Triggering cell detachment from patterned electrode arrays by programmed subcellular release

Abstract

Programmed subcellular release is an in vitro technique for the quantitative study of cell detachment. The dynamics of cell contraction are measured by releasing cells from surfaces to which they are attached with spatial and temporal control. Release of subcellular regions of cells is achieved by plating cells on an electrode array created by standard microfabrication methods. The electrodes are then biochemically functionalized with an arginine-glycine-aspartic acid (RGD)-terminated thiol. Application of a voltage pulse results in electrochemical desorption of the RGD-terminated thiols, triggering cell detachment. This method allows for the study of the full cascade of events from detachment to subsequent subcellular reorganization. Fabrication of the electrode arrays may take 1-2 d. Preparation for experiments, including surface functionalization and cell plating, can be completed in 10 h. A series of cell release experiments on one device may last several hours.

Figure 1

Schematic illustration of the concept of programmed subcellular release. A cross-section of a cell on an electrode array functionalized with RGD-thiol molecules. The integrins of the cell bind to the RGD, promoting cellular attachments on the gold electrodes. Application of a sufficiently negative voltage pulse causes electrochemical desorption of the thiol molecules, which results in the cleavage of the gold-thiol bond.

Figure 2

Experimental setup for programmed subcellular release. (a) Schematic illustration of a subcellular release device comprising the electrode array and Teflon well. (b) Microscope, live cell chamber, potentiostat and computer. (c) Phase microscopy images of NIH 3T3 fibroblast cells on an electrode array. Bar, 50 μ m. (d) Higher magnification phase microscopy images of NIH 3T3 fibroblast cells. The white arrows show ideal electrodes for release in which cells terminate on the electrode. Bar, 10 μm.

Figure 3

Cyclic voltammograms of electrodes. RGD-terminated thiol on a single electrode (PBS, 7.4, 100 mV s⁻¹) (red). U_i indicates the potential at which the thiol desorption begins and U_f indicates the end of the thiol desorption process. The thiol monolayer blocks current flow from 0 V to approximately − 0.7 V. A characteristic desorption peak (or shoulder) is seen at about − 1.2 V. A cyclic voltammogram for a bare gold electrode is shown for comparison (blue).

Figure 4

Programmed subcellular release. (a) Typical release experiment of an NIH 3T3 fibroblast cell monitored under phase-contrast microscopy. A voltage was applied to an electrode (red arrow) at t = 0 s. After release of the cell from the uppermost gold line, there was an induction time before the cell began to contract. Magnification, ×60. Bar, 5 μm. (b) Plot of normalized cell contraction verse time for the cell shown in a. The induction time (t₀) and contraction time (τ) are indicated by arrows. The solid line is a fit to the equation ΔL(t)/ΔL_m = 1 − exp(− (t −t₀)/τ), with t₀ = 37.1 s and τ = 34.2 s. (c) Plot of the normalized cell contraction curves for 22 cells.

Figure 5

Immunofluorescence staining of a cell during cell release. (a) Phase-contrast image of an NIH 3T3 fibroblast cell 175 s after release from electrode labeled with red arrow. (b) Fluorescence microscopy image of the cell in panel a stained with FITC–anti-vinculin. (c) Fluorescence microscopy image of the cell in panel a stained with phalloidin. (d) Overlaid fluorescence images of a fixed NIH 3T3 fibroblast spanning multiple electrodes (dark diagonal lines) stained for actin fibers (Alexa Fluor 568 phalloidin, red), vinculin (FITC–anti-vinculin, green) and cell nucleus (DAPI, blue).

Figure 6

Effect of blebbistatin concentration on cell contraction. Normalized cell contraction curves and mean induction and contraction times with 0 μM (n = 18), 2.5 μM (n = 4), 10 μM (n = 3) and 50 μM (n = 3) blebbistatin.

Figure 7

Live cell imaging of Lifeact-GFP actin during release. (a) Overlay of phase-contrast and fluorescent Lifeact-GFP images of an NIH 3T3 fibroblast spanning two electrodes. (b) Phase-contrast and (c) fluorescent images during release, of the cell in panel a as indicated by the white box.