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>

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	<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+and+disparate+components+of+the+pathophysiologies+of+fetal+growth+restriction+and+preeclampsia.">Shared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia.</a> American Journal of Obstetrics &amp; Gynecology. 195 (1), 40-49 (2006).</li><li>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=Critical+growth+factors+and+signalling+pathways+controlling+human+trophoblast+invasion.">Critical growth factors and signalling pathways controlling human trophoblast invasion.</a> The International Journal of Developmental Biology. 54 (2-3), 269-280 (2010).</li><li>Huber, A. 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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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenol+red+in+tissue+culture+media+is+a+weak+estrogen:+implications+concerning+the+study+of+estrogen-responsive+cells+in+culture.">Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 83 (8), 2496-2500 (1986).</li><li>Cummings, B. S., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cell+death+in+mammalian+cells.">Measurement of cell death in mammalian cells.</a> Current Protocols in Pharmacology. , (2004).</li></ol>

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,...

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 &#956;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&#945;-fluoro-16&#945;-methyl-11&#946;, 17, 21-triol-3, 20-dione; &#8805;98% TLC)</td>
			<td>Steraloids</td>
			<td>P0500-000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Ham&#8217;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 &#8220;Scratch Wound&#8221; 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 &#181;g/mL)</td>
			<td>Life Technologies</td>
			<td>151-40-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol-red free DMEM/Ham&#39;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 de...

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 ...

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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

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

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 ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

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

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Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

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

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

Live Imaging of Mouse Secondary Palate Fusion

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands