The overall goal of these epithelial cell culture artificial wounding procedures is to study cell migration from both a quantitative and qualitive perspective, allowing assessment of the effects of specific treatments of interest on wound healing. This method can help answer key questions in the wound-healing field, as they allow the precise characterization of the roles of specific migration processes during epithelial disruption. The main advantage of this approach is that it allows the correlation of cell migratory measurements with changes in the behavior of relevant molecular markers.
Implication of these techniques extend towards a therapy of clinical wounds, as it offers a convenient setup for early drug testing and for wound closure. Generally in to this method will start because of difficulties in obtaining consistent reproduceability in the wounds with an generation. For an artificial wound scratch assay, begin by seeding each well of a culture plate with two milliliters of epithelial cells of interest in complete medium.
Enculture the cells at 37 degrees Celsius and five percent carbon dioxide for two to three days until 100%confluency. When the cells are ready, wash the cells two times with fresh serum-free medium followed by 24 hours of culture in fresh serum-free medium. The next day, place the plate in a sterile work area and drag the narrow end of a sterile micro-pipette tip across the bottom of each well two times to generate a cross-shaped wound.
A consistent wound gap is achieved by applying constant, firm pressure while smoothly moving the tip along the epithelial surface. Excessive pressure will deform the tip, causing an irregular wound radius. Gently remove the supernatant to discard the detached cells and carefully add fresh serum-free medium to the wells.
Using in inverted phase contrast microscope connected to a charge coupled device camera, align the edge of the microscope field with the adjacent intersection of the scratches to obtain up to four pre-treatment reference images of the scratch gap in each well. When all of the images have been acquired, replace the medium in the designated treatment wells with fresh medium, supplemented with selected treatments of interest and return the plates to the cell culture incubator. When at least one condition reaches about 90%scratch gap closure, gently replace the supernatant with one milliliter of four percent formalin in PBS for a 15 minute incubation at room temperature.
Then gently wash the cells with PBS at least three times to remove any excess formalin and image the wells as just demonstrated, using the same imaging parameters in the original reference areas captured after scratching. To analyze the images using the appropriate image processing software, open the recorded, pre-treatment image of interest and under the Image menu, set the image type to 8-bit. Under the Process menu, select the Filters sub-menu and apply Variance filtering.
Under the Image menu, open the Adjust sub-menu and set the Threshold to black and white. In the Process menu, select the Binary sub-menu and apply Fill Holes. Under the Analyze menu, select Set Measurements and activate Area.
Then draw the measurable area, following the migrating edge contour to delimit the gap. Under the Analyze menu, select Analyze Particles and record the total area values for the drawn area surface. To quantify the absolute migration for each set of individual samples as the difference between the gap surface measurements, export the pre and post-treatment total gap surface area values to a spreadsheet and plot the quantification outputs.
For an artificial migration front assay, under sterile conditions, add one layer of up to 33 sterilized round coverslips into empty 10 centimeter culture plates. When the plate surface is completely covered, gently seed the epithelial cells of interest at a concentration that allows for 100%confluence after two or three days of culture. If necessary, gently depress the coversips with a sterile micropipette tip to prevent floating.
When the cells reach confluency, replace the supernatant with serum-free medium for another 24 hours of culture. Then, use sterile tweesers to carefully transfer one coverslip into a new 10 centimeter plate containing fresh serum-free medium, taking care to hold the coverslip along its periphery. To create the artificial wounds, drag a sterilized razor blade back and forth across each coverslip to make a three to four millimeter transverse line over the center of each cell culture to completely remove the central monolayer strip.
Take care to place the razor blade edge heavily across the coverslip surface when making the wound to safeguard against an irregular edge shape and the generation of flopping epithelial portions. Transfer up to four wound coverslips, cell side up, into individual wells of a six well plate, containing at least two milliliters of serum-free medium and gently wash each well two times with fresh serum-free medium to remove any detached cells. When all of the detached cells have been discarded, gently replace the wash medium with fresh medium supplemented with the treatments of interest, and place the plate in the cell culture incubator.
At the end of the appropriate experimental period, fix the cells with four percent formalin in PBS as just demonstrated, and stain the cells with the appropriate fluorescent conjugated antibodies of interest. Then image the structural changes occurring at the migrating front edge by fluorescence microscopy according to the experimental parameters of interest. In this experiment, the wound gaps were measured before and after treatment with epidermal growth factor, a well-known inducer of epithelial cell proliferation and migration.
The absolute migration was then calculated as the difference between the pre and post-treatment gap surface areas. Treatment with epidermal growth factor potentiates cell cytoskeletal dynamics as observed in these laser scanning microscopy images of F-Actin staining of control and growth factor stimulated cells. Further, c-Jun over-expression is observed with an increased expression of the protein apparent in the cells adjacent to the migrating front.
While attempting this procedure, it's important to remember to check the integrity of the wound edges for the presence of epithelial cell flaps that may disrupt the migration quantification. After watching this video, you should have a good understanding of how to both quantitatively and qualitatively assess the migratory behavior of the
