The authors thank women who donated their placentas for this study. We also thank the Labor and Delivery Department at OHSU and the Maternal and Fetal Research Team for coordinating the collection of placentas. We are grateful to Eric Wang, Ph.D., and Kelly Kuo, MD for support and help with experimental methods and optimization.
This work was funded by NIH HD076259A (AM) and AHA GRNT29960007 (AM).

Placentae were collected from the Labor and Delivery Unit at University Hospital under a protocol approved by the Institutional Review Board of Oregon Health and Science University in Portland, Oregon, with informed consent from the patients.
1. Collection of Placental Tissue
<ol>
	<li>Preparation 
	NOTE: All equipment that encounters tissue must be sterile.
	<ol>
		<li>Sterilize the dissecting equipment by autoclaving for 60 min at 121 &#176;C.</li>
		<li>Use personal protective equipment (PPE): lab coats/gowns/scrubs, gloves, and mask with a face shield or goggles.</li>
		<li>Turn on water bath and set to 37 &#176;C.</li>
		<li>Warm two 50 mL conical tubes, each containing 25 mL of complete media (Iscove&#39;s Modified Dulbecco&#39;s Medium supplemented with 10% FBS and 1% Penicillin/Streptomycin, Table 3) in a 37 &#176;C water bath.</li>
		<li>With informed patient consent, obtain a placenta immediately following delivery by cesarean section.</li>
		<li>Become familiar with the placenta. The umbilical cord is inserted on the fetal side (chorionic plate) and blood vessels can be seen radiating out from the umbilical cord insertion site. The opposite side is the maternal side, or basal plate. It includes the decidua and structures that contain vessel trees, called cotyledons.</li>
	</ol>
	</li>
	<li>Sampling of Villous Tissue 
	NOTE: Villous tissue should be sampled from the placenta as soon as possible following delivery, preferably in 30 min or less.
	<ol>
		<li>With the chorionic plate facing upwards, excise 2-3 full-thickness sections (approximately 2.5 cm x 2.5 cm in size) 2-3 cm in from the periphery of the placenta at random, using forceps and scissors (Figure 1A). 
		NOTE: Avoid parts of the placenta that appear abnormal (i.e. white calcifications).</li>
		<li>Trim away the chorionic and basal plates and any large blood vessels.</li>
		<li>Place the resulting villous tissue (Figure 1B, approximately 80 - 120 g total) in warm complete media and begin trophoblast isolation within 30 min of sampling. 
		NOTE: Some downstream assays and applications are affected by the length of latency period between delivery, sampling, and trophoblast isolation. It is best to keep these periods as short as possible and consistent between isolations.</li>
		<li>Using the techniques described in steps 1.2.1 - 1.2.2, sample 5 random 1 cm x 1 cm sections of villous tissue from across the placenta (including the center).</li>
		<li>Cut each 1 cm x 1 cm villous tissue sample into 4-5 smaller pieces (approximately 30 mg each), place in 2 mL microcentrifuge tubes, and flash freeze in liquid N2. Store at -80 &#176;C for use in later analyses.</li>
	</ol>
	</li>
</ol>
2. Isolation of Trophoblasts from Villous Tissue
<ol>
	<li>Preparation 
	NOTE: Use sterile equipment and perform procedures involving cells in a laminar flow hood.
	<ol>
		<li>Thaw four frozen density gradients (Table 1, see table of the essential supplies, reagents, and equipment, supplementary material) at 4 &#176;C the night before the placenta arrives. 
		NOTE: Alternatively, density gradients can be made on the day of the isolation.</li>
		<li>Turn on centrifuge and set to 20 &#176;C. 
		NOTE: All centrifugation steps are at maximum acceleration and deceleration unless specified otherwise.</li>
		<li>Prepare 1x HEPES-buffered salt solution (HBSS) supplemented with Ca+2 and Mg+2 (Table 3).</li>
		<li>Warm trypsin to 37 &#176;C.</li>
		<li>Dilute DNase to approximately 2720 kilounits/mL in sterile supplemented HBSS.</li>
		<li>Warm 50 mL of Newborn Calf Serum (NCS) to 37 &#176;C.</li>
		<li>Warm complete media to 37 &#176;C.</li>
		<li>Warm freezing media (90% FBS, 10% DMSO, Table 3) to 37 &#176;C. 
