Magnetic microposts as an approach to apply forces to living cells

Abstract

Cells respond to mechanical forces whether applied externally or generated internally via the cytoskeleton. To study the cellular response to forces separately, we applied external forces to cells via microfabricated magnetic posts containing cobalt nanowires interspersed among an array of elastomeric posts, which acted as independent sensors to cellular traction forces. A magnetic field induced torque in the nanowires, which deflected the magnetic posts and imparted force to individual adhesions of cells attached to the array. Using this system, we examined the cellular reaction to applied forces and found that applying a step force led to an increase in local focal adhesion size at the site of application but not at nearby nonmagnetic posts. Focal adhesion recruitment was enhanced further when cells were subjected to multiple force actuations within the same time interval. Recording the traction forces in response to such force stimulation revealed two responses: a sudden loss in contractility that occurred within the first minute of stimulation or a gradual decay in contractility over several minutes. For both types of responses, the subcellular distribution of loss in traction forces was not confined to locations near the actuated micropost, nor uniformly across the whole cell, but instead occurred at discrete locations along the cell periphery. Together, these data reveal an important dynamic biological relationship between external and internal forces and demonstrate the utility of this microfabricated system to explore this interaction.

Fig. 1.

Microfabricated arrays of magnetic and nonmagnetic posts for applying external forces and measuring traction force response. (A) External force F_Mag is applied to the adherent cell through magnetic posts embedded with Co nanowires that bend under the influence of a magnetic field, ⇑B (not drawn to scale). Nonmagnetic posts report local traction forces through post deflections δ. (B) SEM micrograph of a Co nanowire. (C) Magnetic moment components per wire μ_⊻ and μ_⫠ for 15-μm-long Co nanowires versus applied magnetic field μ₀H as measured by vibrating sample magnetometer for ⇑H oriented at θ = 85° to the nanowires (Inset). ⇑μ scales simply with nanowire length (32), so these results are representative of the wires used in the magnetic posts. (D) Process flow diagram for embedding nanowires into the micropost. (E) SEM backscattering micrograph showing a nanowire in the micropost array. (F) Energy-dispersive x-ray microanalysis measurements for bright material (red curve; Inset, red cross-hairs) within the magnetic posts observed under backscattering SEM (Inset). Areas nearby do not contain Co (blue curve; Inset, blue cross-hairs). The red curve is offset by 25 counts per sec to clarify between curves.

Fig. 2.

Characterization of magnetic post actuation. (A) The magnetic torque, τ, on a magnetic post of length L depends on the applied field, B⃑, the nanowire length, L_W, and dipole moment, μ⃑. (B and C) Phase-contrast micrographs of a magnetic post deflected under no field and a 0.31-T field. (D) Plots of post displacement versus applied field with arrows indicating direction of driving magnetic field. Actuations caused negligible mechanical displacement in adjacent nonmagnetic post (blue curve).

Fig. 3.

FA protein recruits to site of external force application. (A) Representative immunofluorescent micrograph of FAs (green), microposts (red), and nucleus (blue) after force actuation. The direction and magnitude of the field are shown. The cell is outlined, and the location of the magnetic post is marked by the asterisk (*). (B) Vector plot of traction forces at each post are shown with white arrows. The cell is outlined, and the location of the magnetic post is marked by the asterisk (*). (C) Plot of average FA area for all posts underneath cells (white bars) and average FA area at magnetic posts (blue bars) when cells are subjected to no actuation and single actuation (†, P = 0.0875). (D) Plot of average FA area for cells subjected to no actuation and multiple actuations (∗∗, P = 0.0041). (Error bars on all graphs denote standard error of the mean.)

Fig. 4.

Changes in traction forces in cells after force application. (A) Immunofluorescent micrograph of cell A, force-stimulated with a magnetic post, and cell B, unstimulated control. Labeling for actin (cyan), nuclei (blue), and PDMS (brown) were performed immediately after traction force video observation. Posts of interest are marked with colored circles and labeled according to which cell they were attached to (A_i or B_i) or background (Bkg). (B) Plot of displacement and force versus time for all posts for cell A (light red). A subset of posts of interest are designated (A_I, red; B_I, green; magnetic post A₂, yellow; and background post, blue). Onset of force stimulation is indicated by dashed line (t = 0). The force reported by the deflection also reflects cellular traction forces, except for A₂, which has an additional magnetic force component of 1.3 nN introduced at t = 0. (C) Plot of average strain energy per post (u) versus time for force-stimulated cells. Each cell is categorized by their response: sudden (red), gradual (purple), and no apparent (black) response. The cell marked with asterisk (*) has large strain energy and is shown with a scaling reduction of 3. (D) Plot of u versus time for unstimulated control cells (green) and background posts for all experiments (blue). (B–D) Error bars for all graphs indicate uncertainty in analysis. (E) Spatial plots of change in strain energy (Δu_i) in posts immediately before and after force application for stimulated cells that have sudden responses. Posts underneath each cell are shown in each plot (+). For each spatial plot, the direction of applied field (B = 0.2 T) is upward. For cell A, F_Mag = 1.3 nN, and for cell C, F_Mag = 3.3 nN. For posts with Δu_i outside the range of the scale, they are colored as the nearest extrema. (F) Spatial plots of Δu_i in posts immediately before and 10 min after for stimulated cells responding with gradual changes in energy. For cell D, F_{Mag, top} = 5.4 nN and F_{Mag, bottom} = 6.2 nN, and for cell E, F_Mag = 3.9 nN. (G) Spatial plots of control cells comparing Δu_i immediately before and after force application.