The authors thank Dr. Gil Mor for providing the Sw.71 cells. This research was supported by an Albert McKern Scholar Award to S.W.

1. Cell Preparation
<ol>
	<li>Maintain cells in T-75 flasks in standard growth media at 37 &#176;C, 5% CO2, and 95% humidity. 
	NOTE: The cells used in these experiments were the immortalized first-trimester trophoblast cell lines Swan.71 (Sw.71) and HTR-8/SVneo9,10. The Sw.71 cells were maintained in DMEM/F-12 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1.0 mM sodium pyruvate, 10 mM HEPES, 0.10 mM MEM non-essential amino acids, 100 units/mL penicillin, and 100 &#181;g/mL streptomycin. The HTR-8/SVneo cells were maintained in RPMI-1640 supplemented with 5% heat-inactivated FBS.</li>
	<li>Allow the cells to replicate until they reach 90-100% confluence. Using a sterile tissue-culture flow hood, aspirate the media from the flask and wash cells with 10 mL of sterile 1x phosphate-buffered saline (PBS).</li>
	<li>Aspirate the PBS and add 5 mL of 1x (0.05%) trypsin-EDTA to the flask, making sure that cells are covered by trypsin. Place flask in the laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity until all cells have detached from the bottom of the flask (approximately 5-10 min).</li>
	<li>Add 10 mL of pre-warmed growth media to the flask to deactivate the trypsin-EDTA.</li>
</ol>
2. Scratch Assay
<ol>
	<li>Following step 1.4, seed the appropriate number of cells into a 24-well plate to achieve 100% confluence in 48 h. 
	NOTE: It is possible to perform the scratch assay with a variety of different plate layouts. The maximum number of wells allowed by the wound maker tool is a 96-well format.</li>
	<li>The next day (day prior to scratch assay), change media to phenol-red free media supplemented as required, including heat-inactivated, charcoal dextran-stripped FBS. 
	NOTE: Other studies have recommended the use of media with low concentration FBS (e.g., 1%) to minimize cell proliferation, which can be used as an alternative in this protocol11.</li>
	<li>On the day of the scratch assay, pretreat cells for 30 min by adding desired treatment to the media in each well. The outlined experiments utilized vehicle (1x PBS) and 100 nM dexamethasone diluted in 1x PBS.</li>
	<li>To create uniform wounds, place sterile 10 &#181;L 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.
	<ol>
		<li>Move the plate back to the starting position, allowing the pipette tips to continuously touch the bottom of the plate to ensure a constant wound across the diameter of the well. Remove and replace the sterile 10 &#181;L pipette tips and lower the tips into the next row of wells.</li>
	</ol>
	</li>
	<li>Repeat step 2.4 until all wells are scratched. 
	NOTE: If a wound maker tool is not available, wounds can be created by manually scraping the center of the wells with a 10 &#181;L pipette tip.</li>
	<li>Carefully aspirate media and gently wash cells twice with 1x PBS. Following a final wash, add supplemented phenol-red free, stripped or low-FBS media with desired treatment or vehicle (as directed in step 2.3). 
	NOTE: HTR-8/SVneo cells should be washed with supplemented phenol-red media, as washing with 1x PBS will cause cells to detach.</li>
	<li>Place the 24-well plate containing scratched wells in the automated, time-lapse imager (see Table of Materials) housed in a laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity. Image wells at regular intervals as needed to allow cells to complete wound healing (e.g., every 2 h for 72 h).</li>
	<li>Calculate the wound density and width by the software associated with the automated, time-lapse imager (see Table of Materials). To calculate the percentage change in wound size relative to the size of the starting wound, set the wound width at time 0 equal to 100%. Divide the wound size in each subsequent timepoint by the wound size at time 0 and multiply by 100 to obtain the percentage (Figure 1).</li>
</ol>
3. Invasion Assay
<ol>
	<li>Following step 1.4, seed the appropriate number of cells into 6-well plates to allow cells to reach 90% confluence in 48 h. Maintain cells in a laboratory incubator set to 37 &#176;C, 5% CO2, and 95% humidity.</li>
	<li>The next day, change media to phenol-red free media supplemented as required, including heat-inactivated, charcoal dextran-stripped FBS. 
	NOTE: Cells can be pretreated at this time depending on experimental design.</li>
	<li>At this time, place the vial of the 10 mg/mL extracellular matrix (see Table of Materials) on ice, and let it thaw in the refrigerator overnight.</li>
	<li>The next day, pre-cool invasion assay supplies by placing 24-well plates, cell culture inserts, microcentrifuge tubes, and pipette tips in the refrigerator (4 &#176;C) for at least 2 h prior to working with the extracellular matrix.</li>
	<li>Using chilled supplies in the tissue culture hood, dilute 10 mg/mL extracellular matrix 1:1 with phenol-red-free, serum-free media to a final concentration of 5 mg/mL. 
	NOTE: Always keep the stock and diluted extracellular matrix on ice. The ratio of extracellular matrix to media can be adjusted based on the properties of the cell line used in the assay.</li>
	<li>Using chilled supplies in the tissue culture hood, add 100 &#956;L of diluted extracellular matrix to the inserts (see Table of Materials) that have been placed in the 24-well plate. Make sure that the matrix is level without any bubbles. Place the 24-well plate containing matrix-coated inserts in the incubator at 37 &#176;C to allow the matrix to harden.</li>
	<li>To prep cells for invasion assay, wash cells with 1 mL of 1x PBS, aspirate the PBS, and add 500 &#181;L of 1x trypsin-EDTA to each well. Place the 6-well plate in the laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity until all cells have detached from the bottom of the plate.</li>
	<li>Once cells have detached, add 500 &#181;L of phenol-red-free, serum-free media to each well of the trypsinized cells.</li>
	<li>Transfer 1,000 &#181;L of detached cells to microcentrifuge tube and spin down for 5 min at 400 x g at 4 &#176;C.</li>
	<li>Aspirate media and resuspend cells in phenol-red-free, serum-free media (volume will depend on concentration of cells). Count cells using an automated cell counter (see Table of Materials) and adjust volume of cell suspension for a final concentration of 5 x 105 cells per mL. 
	NOTE: A hemocytometer and microscope may also be used to count cells.</li>
	<li>Add 600 &#956;L of complete growth media to the wells of the 24-well plate that will be used for the invasion assay. 
	NOTE: Additional chemoattractants or treatments may be added to the complete growth medium in the well (lower chamber). The outlined experiments added vehicle (1x PBS) or 100 nM dexamethasone diluted in 1x PBS to the lower chamber.</li>
	<li>Place the pre-coated insert into each well containing complete growth medium that will be used for the invasion assay. Then add 200 &#956;L of cells on top of each pre-coated cell insert (upper chamber) for a total of 1 x 105 cells. 
	NOTE: As a negative control for this assay, an equal volume of phenol-red-free, serum-free media (without cells) can be added to the upper chamber.</li>
	<li>Place 24-well plate in the laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity for 24 h. 
	NOTE: The 24 h incubation period was optimized for the immortalized first-trimester trophoblast cells and further tests may be required to determine the required incubation period in other cell lines.</li>
	<li>Following overnight incubation, suction the remaining media from the upper and lower chamber without disturbing the extracellular matrix. Then, carefully remove the extracellular matrix and non-invaded cells from the upper portion of the insert with a cotton swab, swabbing as many times as necessary to remove extracellular matrix.</li>
	<li>Wash each insert 3x with 1x PBS, adding PBS to both the upper and lower chambers of the well. Aspirate PBS after each wash.</li>
	<li>Fix cells attached to the inserts by placing inserts into 100% ice-cold methanol for 30 min at 4 &#176;C. Wash inserts 3x with 1x PBS and remove PBS after the final wash. Stain the cells attached to the inserts for 10 min with 0.2% crystal violet in 20% ethanol.</li>
	<li>Rinse 3x with deionized water and aspirate deionized water after the final wash.</li>
	<li>Air-dry inserts for 1 h at room temperature. Using forceps and a razor blade, carefully cut the bottom membrane of the insert and mount on a glass slide with the bottom side facing up. Allow mounting media to dry at room temperature.</li>
	<li>Obtain at least 4 unique images of invaded cells per sample using a light microscope with a 20x objective. Count the total number of cells in each image and normalize the number of cells to control samples and plot data (Figure 2).</li>
</ol>
4. Proliferation Assay
<ol>
	<li>Following step 1.4, count trypsinized cells from the flask using an automated cell counter and seed into 6-well plates at a density of 2 x 105 cells per well in standard growth media.</li>
	<li>Place cells in a laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity for 4 h or until cells have adhered.</li>
	<li>Change media to phenol-red free media supplemented as required, including heat-inactivated, charcoal dextran-stripped FBS and add desired treatment. The outlined experiments added vehicle (1x PBS) or 100 nM dexamethasone diluted in 1x PBS. Place the 6-well plates in a laboratory incubator maintained at 37 &#176;C, 5% CO2, and 95% humidity.</li>
	<li>Remove media and replace with new media and treatment every 48 h.</li>
	<li>After 24, 48, or 72 h treatment, wash cells with 1 mL of 1x PBS and add 500 &#181;L of trypsin-EDTA to each well. Place 6-well plate in incubator until cells have detached from plate.</li>
	<li>Once cells have detached from the plate, add 500 &#181;L of the supplemented phenol-red free media to deactivate trypsin.</li>
	<li>Add 5 &#181;L of trypsinized cells to 5 &#181;L of Trypan blue in a separate tube and mix by pipetting.</li>
	<li>Count cells from each well in duplicate using an automated cell counter.</li>
	<li>Average the number of cells per well at each time point and plot as a function of time (Figure 3).</li>
</ol>

<ol>
	<li>Ji, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+trophoblast+cell+differentiation:+Physiological+regulation+and+pathological+relevance+to+preeclampsia.">Placental trophoblast cell differentiation: Physiological regulation and pathological relevance to preeclampsia.</a> Molecular Aspects of Medicine. 34 (5), 981-1023 (2013).</li><li>Caniggia, I., Winter, J., Lye, S., Post, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+and+placental+development+during+the+first+trimester:+implications+for+the+pathophysiology+of+pre-eclampsia.">Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia.</a> Placenta. 21, S25-S30 (2000).</li><li>Norwitz, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+implantation+and+placentation:+laying+the+blueprint+for+pregnancy+complications.">Defective implantation and placentation: laying the blueprint for pregnancy complications.</a> Reproductive Biomedicine Online. 13 (4), 591-599 (2006).</li><li>Ness, R. B., Sibai, B. 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V., Saleh, L., Bauer, S., Husslein, P., Kn&#246;fler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF&#x3b1;-Mediated+Induction+of+PAI-1+Restricts+Invasion+of+HTR-8/SVneo+Trophoblast+Cells.">TNF&#x3b1;-Mediated Induction of PAI-1 Restricts Invasion of HTR-8/SVneo Trophoblast Cells.</a> Placenta. 27 (2), 127-136 (2006).</li><li>Nadeem, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+Signals+through+Activin+Receptor-Like+Kinase+7+to+Inhibit+Trophoblast+Migration+and+Invasion.">Nodal Signals through Activin Receptor-Like Kinase 7 to Inhibit Trophoblast Migration and Invasion.</a> The American Journal of Pathology. 178 (3), 1177-1189 (2011).</li><li>Onogi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+inhibits+invasion+of+extravillous+trophoblast+cells+through+reduction+of+matrix+metalloproteinase+(MMP)-2+activation+in+the+early+first+trimester+of+human+pregnancy.">Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy.</a> Placenta. 32 (9), 665-670 (2011).</li><li>Straszewski-Chavez, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+a+novel+telomerase+immortalized+first+trimester+trophoblast+cell+line,+Swan+71.">The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71.</a> Placenta. 30 (11), 939-948 (2009).</li><li>Graham, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+first+trimester+human+trophoblast+cells+with+extended+lifespan.">Establishment and characterization of first trimester human trophoblast cells with extended lifespan.</a> Experimental Cell Research. 206 (2), 204-211 (1993).</li><li>Liang, C. C., Park, A. Y., Guan, J. 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The authors have nothing to disclose.

This procedure builds on the use of migration and invasion assays to include the rate of cell proliferation as a potential contributor to measured differences in trophoblast cell movement. Used together, these in vitro assays are simple and effective methods that can be used to identify the cellular pathways that regulate trophoblast migration. As described above, trophoblast cells may be treated with hormones, cytokines, growth factors, or other molecules to determine their impact on trophoblast movement. Alternatively, target proteins may be depleted from cells to elucidate their functional role in trophoblast motility. Identifying key modulators of trophoblast migration may provide a better understanding of the basic mechanisms underlying complications of pregnancy related to placental defects, including preeclampsia, intrauterine growth restriction, and preterm birth.
There are several critical steps in this protocol that are required to obtain accurate results. Because phenol-red has estrogenic activity at concentrations used in cell culture media13, it is important that phenol-red free media is used for experimental endpoints to prevent undesired activation of the estrogen receptor in cells. During the transwell invasion assay, it is important to ensure that the extracellular matrix is homogenous, level, and that cold supplies are used to prevent premature polymerization of the matrix. When conducting the cell proliferation and invasion assays, it is important to count samples immediately to prevent re-adherence of the cells to tissue culture plates. It should also be taken under consideration that cell death due to experimental treatment may influence the results of all three assays. Therefore, markers of cell death (e.g., lactate dehydrogenase release, phosphatidylserine externalization, or DNA degradation) in response to treatment should be evaluated in the experimental cell line prior to evaluating migration, invasion, and proliferation14. Some limitations exist for these assays, for example, the process of creating a wound in a cell monolayer induces a cell injury response than may confound the cell migration process. Furthermore, manual scraping may introduce interexperiment variability, although utilizing a wound-maker tool that creates uniform wound widths can partially mitigate this limitation. The disadvantage of the invasion assay is that it is an endpoint assay, and the kinetics of movement are not easily attained. Despite these limitations, the assays described here provide reproducible, quantitative data at relatively low-cost.
The assays described here can also be modified to measure cell migration in other cell types, although these assays would not be suitable for non-adherent cell types. In the scratch assay, the imaging intervals for time-lapse microscopy may be adjusted to sufficiently capture differences in the rate of cell migration. Automated systems for live cell imaging can capture wound size images as frequently as every 15 min. Images taken at least every 30 min can be stitched together to create a movie of wound healing. In the invasion assay, the concentration of extracellular matrix, the total number of cells seeded, and the length of incubation prior to fixing and counting cells may be adjusted to account for the specific rate of invasion in different cell types. In the cell proliferation assay, the number of cells seeded onto the plates and the time points for cell counting may be adjusted to account for different rates of cell growth. Overall, these assays are flexible and highly accessible tools that can be used in a wide range of cell types to identify the mechanisms underlying cell migration.