		CAUTION: DMSO is toxic and must be handled with gloves.</li>
		<li>Prepare digestion buffer (Table 2 and 3) by mixing 308 mL of supplemented HBSS, 50 mL of Trypsin (3751.7 BAEE units/mL), and 0.5 mL of DNase (379.4 kilounits/mL) in a sterile bottle. 
		NOTE: The approximate time required to isolate cells from villous tissue is 7 h.</li>
	</ol>
	</li>
	<li>Processing the Villous Tissue
	<ol>
		<li>Rinse each piece of villous tissue in a 50 mL conical tube filled with room temperature phosphate buffered saline (PBS). Repeat and replace the PBS as necessary until the excess blood is removed (PBS rinse will be light red or pink when the tissue is thoroughly rinsed).</li>
		<li>Place the villous tissue in a sterile Petri dish and remove as many blood vessels as possible by gently scraping off the soft villous tissue from the vessels using a microscope slide.</li>
		<li>Finely mince the resulting villous tissue using scissors.</li>
	</ol>
	</li>
	<li>Villous Tissue Digestion and Crude Isolation of Trophoblasts
	<ol>
		<li>Transfer the minced villous tissue to a sterile bottle with digestion solution according to the calculated volumes based on the specific activity of trypsin and DNase (165 mL, Table 2).</li>
		<li>After 35 min of incubation in a 37 &#176;C water bath with shaking at 70 revolutions per minute (rpm), tilt the digestion bottle on its side and allow the undigested pieces of tissue to settle at the bottom of the bottle. Carefully draw up the supernatant with a serological pipette, avoiding the settled tissue.</li>
		<li>Dispense the supernatant through a 100 &#181;m cell strainer equally between 50 mL conical tubes. 
		NOTE: To save time, it is advisable to begin the second digestion by adding digestion solution (110 mL, Table 2) to the remaining settled tissue and resuming incubation as described in step 2.3.2.</li>
		<li>Gently layer 3 - 5 mL of NCS underneath the strained supernatant by slowly dispensing from a serological pipette at the bottom of the tube. A meniscus between the strained supernatant (containing trophoblasts) and NCS should be visible (Figure 2A).</li>
		<li>Centrifuge the supernatants over NCS at 1,250 x g for 15 min at 20 &#176;C. The resulting pellet will include red blood cells in the lower-most layer followed by a white layer containing the trophoblast cells (Figure 2B).</li>
		<li>Repeat steps 2.3.2 - 2.3.5 for each of the second and third digestions (adding 110 mL and 83.5 m, respectively, of digestion solution to the bottle of tissue, Table 2).</li>
		<li>Once all supernatants have been centrifuged, resuspend each pellet in 5 mL of warm complete media, and then pool the suspensions together.</li>
		<li>Split the cell suspension equally between two 50 mL conical tubes and centrifuge at 1,250 x g for 15 min at 20 &#176;C.</li>
		<li>Gently remove the supernatant and resuspend each of the cell pellets in 6 mL of warm complete media.</li>
	</ol>
	</li>
	<li>Density Centrifugation
	<ol>
		<li>Divide the cell suspension equally between four density gradients (3 mL each) by slowly and carefully layering the cell suspension on the top of the density gradients with a transfer pipet.</li>
		<li>Centrifuge the density gradients at 1,250 x g for 20 min at 20 &#176;C with minimum acceleration and deceleration. This should produce distinguishable bands of sedimented cells (Table 4).</li>
		<li>Slowly and carefully remove the top layers of density gradient media (DGM) until the opaque band(s) containing trophoblast cells (between 35&#160;- 50% DGM) is reached (Table 4).</li>
		<li>Transfer the trophoblast bands into two 50 mL conical tubes and fill with warm complete media.</li>
		<li>Gently invert the tubes 3 - 6 times to mix and centrifuge at 1,250 x g for 15 min at 20 &#176;C.</li>
		<li>Remove the supernatant and resuspend each cell pellet in 5 mL of warm complete media. Combine the cell suspensions and count viable cells using a hemocytometer and Trypan blue (or preferred cell counting method).</li>
	</ol>
	</li>
	<li>Counting Cells with a Hemocytometer
	<ol>
		<li>Mix the cell suspension by pipetting up and down with a serological pipet or by gently inverting the tube several times.</li>
		<li>Combine equal parts of cell suspension and Trypan blue (i.e. 20 &#181;L each) in a separate tube and mix gently.</li>
		<li>Incubate for 1 - 2 min at room temperature.</li>
		<li>Gently dispense 15 - 20 &#181;L of the cell-Trypan blue mixture in between the coverslip and the hemocytometer and allow the cells to diffuse across the grid by capillary action.</li>
		<li>Count viable cells (dead cells will be stained deep blue) in each of the four 4 x 4 quadrants with a tally counter. Employ a system of counting to ensure cells are not counted more than once (i.e. do not count cells that touch the bottom or left boundaries). 
