Assessment of cell migration by ECIS-based wound-healing assay

Electric cell-substrate impedance sensing technology was used to monitor the migration of CMVECs in a wound-healing assay as reported (Applied BioPhysics Inc., Troy, NY). Briefly, CMVECs (2.5 × 10⁵ cells/well) were seeded in 96-well array culture dishes (electric cell-substrate impedance sensing (ECIS), 96W1E) and placed in an incubator (37 °C), and changes in resistance and impedance were continuously monitored. When impedance reached a plateau, cells in each well were subjected to an elevated field pulse (“wounding”) of 5 mA applied for 20 s at 100 kHz, which killed the cells present on the small active electrode due to severe electroporation. The detachment of the dead cells was immediately evident as a sudden drop in resistance (monitored at 4000 Hz) and a parallel increase in conductance. VEGF (100 ng/mL) was immediately added to each well. CMVECs surrounding the active electrode that had not been subjected to the wounding then migrated inward to replace the detached dead cells resulting in resistance recovery (continuously monitored at 4000 Hz for up to 24 h). The time to reach 50% resistance recovery (corresponding to 50% confluence on the active electrode) was determined for cells in each experimental group, and this parameter and the known physical dimensions of the electrode were used to calculate the migration rate (expressed as μm/h).