Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

The overall goal of this procedure is to isolate the infiltrating leukocytes of the murine maternal fetal interface. This is accomplished by first harvesting the uterus, placenta, and eua. The tissues are broken down by mechanical dissociation in an enzymatic solution, followed by a subsequent enzymatic digestion.

The resulting cell suspension is then filtered and washed, at which point the immune cells of interest are separated of cell debris such as non-viable cells by a fetal bovine serum gradient. Ultimately, the isolated leukocytes can be used for immunophenotyping cell culture or cell sorting. We first had the idea for this method when we were trying to establish a protocol for isolating leukocytes from the maternal fetal interface, and we realized that the published methods did not facilitate the isolation of the rare immune cells that are of interest to us.

A demonstration The procedure will be performed by Mile El Sanchez Rodriguez Marcina Hernandez, who are research associates from my laboratory To collect the maternal fetal interface tissues begin by using a pair of sterile surgical scissors to completely remove the lower abdominal portion of the skin and muscular tissue. Next, use forceps to move aside all of the other organs until the uterus and ovaries are visible. Then move the uterus outside of the peritoneal cavity.

Locate the ovaries distal to the UCT and the utero tubal junction of each uterine horn. Then make an incision at the utero tubal junction to separate the horns from the mesentary containing the uterine vessels. Excise at the cervix to detach the uterine horns from the peritoneal cavity.

Then use surgical scissors to make an incision between the cervix and the lower segment of the horns, leaving a portion of the cervix attached to avoid opening the uterine horn. Immerse the horns and cervix in a large Petri dish filled with 10 to 15 milliliters of PBS to hydrate the implantation sites. Trim away any fat from the tissues.

Then holding the horns in place with the forceps. Make a transverse cut through an inter implantation region to remove one of the implantation sites from the uterus. Now hold the site in place and make a small incision in the uterine wall adjacent to the placental and residual tissues.

Trim around the perimeter of the placenta and decidua until the tissues become separated from both the uterine wall and the chorio toic membrane. Then transfer the placenta and the attached decidual tissues to a fresh Petri dish containing three to five milliliters of PBS. Transfer the fetus into another Petri dish with PBS and use fine tip tweezers to gently peel the white gray deci tissue from the placental surface.

Taking care to maintain the integrity of the placental and ECI tissues. Split the placenta and ECI tissues into two individually labeled Petri dishes filled with PBS. Then use the fine tip tweezers to gently remove the uterine wall of the chorio Allen toic membrane and place these uterine tissues into a new Petri dish filled with PBS to mechanically dissociate the tissues.

Next, transfer two 100 to 150 milligrams of the uterine disci and placental tissues and individually labeled two milliliter conical tubes. Add 500 microliters of cold enzymatic solution one to each tube. Then use sterile scissors to mechanically dissociate each of the tissues for no more than two minutes until the suspensions.

Develop a milky appearance. Add 500 more microliters of cold enzymatic solution, one to each tube, and place the tubes on ice. Then incubate all of the tissues at 37 degrees Celsius with a gentle orbital agitation at 80 RPM, placing the tubes on ice after 30 to 35 minutes to stop the digestion.

Now strain each tissue slurry through a 100 micron strainer into individually labeled 50 milliliter conical tubes, and wash each two milliliter tube and strainer two to three times with 20 milliliters of PBS to collect all the cells. When all of the cells have been filtered, spin them down and carefully aspirate the SUP natin without disturbing the pellets. Gently resus suspend the isolated leukocytes and stromal cells in one milliliter of RPMI culture, medium without vortexing.

Then for each tissue, add 500 microliters of fetal bovine serum into a five milliliter polystyrene tube and slowly overlay the cell suspension on serum. Finally, when all of the cells have been overlaid centrifuge's cells without break and aspirate the supinate without touching the cell pellets. In these images, the morphology of F four 80 positive macrophages isolated from decid and uterine tissues.

At 16.5 days post coum are shown cells from both maternal and fetal interface tissues. Exhibit a viability of more than 70%after isolation by this method and contain neutrophil macrophage, T cell, NK cell, and dendritic cell populations with a high proportion of macrophages that coex express CD 11 C leukocyte subpopulations isolated from tissues at the mirror and maternal fetal interface. Also include B cells and CD four positive T cells, CD eight positive T cells, and CD three positive CD four negative CD eight negative T cells are also isolated as observed in this density plot.

Okay, while attempting this procedure, it is important to remember to keep the tissues and enzyme cocktail on ice to set the correct temperature in the incubator and to not over manipulate your tissues during the digestion following this procedure. Other methods like immunophenotyping, cell culture, cell sorting and D-N-A-R-N-A isolation can be performed. After watching this video, you should have a good understanding of how to isolate the infiltrating leukocytes at the maternal fetal interface, the uterus, the and placenta.