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 &amp; Human Development of the National Institutes of Health, grant number 
HD055231

1. Dissection of the mouse placenta
<ol>
 <li>Set up mating pairs of mice.</li>
 <li>On each of the following days, check for the presence of a copulatory plug. The day in which a plug is detected is considered 0.5 days post-coitus 
 (dpc)7.</li>
 <li>Placentas may be collected at the desired stage of pregnancy, with a clean dissection of the placenta possible beginning with the development of the mature placenta on d 10.5. Here, we demonstrate 14.5 dpc. At the given volumes, 40-60 placentas on 10.5 dpc, and 10-20 placentas on 14.5 dpc will yield a clearly visible trophoblast cell layer. </li>
 <li>Euthanize the pregnant mouse, and remove the uterus, cutting at the cervix, and along the mesometrium.</li>
 <li>Using sharp scissors, cut between each conceptus (Figure 1A).</li>
 <li>Using two pairs of forceps, grasp the uterine wall at the cut edge, and pull apart, removing the myometrium (Figure 1B). One pair serrated, moderately fine (curved or straight) forceps plus one pair very fine forceps (#5, for example) is recommended. </li>
 <li>Opposite the placenta, open the decidua with forceps or scissors, exposing placenta. Grasping uterine tissue with one forceps pair, slide another forceps beneath the placenta to separate (Figure 1C). If necessary, separate fetus and fetal membranes from placenta. It is not possible to remove all decidual tissue by dissection without also losing placental tissue, but additional decidual tissue (pale) can be removed from the surface of the placenta by gently peeling with forceps. We will rely on the Percoll gradient for further purification.</li>
 <li>Place each dissected placenta in 25 ml ice cold dissociation buffer in a 50 ml conical tube until all dissections are complete. At later stages, coarsely mince placental tissue with scissors or razor blade prior to transfer into dissociation buffer. </li>
</ol>
2. Isolation of trophoblast cells
<ol>
 <li>Loosely cap conical tube and place in a 37&deg;C bath for 45-60 minutes. Mix the dissociation solution vigorously by pipetting approximately every 10 min. The total digestion time should be monitored closely and will likely require some trial and error to achieve optimal cell recovery. Tissues should be able to be passed through a 10 ml pipet, but visible pieces of tissue will remain. Overdigestion will result in poor cell survival and plating efficiency. Optimal cell survival and plating efficiency will occur when clumps of a few cells are present, rather than when complete dissociation is achieved. Conversely, underdigestion will lead to reduced purity following percoll separation. When the proper digestion point has been reached, place tube on ice to stop collagenase activity.</li>
 <li>Pass the solution through a cell strainer to remove undigested materials, and centrifuge @ 500 x g for 5 minutes.</li>
 <li>To wash, remove supernatant and resuspend cells in 10 ml wash solution. Centrifuge @ 500 x g for 5 minutes.</li>
 <li>During the centrifugation, prepare percoll solution by mixing 9.6 ml percoll, 13.4 ml wash solution and 1.1 ml 10x medium 199 in a 50 ml tube appropriate for high speed centrifugation. </li>
 <li>Remove supernatant and resuspend cell pellet in 2 mL wash solution.</li>
 <li>Add cells to percoll solution and centrifuge @ 30,000 x g for 30 minutes at 4&deg;C.</li>
 <li>Remove tube from centrifuge without disturbing the gradient. To visualize the gradient, place tube in front of a bright light source. Locate the trophoblast band (Figure 2), and aspirate with a pipet, taking care not to disturb the red blood cell band below. Volume of the trophoblast band will be 5-8 ml.</li>
 <li>Transfer trophoblast cells to a 50 ml conical tube. Add wash solution to a total volume of 45ml.</li>
 <li>Centrifuge @ 500 x g for 5 minutes. Remove supernatant, and resuspend in culture medium (NCTC-135). Cells can be cultured on thin-layer matrigel or tissue culture plastic at 37&deg;C and 5% CO2. At day 10.5, each placenta yields roughly 50,000 live cells, of which 80-90% are cytokeratin positive. </li>
</ol>

3. Trophoblast invasion Assay
<ol>
 <li>Add 1-2 ml culture medium (NCTC-135) to each matrigel chamber and incubate at 37&deg;, 5% CO2 for 30-90 min. </li>
 <li>Remove medium, and add 1.5 ml x 105 freshly isolated cells in 2ml medium to upper chamber. Add 1 ml medium to lower chamber to cover lower membrane surface.</li>
 <li>Culture cells in inserts for 18-24 hours.</li>
 <li>Scrape cells and matrigel from the insert using a foam swab.</li>
 <li>Rinse the top and bottom of the chamber in 2 ml PBS.</li>
 <li>Fix for 5 min in 1 ml 4% paraformaldehyde in PBS.</li>
 <li>Stain by filling lower chamber with 400 &mu;l DAPI (300nM) and incubate for 30 min. </li>
 <li>Wash three times briefly in PBS.</li>
 <li>Remove membranes from inserts with a sharp scalpel blade.</li>
 <li>Add a 10 &mu;l drop of mounting medium (50% glycerol in PBS) to slide. Using forceps, lay membrane on drop. Add another 10 &mu;l drop of medium, then coverslip carefully.</li>
 <li>Photograph and count cells. Be sure to orient the membrane appropriately (cell-side up or cell-side down) for the microscope being used. Image J software 
 (National Institutes of Health) includes a manual counting feature that can be used for this purpose, as can Adobe Photoshop or a manual counter. </li>
</ol>

4. Representative Results

<img src="/files/ftp_upload/3202/3202fig1.jpg" alt="Figure 1" /> 
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.

<img src="/files/ftp_upload/3202/3202fig2.jpg" alt="Figure 2" /> 
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. 

<img src="/files/ftp_upload/3202/3202fig3.jpg" alt="Figure 3" /> 
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. 

<img src="/files/ftp_upload/3202/3202fig4.jpg" alt="Figure 4" /> 
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.

<ol>
	<li>Thordarson, G., Folger, P., Talamantes, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+placental+cell+culture+system+for+studying+the+control+of+mouse+placental+lactogen+II+secretion.">Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion.</a> Placenta. 8, 573-585 (1987).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods. Mol. Med. 121, 203-217 (2006).</li><li>Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+in+vitro+differentiation+of+cytotrophoblasts+from+human+term+placentae.">Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.</a> Endocrinology. 118, 1567-1582 (1986).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Librach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=92-kD+type+IV+collagenase+mediates+invasion+of+human+cytotrophoblasts.">92-kD type IV collagenase mediates invasion of human cytotrophoblasts.</a> J. Cell. Biol. 113, 437-449 (1991).</li><li>Schulz, L. C., Widmaier, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leptin+on+mouse+trophoblast+cell+invasion.">The effect of leptin on mouse trophoblast cell invasion.</a> Biol. Reprod. 71, 1963-1967 (2004).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

We have nothing to disclose.

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 wi...

Specific reagents and equipment:
Wash Solution (filter sterilize after mixing)
<table border='1'>
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>50ml</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>2.383 g</td>
 <td>0.02M</td>
 </tr>
 <tr>
 <td>sodium bicarbonate</td>
 <td>0.42 g</td>
 <td>0.01M</td>
 </tr>
 <tr>
 <td>Penicllin-Streptomycin</td>
 <td>5 mL</td>
 <td>100 U- 100 &mu;g /ml</td>
 </tr>
 <tr>
 <td>Sterile dH20</td>
 <td>to 500 mL</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

Dissociation solution (make fresh and filter sterilize)
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>Wash Solution</td>
 <td>100 mL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>1 vial (2000 U)</td>
 <td>20 U/mL</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>100 mg</td>
 <td>125 digestion units/mL</td>
 </tr>
 </tbody>
</table>


<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma</td>
 <td>C9891</td>
 <td>This is Clostridium collagenase.
Bovine pancreatic collagenase would likely work as well</td>
 </tr>
 <tr>
 <td>NCTC-135</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>Add FBS to 10%</td>
 </tr>
 <tr>
 <td>Matrigel invasion chambers</td>
 <td>BD Biosciences</td>
 <td>354481</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>Sigma</td>
 <td>M9163</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m Cell strainer</td>
 <td>Fisher</td>
 <td>22363549</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibody to cytokeratin 7</td>
 <td>DAKO</td>
 <td>M7018</td>
 <td>Used at 1:100 dilution</td>
 </tr>
 </tbody>
</table>

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.

isolation of primary mouse trophoblast cells and trophoblast 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.

Watch this Scientific Journal Video about Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay at JoVE.com

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

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 ...

isolation-primary-mouse-trophoblast-cells-trophoblast-invasion

Research

JoVE Journal

Biology

23.7K vistas.  University of Missouri. En este protocolo, se describe la disección de placenta de ratón en el embarazo D10.5, seguido por el aislamiento de las células trofoblásticas utilizando un gradiente de Percoll. A continuación, demostrar el uso de las células aisladas en un ensayo de invasión de Matrigel.

Video: Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

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In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

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Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

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In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

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The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

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This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

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Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay