Cell-substrate separation: effect of applied force and temperature.

We measure the change in cell-substrate separation in response to an upward force by combining two relatively new techniques, Electric Cell-substrate Impedance Sensing (ECIS) to measure average cell-substrate separation, and collagen-coated magnetic beads to apply force to the top (dorsal) surface of cells. The collagen-coated ferric oxide beads attach to integrin receptors in the dorsal surfaces of osteoblastlike ROS 17/2.8 cells. Magnetic force is controlled by the position and the number of permanent magnets, applying an average 320 or 560 pN per cell. Comparing model calculations with experimental impedance data, the junctional resistivity of the cell layer and the average distance between the lower (ventral) cell surface and substrate can be determined. The ECIS analysis shows that these forces produce an increase in the distance between the ventral cell surface and the substrate that is in the range of 10 to 25%. At temperatures of 4 degrees, 22 degrees and 37 degrees C, the measured cell surface-substrate distances without magnetic beads are 84 +/- 4, 45 +/- 2 and 38 +/- 2 nm. The force-induced changes at 22 degrees are 11 +/- 3 and 21 +/- 3 nm for 320 and 560 pN, and at 37 degrees they are 5 +/- 2 and 9 +/- 2 nm. The resulting cell-substrate spring constants at 22 degrees and 37 degrees are thus about 28 and 63 pN nm-1 (dyne cm-1). Using a reasonable range for the number for individual integrin-ligand adhesion bonds gives a range for the spring constant of the individual adhesion bond of from about 10(-3) to 10(-1) pN nm-1. These data also provide evidence that the number of adhesion bonds per cell increases with temperature.