A standardized objective method for continuously measuring the kinematics of cultures covering a mechanically damaged site

The kinematics of cell migration is frequently being studied in the context of wound healing. Scratch wound assays in vitro are particularly popular, being a cost-effective method for characterizing the kinematic parameters of cultures. However, currently there are no objective and standardized measures of these kinematic parameters. We addressed these issues by developing an automatic and quantitative method for determining time-dependent damage areas in “wound healing” monolayer culture experiments by means of image processing. “Wound” area over time data are then fitted to a Richards function (non-symmetric sigmoid) from which we determine the migration rate, time for onset of mass cell migration and time for end of mass cell migration. We demonstrate the utility of our present method by conducting “wound healing” experiments in 8 cultures of NIH3T3 fibroblast cells which were monitored by time-lapse microscopy. The measures derived from Richards function fits to the area–time curves (normalized root mean squared errors ≤3.8%) are calculated based on the entire time course of the data. Accordingly this method is substantially more reliable than the common practice where migration rates are determined based on two time points (start and end stages of migration).