The overall goal of this procedure is to isolate primary placental trophoblast cells from mice for cell culture. We will also demonstrate one use of these cells, a matri gel invasion assay. The uterus will be removed from the pregnant mouse and the placenta will be dissected.
The placental tissue will be enzymatically digested to obtain individual cells or small clumps of cells, the cells will be separated on a protocol density gradient. The band containing trophoblast cells will be aspirated and the resulting cell population can then be washed and plated. We will next demonstrate the invasion assay cells are plated on a layer of matrigel that covers a thin membrane.
Invasive cells will digest through the matrigel layer and pass through eight micrometer pores in the membrane. The cells are then fixed in place and the membrane removed. The invaded cells can then be stained and counted.
Hello, I'm Kathleen Pennington from the Schultz Laboratory in the Department of Obstetrics Gynecology and Women's Health at the University of Missouri in Columbia, Missouri. Today we are going to show you the procedure we use to isolate primary trophoblast cells from pregnant mice. We'll then demonstrate the invasion assay that we use to test the cells.
We use these procedures to study factors that influence the development of placental trophoblast cells and their ability to invade an extracellular matrix. Let's get started. First, set up mating pairs of mice on each of the following days.
Check for the presence of a copul plug. The day in which a plug is detected is considered. Day 0.5.
Post coist placenta may be collected at the desired stage of pregnancy. Here we will demonstrate dissection of a day, 14.5 placenta on the benchtop. However, a microscope may be required to visualize tissues At earlier stages, locate the uterus, then cut at the utero tubule junction.
Continue to trim away fat and blood vessels cutting at the cervix and the other tubule junction. To remove the uterus, Transfer the uterus to a clean Petri dish using sharp scissors. Separate each conceptus Once separated, Transfer each conceptus to a clean dish.
The fetus lies towards the anti myometrial side from the placenta. The placenta lies along the meso side of the uterus. Use one pair of forceps to grasp the uterus near the cut face.
Carefully pull the two pairs of forceps away from each other, tearing the uterine wall along the anti myometrial side. This will usually rupture the membranes as well. Leaving a clearly visible fetus on top with the red placenta below and the white uterine tissue.
Below that, use fine forceps to separate the fetus cord and amniotic tissue from the placenta. Hold the uterine tissue with one pair of forceps and run the other pair between the placenta and underlying uterine tissue. At this time, excess deci tissue can be removed from the placenta.
However, some deci cells will likely remain. Place each dissected placenta in an ice cold dissociation buffer until all dissections are complete. When dissections are complete loosely, cap the conical tube containing placenta and dissociation solution and place it in a 37 degree Celsius water bath for 45 to 60 minutes.
After 10 minutes, mix the dissociation solution by pipetting vigorously up and down carefully monitor the progress of the digestion. The proper length of the collagenase dissociation step must be determined with each experiment and will be learned with. Experience over digestion will severely reduce cell recovery, whereas under digest may lead to reduced purity as well as reduced cell numbers.
Stop the reaction when few pieces of undigested tissue remain. Pass the solution through a cell strainer to remove undigested materials and centrifuge at 500 times G for five minutes to pellet the placenta cells. Next, wash the cells.
To remove the dissociation solution. Remove snat and resuspend cells in 10 mls of wash solution centrifuge at 500 times G for five minutes. Meanwhile, prepare per call solution by mixing 9.6 mls per call and 13.4 MLS wash solution in a tube suitable for high speed centrifugation.
Add 1.1 MLS of 10 x medium, 1 99 to make the per call solution, isotonic invert the tube to mix After the centrifugation and wash solution, remove the supernatant and resuspend the cell pellet. In two ml's, wash solution, add cells to the protocol solution and mix to create the gradient centrifuge at 20, 000 times. G for 30 minutes at four degrees Celsius, remove the tube from the centrifuge without disturbing the gradient.
The gradient can be visualized more easily by holding the tube in front of a light source. A white band containing mostly cellular debris will be visible near the top of the tube and a band of red blood cells will most likely be visible near the bottom of the tube. Just above.
This is the trophoblast band. Aspirate the medium until just above the trophoblast band and discard. Then using a pipette transfer the Trophoblast Band to a clean centrifuge tube containing 35 mls of wash solution centrifuge at 500 times G for five minutes.
To pellet the trophoblast cells re suspend the pellet in trophoblast culture medium. Here the cells will be cultured in invasion chambers In a six well plate. The matri gel chambers have previously been washed according to the manufacturer's instructions, pipette 0.5 to one ml of culture medium into the well below each insert.
Then add 1.5 times 10 to the fifth cells in one amount of medium to the chamber. Incubate at 37 degrees Celsius and 5%CO2. Remove the invasion chambers from the incubator after 18 to 24 hours.
Remove the culture medium and rinse the chambers three times in PBS for five minutes each time, gently but thoroughly. Scrape the cells and major gel from the upper surface of the insert. Using a cotton swab, fix the invaded cells on the lower surface of the membrane by filling the well with 4%para formaldehyde solution and incubating for five minutes.
Rinse three times in PBS. Stain the invaded cells by filling the well with a nuclear stain such as dappy. Incubate for 20 minutes.
Again, wash three times in PBS. Add a drop of mounting medium to a glass slide using a scalpel blade, carefully remove the membrane from the invasion chamber using forceps. Transfer the membrane to the slide with the cells facing upward.
Lay the membrane across the drop. Taking care to avoid air bubbles. Add another drop of mounting medium, then cover slip.
Carefully view and photograph the invaded cells on the membrane surface. Figure one invasive trophoblast cells will pass through the matrigel layer and the membrane pores appearing on the bottom surface of the membrane. The pores may also be visible.
The number of invaded cells can be counted and compared among treatment groups manually or using computer software. Figure two, in addition to nuclear stains, immuno cyto chemistry may be used to characterize the invaded cells. Here we have used an antibody to cytokeratin seven to confirm that the cells are trophoblast.
We have demonstrated the isolation of trophoblast cells from the mouse placenta. We then showed an in vitro assay for trophoblast invasion. Using this procedure largely but not entirely, pure population of trophoblast cells can be obtained.
If further purity is required, magnetic bead separation or flow cytometry could be performed as has been described for primary human trophoblast cells. Although we showed the use of commercially prepared matri gel invasion chambers, they may also be prepared by adding matri gel to uncoated transwell chambers at the desired thickness. Thank you for watching and good luck with your experiments.
