These methods can be utilized to determine factors that influence trophoblast invasion which may provide insight into major adverse obstetrical outcomes. The advantage of this technique is that it provides a visual and quantitative analysis of several factors that may influence trophoblast invasion including proliferation and migration. To begin the scratch assay, seed the appropriate number of cells into a 24-well plate to achieve 100%confluence in 48 hours.
The next day, change the media to phenol red free media supplemented with heat inactivated charcoal dextran stripped FBS. On the day of the scratch, add desired treatment to the media in each well to pretreat the cells for 30 minutes. To create uniform wounds, place sterile 10 microliter pipette tips on the pins of the wound maker tool.
Lower the tips into the first row of wells and move the plate along the track of the wound maker until the pipette tips reach the other side of the well. Then move the plate back to the starting position to ensure constant wound across the diameter of the well. Lower the tips into the next row of wells and repeat until all wells are scratched.
Confirm that the scratch covers the width of the well by observing the plate under a microscope. Next, carefully aspirate the media and gently wash the cells with 1x PBS twice. Following a final wash, add supplemented phenol red free stripped or low FBS media with a desired treatment.
Next, place the plate with the scratched wells in an automated timelapse imager housed in an incubator which contains 37 degrees Celsius temperature and 5%carbon dioxide at 95%humidity atmosphere. Image the cells at regular intervals as needed to allow cells to complete wound healing. To begin the invasion assay, seed the appropriate number of cells into six-well plates in an incubator which maintains 37 degrees Celsius temperature and 5%carbon dioxide at 95%humidity atmosphere.
Allow cells to reach 90%confluence in 48 hours. The next day, change the media to phenol red free media supplemented with heat inactivated charcoal dextran stripped FBS. Place a vial of 10 milligrams per milliliter extracellular matrix in a refrigerator on ice and let is thaw overnight.
The day after, use chilled pipette tips to dilute 10 milligrams per milliliter of the extracellular matrix with the phenol red free serum free media to a final concentration of five milligrams per milliliter. Then add 100 milliliters of the diluted extracellular matrix to chilled inserts that have been placed in the chilled 24-well plate. Incubate the plate at 37 degrees Celsius to allow the matrix to harden.
To prep cells for the invasion assay, wash cells with one milliliter of 1x PBS. Then aspirate the PBS and add 500 microliters of 1x Trypsin EDTA to each well. Place the six-well plate in the incubator which maintains 37 degrees Celsius temperature and 5%carbon dioxide at 95%humidity atmosphere until all cells have detached from the bottom of the plate.
Once cells have detached, add 500 microliters of the phenol red free serum free media to each well of the trypsinized cells. Then transfer 1, 000 microliters of detached cells to a microcentrifuge tube and spin down for five minutes at 400 times g at four degrees Celsius. Next, aspirate the media and resuspend the cells in the phenol red free serum free media.
Count cells using an automated cell counter and adjust volume of the cell suspension for a final concentration of 0.5 million cells per milliliter. Add 600 microliters of the complete growth media to each well of the 24-well plate and place the pre-coated insert into it. Then add 200 microliters of cells on top of each pre-coated cell insert for a total of 0.1 million cells.
Place the 24-well plate in the incubator which maintains 37 degrees Celsius temperature and 5%carbon dioxide at 95%humidity atmosphere for 24 hours. Following overnight incubation, suction the remaining media from the upper and lower chambers without disturbing the extracellular matrix. Then carefully remove the extracellular matrix and non-invaded cells from the upper portion of the inserts with a cotton swab.
Wash each insert three times by adding 1x PBS to both the upper and lower chambers of the well. Aspirate PBS after each wash. To fix cells attached to the inserts, place the inserts into 100%ice cold methanol at four degrees Celsius for 30 minutes.
Wash inserts three times with 1x PBS and remove PBS after the final wash. Stain the cells attached to the inserts with 0.2%crystal violet in 20%ethanol for 10 minutes. Rinse with deionized water three times and aspirate deionized water after the final wash.
Air dry inserts at room temperature for one hour. Finally using forceps and a razor blade, carefully cut the bottom membrane of the insert and mount it on a glass slide with the bottom side facing up. Using a light microscope with a 20X objective, obtain at least four unique images of the invaded cells per sample.
To begin the proliferation assay, use an automated cell counter to count the trypsinized cells from the flask. Seed cells into six-well plates at a density of 0.2 million cells per well in standard growth media. Then place cells in the incubator which maintains 37 degrees Celsius temperature and 5%carbon dioxide at 95%humidity atmosphere for four hours or until cells have adhered.
Next, change the media to phenol red free media supplemented with heat inactivated charcoal dextran stripped FBS and desired treatment. Place the six-well plate in the incubator which maintains 37 degrees Celsius temperature and 5%carbon dioxide at 95%humidity atmosphere. Then replace the media with the fresh media supplemented with the desired treatment every 48 hours.
After 24, 48, or 72 hours treatment, wash cells with one milliliter of 1x PBS and add 500 microliters of Trypsin EDTA to each well. Place a six-well plate in the incubator until cells have detached from the plate. Once cells have detached from the plate, add 500 microliters of the supplemented phenol red free media to deactivate Trypsin.
Finally add five microliters of the trypsinized cells to five microliters of Trypan Blue in a separate tube and mix by pipetting. Use an automated cell counter to count cells from each well in duplicate. Immortalized human first trimester trophoblast Swan.
71 cells were treated for zero, eight, and 18 hours with vehicle 1x PBS or 100 micromolar of the synthetic glucocorticoid dexamethasone in the scratch assay. Measurement of the wound size and the wound density in Swan. 71 and HTR-8/SVneo cells treated with vehicle or 100 nanomolar dex showed that dex treatment reduced the rate of wound closure indicating that glucocorticoids could inhibit cell migration.
Following the invasion assay, glucocorticoid treatment reduced cell invasiveness as shown by a 15%reduction in the number of invaded Swan. 71 and HTR-8/SVneo cells treated with 100 nanomolar dex for 24 hours compared to cells treated with vehicle. Following the cell proliferation assay, glucocorticoid treatment reduced cell proliferation as indicated by fewer Swan.
71 and HTR-8/SVneo cells treated with 100 nanomolar dex compared to cells treated with vehicle. Methods to evaluate cell death could be used in conjunction with this procedure to determine whether apoptosis or necrosis contribute to altered trophoblast functions. We have uniquely adopted the use of standard techniques routinely used in cancer biology for the study of trophoblast function.
There are no hazards associated with these methods. The only precaution viewers should take is using sterile technique for tissue culture.
