cell on each post superimposed. (C) Average strain energy per post for several cells over the time course of the experiment. Some cells show a sudden increase in energy upon magnetic actuation (solid symbols), while other cells show no responsiveness to this actuation (open squares, circles, and triangles). The apparent energy of the posts not in contact with the cell is nearly zero (open stars in upper panel). (D) Vector force map of the cell shown in Panels (A) and (B) after 15 min. of magnetic stimulation, showing a increase in cellular contractility.

Figure 2

Spatial maps of the change in strain energy δE for each post under a cell. Each hexagon represents a post, and the boundary of the cell is shown by the thick white line. (A) The energy shows little change prior to stimulation. (B) By contrast, some posts show a sudden energy increase 1 min. after application of magnetic stimulation; these posts are not localized near the nanowires.

Figure 3

The increase in strain energy δE_i at individual posts upon magnetic stimulation positively correlates to the baseline strain energy before the actuation (red symbols); this applies to both posts near the nanowires (solid symbols) and posts not near the nanowires (open symbols). The strain energy fluctuations δE_i,base before the actuation are small (black symbols).

Figure 4

(A) Some cells show the average energy per post increase in a gradual manner over the time course of experiment upon actuation with internalized magnetic nanowires (solid symbols), and other cells show no responsiveness to this actuation (open squares, circles, and triangles). The apparent energy of the posts having no cells on the top is nearly zero (open stars in upper panel). (B) Comparison of cellular response to externally bound nanowire stimulation (open symbols) and internally bound nanowire stimulation (solid symbols) by scaling the baseline average energy to zero. For the external stimulation, contractile energy increases in the first 2 minutes then reaches a static state, while the energy of internally stimulated cells gradually increases during the whole experimental time course. (C) The change in energy $\delta E_i^{\prime }$ over the 15 min. time course of the internal stimulation is not localized near the magnetic nanowire. As in Fig. 2, each hexagon represents a post, and the cell boundary is a thick, white line. (D) As for external stimulation, the increase in strain energy $\delta E_i^{\prime }$ at individual posts upon internal magnetic stimulation correlates to the baseline strain energy before the actuation (red symbols). The energy fluctuations δE_i,base before the actuation are small (black symbols).

Figure 5

SMC response to magnetic stimulation after treatment with the calcium inhibiter ruthenium red (RuR). (A) Cells treated with RuR did not respond to the externally bound nanowire actuation (e.g. red open symbols). The response of an untreated cell to external stimulation is shown (gray open symbols) for comparison. In contrast, some RuR-treated cells still show modest response to stimulation by internalized nanowires (red solid symbols), but this response is smaller than for untreated cells (grey solid symbols). (B) Summary of response rates for internal and external stimulation, with and without RuR treatment.

Figure 6

The cellular contractile responsiveness depends on the frequency of the internally applied stimulation: a greater percentage of the cells show enhanced reinforcement at frequencies of 0.5 Hz and 1 Hz.