Placental development is a crucial step in the establishment of pregnancy, impacting both maternal and fetal health. However, the mechanistic basis by which this process occurs is not fully understood. Cell migration is an important biological process that contributes to the establishment and functions of the placenta during pregnancy. Following blastocyst implantation, the trophectoderm differentiates into villous trophoblasts, which cover the surface of the chorionic villi and are involved in gas and nutrient exchange, and extravillous trophoblasts (EVTs), which migrate out from the villi and invade the maternal decidua and vasculature1. Migration of the EVTs is essential for remodeling maternal spiral arteries and establishing uteroplacental circulation to support fetal growth2. Inadequate trophoblast invasion during pregnancy results in abnormal placental development and may contribute to pregnancy complications such as preeclampsia, gestational hypertension, intra-uterine growth restriction, and preterm birth3,4. Therefore, understanding the factors that affect trophoblast motility is essential to determining the pathways required for normal placentation.
Trophoblast migration is controlled by a complex network of signaling molecules, including growth factors, cytokines, hormones, and angiogenic factors5. Due to the limitations of studying the placenta in vivo, in vitro assays utilizing immortalized human trophoblast cell lines have been crucial to identifying factors that contribute to trophoblast motility. Scratch and invasion assays have been used extensively to quantitatively assess the role of individual molecules on trophoblast cell migration6,7,8. However, while useful in tandem to investigate how changes in migration may be caused by changes in cell invasiveness, these two assays do not account for additional mechanisms that may contribute to altered rates of cell migration. For example, reductions to the rate of cell proliferation may result in fewer cells available to migrate.
Here, we describe quantitative in vitro methods for assessing trophoblast migration using scratch, cell proliferation, and cell invasion assays. In the scratch assay, a uniform wound is created in a cell monolayer, and the migration of cells to fill the gap is measured by automated, time-lapse imaging of the wound size and density of cells within the wound. The cell proliferation assay is based on calculating the ratio of cells at each time point compared to a known starting quantity of cells. In the cell invasion assay, cells are seeded atop an extracellular matrix-coated cell culture insert chamber (e.g. Transwell), and the number of cells that invade through the extracellular matrix in response to chemo-attractant are counted.
The scratch assay is a simple and effective tool that may be used to determine how different environmental conditions affect cell migration. The proliferation and invasion assays may subsequently be used to determine the contribution of cell proliferation and invasiveness to overall changes in cell migration. Collectively, these assays provide robust measurements of biological processes that may contribute to cell motility. Two immortalized first-trimester trophoblast cell lines were used in the described assays, Swan.71 (Sw.71) and HTR-8/SVneo9,10. However, these assays may also be optimized for use in other cell types to identify key modulators of cell migration and invasion.

<table><tbody><tr><td>0.4% Trypan blue stain</td><td>Thermo Fisher Scientific</td><td>15250-061</td><td></td></tr><tr><td>0.5% Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>1.0 M HEPES</td><td>AmericanBio</td><td>AB06021-00100</td><td></td></tr><tr><td>10 mM MEM non-essential amino acids</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>100 mM sodium pyruvate</td><td>Life Technologies</td><td>11360-070</td><td></td></tr><tr><td>24-well plate transwell inserts</td><td>Corning</td><td>3422</td><td>Contain polycarbonate filters with an 8 &amp;mu;m pore size</td></tr><tr><td>Charcoal dextran-stripped FBS</td><td>Gemini Bio-Products</td><td>100-119</td><td></td></tr><tr><td>Countess II Automated Cell Counter</td><td>Thermo Fisher Scientific</td><td>AMQAX1000</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma Aldrich</td><td>C0775</td><td></td></tr><tr><td>Cytoseal 60</td><td>Thermo Fisher Scientific</td><td>8310-4</td><td></td></tr><tr><td>Dexamethasone (Dex; 1, 4-pregnadien-9&amp;alpha;-fluoro-16&amp;alpha;-methyl-11&amp;beta;, 17, 21-triol-3, 20-dione; &amp;ge;98% TLC)</td><td>Steraloids</td><td>P0500-000</td><td></td></tr><tr><td>DMEM/Ham&amp;rsquo;s F12</td><td>Life Technologies</td><td>11330-032</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma Aldrich</td><td>F2442</td><td></td></tr><tr><td>IncuCyte 24-well ImageLock Plates</td><td>Essen BioScience</td><td>4365</td><td>If using the IncuCyte Zoom imaging system, seed cells onto IncuCyte ImageLock Plates. These plates have fiducial markers on the bottom of the plate that are used as a reference for repeat imaging in a constant field of view.</td></tr><tr><td>IncuCyte software</td><td>Essen BioScience</td><td>2010A Rev2</td><td></td></tr><tr><td>IncuCyte ZOOM system</td><td>Essen BioScience</td><td>N/A</td><td>Scratch assay images were captured and analyzed using the preset &amp;ldquo;Scratch Wound&amp;rdquo; parameters on the IncuCyte ZOOM system. Other time-lapse microscopes may also be used.</td></tr><tr><td>Matrigel Membrane Matrix</td><td>Becton Dickinson</td><td>356231</td><td>Growth factor reduced, phenol-red free</td></tr><tr><td>Micro Slides</td><td>VWR International, LLC</td><td>48311-7003</td><td></td></tr><tr><td>Microscope cover glass</td><td>Fisher Scientific</td><td>12-545-E</td><td></td></tr><tr><td>Olympus IX71 inverted microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Penicillin (10,000 units/mL)/streptomycin (10,000 &amp;micro;g/mL)</td><td>Life Technologies</td><td>151-40-122</td><td></td></tr><tr><td>Phenol-red free DMEM/Ham&#039;s F12</td><td>Life Technologies</td><td>11039-021</td><td></td></tr><tr><td>Semi-automatic wound maker tool</td><td>Essen BioScience</td><td>N/A</td><td></td></tr></tbody></table>

in vitro assays to evaluate the migration, invasion, and proliferation of immortalized human first-trimester trophoblast cell lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

The immortalized human first-trimester trophoblast cell lines Sw.71 and HTR-8/SVneo were used in these experiments to determine the role of glucocorticoids in trophoblast cell migration12. Glucocorticoids were used in these experiments as they have recently been shown to alter trophoblast functions, although alternative treatments could be used12. Both cell lines were treated with vehicle (1x PBS) or 100 nM of the synthetic glucocorticoid dexamethasone (Dex) for all experiments. Wound density and size were measured every 2 h for 72 h using an automated, time-lapse imaging system.&#160;Figure 1 shows an example of images and results obtained from the scratch assay at different timepoints. Sample images from 0, 8, and 18 h timepoints for both treatments have been provided (Figure 1A). The wound density and wound size are graphed over time for samples treated with vehicle control (Veh) or 100 nM Dex (Figure 1B). Dex treatment reduced the rate of wound closure as determined by both wound density and wound size, indicating that glucocorticoids inhibit cell migration in first-trimester trophoblast cells.
Figure 2 shows an example of images taken of the cell culture insert following the invasion assay. Cells were treated for 24 h with Veh or 100 nM Dex. Invaded cells were counted from four independent replicates per treatment using four unique fields of view per replicate. Glucocorticoid treatment reduced cell invasiveness, as shown by a 15% reduction in the number of invaded cells.
Figure 3 shows an example of the cell proliferation assay. Cells were counted at 24, 48, and 72 h following treatment with Veh or 100 nM Dex. Glucocorticoid treatment reduced cell proliferation, as indicated by fewer cells at each time point. Overall, these assays demonstrate that glucocorticoid exposure reduces cell motility by inhibiting both cell proliferation and invasion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58942/58942fig1.jpg" /> 
Figure 1: Cell Scratch Assay. (A) Representative images of wounds for Veh- and Dex-treated Sw.71 cells at 0, 8, and 18 h are shown. Red lines indicate wound size. (B) Wound density and wound size were measured in Sw.71 and HTR-8/SVneo cells treated with vehicle (Veh) or 100 nM Dex. Pictures were taken every 2 h for 72 h using an automated, time-lapse microscope. Data represents the mean of four independent replicates &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58942/58942fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58942/58942fig2.jpg" /> 
Figure 2: Cell Invasion Assay. Invasion assays were conducted with Sw.71 and HTR-8/SVneo cells treated for 24 h with vehicle (Veh) or 100 nM Dex. (A) Representative images from the invasion assay for Veh- and Dex-treated cells after 24 h incubation. Inset image is negative control with no cells added to the upper chamber. Images taken at 200x magnification. Scale bars are 120 &#181;m. (B) Invaded cells were counted and the bar graphs represent the normalized mean of four independent replicates &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58942/58942fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58942/58942fig3.jpg" /> 
Figure 3: Cell Proliferation Assay. Sw.71 and HTR-8/SVneo cells treated with vehicle (Veh) or 100 nM Dex were counted every 24 h using an automated cell counter. Cell counts are graphed as the mean &#177; SEM of at least 11 independent replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58942/58942fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines at JoVE.com

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

in-vitro-assays-to-evaluate-migration-invasion-proliferation

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.1K Views.  Yale School of Medicine. Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Video: In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Genetics

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Bioengineering

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

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.

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 ...

Biology

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Medicine

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

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siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

In vitro Cell Migration and Invasion Assays

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos