In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.
The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained for counting.
1. Dissection of the mouse placenta
2. Isolation of trophoblast cells
3. Trophoblast invasion Assay
4. Representative Results
Figure 1. Dissection of the mouse placenta. (a) The uterus is removed by cutting along the oviducts (yellow), along the mesometrial surface, and at the cervix (bottom). Individual conceptuses can then be separated by cutting with scissors as shown. (b) The uterine wall is removed by placing two forceps in the cut uterine surface, and tearing along the anti-mesometrial side, near the fetus and away from the placenta. (c) The placenta is then isolated by grasping with a forceps and sliding another between the placenta and uterine tissue. Umbilical cord and other extraembryonic membranes can then be separated from the placenta.
Figure 2. Percoll separation of trophoblast cells. After centrifugation, three major bands will be visible as shown in cartoon at left, and photograph at right. The upper band contains cellular debris and fibroblasts and will be most apparent. A tight (red) band near the bottom containing red blood cells will be visible just below the diffuse trophoblast band. Cell aggregates will likely seen in the trophoblast band, as in the two shown just to the left of the arrow in the photograph.
Figure 3. Invasive trophoblast cells (white arrow) will pass through the matrigel layer and the membrane pores, appearing on the bottom surface of the membrane. The pores are also visible (black arrowhead). The number of cells on the entire membrane surface or a representative sample area may be counted. A grayscale image of fluorescent DAPI nuclear staining has been inverted to improve visualization of cells.
Figure 4.Trophoblast cells can be identified, and the purity of the isolated population assessed, by immunostaining for cytokeratin 7 (center,magenta). Cells are counterstained with DAPI (top,blue). Merged image, bottom.
In this video, we demonstrate the isolation of trophoblast cells from the mouse placenta. We then show an in vitro assay for trophoblast invasion. Using this procedure, a 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. 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-digestion may lead to reduced purity, as well as reduced numbers. Although we show the use of commercially prepared matrigel invasion chambers, they may also be prepared by adding matrigel to uncoated transwell chambers at the desired thickness.
The authors have nothing to disclose.
The authors wish to thank the Creative Services team at the University of Missouri Academic Support Center who produced this video. This work was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, grant number HD055231
Specific reagents and equipment:
Wash Solution (filter sterilize after mixing)
Reagent | Amount | Final concentration |
10x Medium 199 | 50ml | 1X |
Hepes | 2.383 g | 0.02M |
sodium bicarbonate | 0.42 g | 0.01M |
Penicllin-Streptomycin | 5 mL | 100 U- 100 μg /ml |
Sterile dH20 | to 500 mL |
Dissociation solution (make fresh and filter sterilize)
Reagent | Amount | Final concentration |
Wash Solution | 100 mL | |
DNase | 1 vial (2000 U) | 20 U/mL |
Collagenase | 100 mg | 125 digestion units/mL |
Name of the reagent | Company | Catalogue number | Comments |
Collagenase | Sigma | C9891 | This is Clostridium collagenase. Bovine pancreatic collagenase would likely work as well |
NCTC-135 | Sigma | D4263 | Add FBS to 10% |
Matrigel invasion chambers | BD Biosciences | 354481 | |
10x Medium 199 | Sigma | M9163 | |
Penicillin-Streptomycin | Invitrogen | 15140 | |
DNase | Sigma | D4263 | |
100 μm Cell strainer | Fisher | 22363549 | |
Antibody to cytokeratin 7 | DAKO | M7018 | Used at 1:100 dilution |
Equipment
Standard cell culture equipment including a CO2 incubator is required. In addition, a centrifuge rotor capable of spinning 50 ml tubes at 30,000 x g is needed. Note that this exceeds the maximum speed of most conical centrifuge tubes and will likely require an Oak Ridge style tube.