		NOTE: Each 4 x 4 quadrant should contain between 50 - 150 cells. Too few or too many cells can lead to an over or underestimation of cell number.</li>
		<li>Average the total cell counts from each of the 4 x 4 quadrants, multiply by 104, and then multiply by the dilution factor (cell suspension to Trypan blue) to calculate number of cells per mL.</li>
		<li>Multiply the number of cells per mL by the total volume of cell suspension to calculate the total cell yield. 
		NOTE: Approximately 100 million cells are expected from isolations starting with 80-120 g of villous tissue.</li>
	</ol>
	</li>
	<li>Plating Cells
	<ol>
		<li>Plate 3 million cells/well (3.3 x 105 cells per cm2) in a 6-well plate (2 mL of suspension per well) and gently rock back and forth and side to side to evenly distribute the cells. 
		NOTE: Trophoblasts require tissue culture treated plates to adhere properly. A monolayer of cells is required to promote syncytialization.</li>
		<li>Leave the plated cells in the laminar flow hood for approximately 30 min to allow cells to evenly distribute, settle, and begin to adhere to the bottom of the wells before placing in the incubator.</li>
		<li>Culture cells for up to 72 h (with daily media changes) in a 37 &#176;C incubator with 5% CO2 and 95% humidity. 
		NOTE: Examine the trophoblasts under a microscope at 10 - 20x every 24 h of culture. Trophoblasts do not proliferate and cannot be passaged. Over the course of 72 h of culture, the round individual trophoblasts fuse to form a syncytium (Figure 3A and B).</li>
	</ol>
	</li>
	<li>Freezing Cells
	<ol>
		<li>Pellet unused cells by centrifugation at 1,250 x g for 10 min at 20 &#176;C.</li>
		<li>Aspirate as much media as possible from the pellet.</li>
		<li>Resuspend the pellet in freezing media (Table 3).</li>
		<li>Freeze the aliquots at -80 &#176;C in a freezing container filled with 100% isopropanol. Transfer the frozen aliquots to liquid N2 the following day for long term storage. 
		NOTE: Cells can be cultured after freezing.</li>
	</ol>
	</li>
	<li>Thawing Cells
	<ol>
		<li>Remove an aliquot of frozen cells and thaw in a 37 &#176;C water bath while swirling. Remove the aliquot from the water bath just before it has fully thawed to a liquid.</li>
		<li>Immediately transfer the thawed cells to a 15-ml conical tube. Beginning slowing at first, add 10 mL of complete media with intermittent mixing.</li>
		<li>Invert tube several times to mix.</li>
		<li>Centrifuge at 200 x g for 10 min at 20 &#176;C.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 2 - 5 mL of warm complete media.</li>
		<li>Count the cells and plate as previously described.</li>
	</ol>
	</li>
</ol>
3. Treatment of Primary Trophoblasts with TNF&#945;, Collection of Cell Lysates, and Western Blotting
<ol>
	<li>Preparation
	<ol>
		<li>Warm complete media to 37 &#176;C.</li>
		<li>Make a 10 &#181;g/mL stock of TNF&#945; in complete media and store at -20 &#176;C until ready for use.</li>
		<li>Make a 1 &#181;g/mL working stock of TNF&#945; in complete media when ready to treat the cells. Serial dilute the TNF&#945; working stock to 104 pg/mL, 103 pg/mL, 500 pg/mL, 250 pg/mL, and 125 pg/mL in complete media. 
		NOTE: The TNF&#945; concentration of 104pg/mL is moderately cytotoxic. Adjustment of the TNF&#945; concentrations tested will depend on the specifics of the desired downstream applications.</li>
	</ol>
	</li>
	<li>Treating Cells with TNF&#945;
	<ol>
		<li>After 24 h of culture, aspirate the culture media from the cells and replace with 2 mL of TNF&#945; supplemented medias per well on 6-well plates (at least two wells per treatment and vehicle control).</li>
		<li>After 24 h of TNF&#945;-exposure (48 h of culture), replace the TNF&#945; supplemented medias with complete media.</li>
	</ol>
	</li>
	<li>Harvesting Cells and Total Protein
	<ol>
		<li>After 72 h of culture (24 h past removal of TNF&#945;), aspirate media, gently rinse cells with room temperature PBS, and add 80 &#181;L of ice cold Radioimmunoprecipitation Assay Buffer (RIPA) containing freshly added protease and phosphatase inhibitors (Table 3) directly to each well.</li>
		<li>Remove the cells from the plate with a cell scraper. Transfer the cells to a 1.5 mL microcentrifuge tube, pooling wells within treatment groups.</li>
		<li>Lyse the cells by vortexing the tubes on high for at least three intervals of 15 s.</li>
		<li>Incubate the cells at 4 &#176;C with rocking for 15 min. 
		NOTE: Alternatively, after step 3.3.3., the cells can be incubated on ice for 15 min with intermittent agitation by flicking the tube, vortexing, or pipetting up and down.</li>
		<li>Repeat step 3.3.3.</li>
		<li>Centrifuge the tubes at 10,000 x g for 5 min at 4 &#176;C to pellet cellular debris.</li>
		<li>Transfer the supernatant (containing cellular protein) to a new 1.5 mL microcentrifuge tube and store at -80 &#176;C. 
		NOTE: The protocol can be stopped here and resumed at a later time. It is advisable to make several aliquots of cellular protein samples to avoid multiple freeze-thaw cycles.</li>
		<li>Determine total protein concentration by a preferred method, such as a bicinchoninic acid assay (BCA, see table of the essential supplies, reagents, and equipment, supplementary material).</li>
	</ol>
	</li>
	<li>SDS-PAGE and Western Blotting for Rubicon or Protein of Interest 
	NOTE: Follow Western blotting protocols according to manufacturer&#39;s instructions using a preferred laboratory system.
	<ol>
		<li>Load between 20-40 &#181;g of total protein in sample buffer (Table 3) per well on a 12% acrylamide gel. Separate proteins by SDS-PAGE in running buffer (Table 3).</li>
		<li>Wet-transfer the proteins from the gel to a polyvinylidene difluoride (PVDF) membrane according to manufacturer&#39;s instructions in transfer buffer (Table 3).</li>
		<li>Incubate the membrane in 5% milk powder in Tris-Buffered Saline with 0.1% Tween 20 (TBST, Table 3) for at least 1 h at room temperature with rocking.</li>
		<li>Incubate the membrane in a Rubicon primary antibody (see table of the essential supplies, reagents, and equipment, supplementary material) at 1:500 in 1% milk powder in TBST overnight at 4 &#176;C with rocking.</li>
		<li>Gently wash the membrane 3 x 5 min in TBST and 1 x 5 min in TBS at room temperature with rocking.</li>
		<li>Incubate the membrane in 1:2000 - 1:5000 secondary antibody (HRP-linked or preferred visible conjugate, see table of the essential supplies, reagents, and equipment, supplementary material) in 5% milk powder in TBST for at least 1 h at room temperature with rocking.</li>
		<li>Repeat the washing procedure outlined in step 3.4.5 and visualize the blot with an appropriate visualization (i.e. chemiluminescent) substrate on an imaging system.</li>
		<li>Repeat the washing procedure outlined in step 3.4.5 and probe the membrane for &#946;-actin or a preferred loading control according to manufacturer&#39;s instructions.</li>
		<li>On preferred image analysis software, analyze the expression of Rubicon by manual quantification of absorbance (band intensities), subtraction of background absorbance, and normalization to the corresponding loading control band intensities. Perform statistical analyses as appropriate to test for statistically significant changes in protein levels.</li>
	</ol>
	</li>
</ol>

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Immunol. 10 (5), 461-470 (2009).</li><li>Sagrillo-Fagundes, 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=Human+Primary+Trophoblast+Cell+Culture+Model+to+Study+the+Protective+Effects+of+Melatonin+Against+Hypoxia/reoxygenation-induced+Disruption.">Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.</a> JoVE. (113), (2016).</li><li>Segovia, S. A., Vickers, M. H., Gray, C., Reynolds, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+obesity,+inflammation,+and+developmental+programming.">Maternal obesity, inflammation, and developmental programming.</a> Biomed. Res. Int. , 418975 (2014).</li><li>Komatsu, M., 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=Tumor+necrosis+factor-alpha+in+serum+of+patients+with+inflammatory+bowel+disease+as+measured+by+a+highly+sensitive+immuno-PCR.">Tumor necrosis factor-alpha in serum of patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR.</a> Clin. Chem. 47 (7), 1297-1301 (2001).</li><li>Matsunaga, K., Noda, T., Yoshimori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+Rubicon+to+cross+the+Rubicon.">Binding Rubicon to cross the Rubicon.</a> Autophagy. 5 (6), 876-877 (2009).</li></ol>

The authors have nothing to disclose.

The placenta, responsible for regulating the growth of the fetus, exhibits compromised function in the obese environment6. Despite the high metabolic demands of trophoblasts, placentas with maternal obesity exhibit dysfunctional mitochondrial respiration6,19. Changes in placental metabolism may contribute to the increased incidence of complications and adverse fetal outcomes observed in obese pregnancies3,6,7. Autophagy is also compromised in placentas with maternal obesity. Placentas from obese pregnancies with male fetuses show dysfunctional autophagic flux as evidenced by the accumulation of autophagosomes13. Maintaining optimal autophagic flux is critical for cellular homeostasis and defective autophagy in placentas with maternal obesity may play an important role in mediating the adverse outcomes associated with obese pregnancies.
The precise cause(s) of the compromised placental function exhibited in maternal obesity are not well understood and may have their origins in inflammatory signaling. Both obesity and pregnancy are proinflammatory states that are characterized by elevated levels of circulating TNF&#945;1,4,20,21. TNF&#945; is an activator of autophagy22,23and may mediate changes in autophagic flux in the placentas from obese pregnancies. TNF&#945; is commonly used to study the effects of inflammation on cells, especially in the context of cancer2. The protocol presented here adapts this approach for studying the effects of obesity-related inflammation on the functional capacity of the placenta by treating primary trophoblasts with exogenous TNF&#945; in culture.
This protocol consists of four main components: sampling of villous tissue from the placenta, isolation of primary trophoblasts from villous tissue, culture of primary trophoblasts followed by TNF&#945; treatment, and collection of total cellular lysates followed by Western blot analysis to measure the expression of a protein(s) of interest. To isolate pure trophoblasts, it is critical that the chorionic and basal plates are thoroughly dissected away from the villous tissue during the sampling of the placenta (Figure 1B). It is also imperative that villous tissue is processed in a timely manner (i.e. within 30 minutes of delivery). This protocol details the use of three digestion steps, each lasting 35 minutes, to release trophoblasts from the extra cellular matrix of the villous tissue. Lengthier digestions risk damaging cells by trypsinization of cell surface proteins. Increasing the number of digests does not appreciably increase the final yield of viable trophoblasts. However, decreasing the time and number of digestions will result in decreased cell yields (Simon, Bucher, and Maloyan, unpublished data). This protocol reliably produces approximately 100 million cells from 80 - 120 grams of villous tissue.
There is variability in the number of distinguishable trophoblast bands observed on the density gradients between isolations. To avoid contamination from other cell types, it is important to strictly collect the band(s) containing trophoblasts on the density gradient (Table 4). The protocol detailed here is optimized from previously established methods that yield relatively pure trophoblasts with less than 5% contamination from other cell types10,11,12. Further purification can be achieved by positive or negative selection24. However, this approach runs the risk of reducing the yield of viable trophoblasts. Immunocytochemical analysis of Cytokeratin-7 in primary trophoblasts isolated using the procedure presented here after 24 versus 72 h of culture time confirmed that the cells underwent syncytialization as evidenced by the presence of multinucleated cell masses at 72 h, a hallmark event in the lifespan of a trophoblast (Figure 3A and B). Furthermore, immunocytochemical analysis of Vimentin in primary trophoblasts cultured for 72 h &#160;revealed very few contaminating fibroblasts (Figure 3C). For routine purposes, the purity of trophoblasts can be monitored in culture by simple morphological assessment. At 24 h of culture time, round individual cytotrophoblasts dominate the culture. Cytotrophoblasts undergo syncytialization after 72 h of culture, resulting in clumps of fused cells. The most common contaminating cell type found in this culture is the fibroblast or endothelial cell, which are elongated and polygonal in shape, respectively. These features can be roughly monitored using a bright microscope.
Treating primary trophoblasts with TNF&#945; provides a controlled system that allows one to measure the effect of TNF&#945; signaling on the regulation of critical pathways in trophoblasts. Naturally, there are factors other than TNF&#945; in the obese maternal environment in vivo that may be responsible for modulating trophoblast function. These include but are not limited to hormonal signals, hyperlipidemia, and a host of other proinflammatory factors25. Combinations of different treatments will further recapitulate the obese intrauterine environment ex vivo in a more physiologically accurate way. The concentrations of exogenous TNF&#945; used to treat trophoblasts in this protocol far exceed those typically circulating in vivo. Serum concentrations have been reported to reach 20 ng/mL or higher in certain inflammatory conditions26. To obtain a measurable response by current laboratory methods (i.e. Western blotting), it is necessary to amplify TNF&#945; concentrations to reveal changes in gene expression and pathways of interest. These responses may exist in vivo to a lesser extent. However, they could nonetheless significantly impact the function of trophoblasts. Exposing trophoblasts to high concentrations of TNF&#945; runs the risk of producing artifacts in gene expression and pathway analyses that may be rooted in cytotoxic effects. Moderate cytotoxicity was observed in trophoblasts exposed to 104 pg/mL TNF&#945; supplemented media for 24 h&#160;as evidenced by LDH cytotoxicity assays (data not shown). However, concentrations at or below 103 pg/mL TNF&#945; did not produce any appreciable cell death.
Testing TNF&#945; concentrations at smaller intervals and/or that are lower than described here may be best followed up by quantitative polymerase chain reaction (qPCR) for gene expression analysis, a technique well suited for detecting small changes in gene expression. While qPCR specifically tests for gene expression levels at the transcriptional level, Western blotting detects the final levels of protein products that are presumably available to perform their cellular task(s). Using both analytical techniques in conjunction is a powerful approach for determining the impact of TNF&#945; treatment on the regulation of gene expression levels as well as for determining if changes in expression levels are a result of transcriptional, post-transcriptional, or post-translational regulation. Inflammatory stress may impact trophoblast behavior, morphology, and syncytialization. Including morphological assessments (i.e. immunocytochemistry) in addition to molecular analytical approaches will provide a more comprehensive analysis of the effects of TNF&#945; exposure on trophoblast health. Adjustment of the TNF&#945; concentrations tested and downstream assays according to experimental demands is advisable.
By implementing the protocol described here, TNF&#945;-mediated inflammation is found to upregulate Rubicon expression in human trophoblasts from lean pregnancies with female fetuses. Western blotting for Rubicon in trophoblast lysates revealed two distinct bands near and above the expected molecular weight of 140 kDa (Figure 4A). There is evidence to suggest that the lower band is non-specific (PD047 Anti-Rubicon pAb, MBL data sheet, http://ruo.mbl.co.jp/bio/g/dtl/A/?pcd=PD027#u-pub). The trophoblasts of females demonstrated the ability to upregulate the expression of Rubicon in response to TNF&#945; treatment of concentrations up to 250 pg/mL. At concentrations higher than this, Rubicon expression levels decreased back towards baseline (untreated control) and even below (Figure 4B, n = 3). Given that treating trophoblasts from lean pregnancies with TNF&#945; simulates inflammation in the obese intrauterine environment, this result is complementary to the finding that Rubicon is significantly upregulated in flash-frozen villous tissue biopsies from placentas from obese pregnancies with female fetuses compared to lean controls (Figure 4C and D, ANOVA, n = 6 per BMI class, P&#60;0.05).
While autophagic flux does not appear to differ in trophoblasts from obese pregnancies with female fetuses compared to lean controls, trophoblasts from obese pregnancies with male fetuses exhibit activation of formation and accumulation of autophagosomes13. Rubicon is a negative regulator of autophagy, where it acts to halt autophagosome-lysosome fusion27. Female trophoblasts may upregulate the expression of Rubicon as a compensatory or protective mechanism against TNF&#945;-mediated inflammation, which can activate autophagy22, allowing them to maintain lean-like autophagic flux in the setting of maternal obesity. This response in the placenta may play a role in how female fetuses fare better than males in the obese intrauterine environment. Modeling the inflammatory milieu associated with maternal obesity ex vivo through treating primary human trophoblasts with TNF&#945; provides a platform to study the effects of the obese intrauterine environment on the regulation of critical pathways in trophoblasts. Amending this protocol with new and combinatorial treatments aimed at recapitulating the obese maternal environment will provide an exciting avenue to study the effects of maternal obesity on placental function.

Obesity is an inflammatory state characterized by chronic low-grade inflammation, stemming from excess adipose tissue and nutrient availability. In obesity, proinflammatory cytokines are elevated in metabolic tissues as well as systemically in circulation. A robust body of evidence has shown that TNF&#945; is significantly elevated in the setting of obesity with implications in insulin resistance and metabolic dysfunction1. Activation of TNF&#945; also contributes to disease pathogenesis in conditions such as cancer and autoimmunity, making it an attractive therapeutic target2.
Inflammation in obesity is compounded by pregnancy, also a proinflammatory state3,4. It has been previously shown that placental TNF&#945; content increases with maternal adiposity in pregnancies with female fetuses. Furthermore, TNF&#945; treatment inhibits mitochondrial respiration in female but not male trophoblast cells, suggesting that TNF&#945; is involved in regulating placental metabolism in a sexually dimorphic manner5. Maternal obesity is associated with the increased incidence of a variety of complications during pregnancy, including stillbirth, with male fetuses being the most susceptible3,6,7,8. Due to its key role at the maternal-fetal interface, changes in the functional capacity of the placenta in the obese intrauterine environment in response to inflammatory signaling may play an important role in mediating the outcomes of obese pregnancies.
Cytotrophoblasts and syncytiotrophoblasts in the villous tissue of the placenta are critical for endocrine signaling and nutrient and oxygen exchange between the mother and developing fetus9. Disruptions in the functional capacity of villous cytotrophoblasts (hereafter referred to as trophoblasts) may jeopardize fetal health and development. This protocol describes a method for sampling of villous tissue from the human term placenta by dissecting away the chorionic and basal plates along with an optimized procedure for the isolation of trophoblasts for primary cell culture. This protocol is derived from established methodologies involving enzymatic digestion of villous tissue to release cells from the extracellular matrix followed by differential density centrifugation to isolate trophoblasts10,11,12. This protocol details an approach in which primary trophoblasts from placentas from lean pregnancies are treated with culture media supplemented with TNF&#945; to simulate one component of the inflammatory milieu associated with maternal obesity. Finally, a simple procedure for harvesting total cell lysates from TNF&#945;-treated trophoblasts followed by Western blotting to detect changes in gene expression is described.
While this model does not recapitulate the obesogenic in utero environment in its entirety, it provides a controlled system that allows one to parse out the individual contribution of TNF&#945;-mediated inflammation in the trophoblasts&#39; response to maternal obesity. This model affords both the opportunity to discover or confirm molecular targets directly regulated by TNF&#945; signaling in trophoblasts as well as allows one to test if changes in gene expression patterns observed in vivo in placentas with maternal obesity may be a result of TNF&#945;-mediated inflammation.
The approach described here was implemented to test the effect of TNF&#945;-mediated inflammation on the regulation of autophagy in human trophoblasts. Trophoblasts from obese pregnancies with male fetuses exhibit disrupted autophagic turnover, or autophagosome maturation13. A protein called Rubicon (RUN domain protein Beclin1-interacting and cysteine-rich containing), which is localized to the lysosomes and late endosomes, has been recently described as a &#34;brake&#34; in the autophagic turnover process because it functions as a negative regulator of autophagosome maturation14,15. In fact, Rubicon is a rare example of a protein that restrains autophagy, which makes it a valuable therapeutic target. Very little information is available about the pathophysiological significance of Rubicon, except for its roles in the innate immune response to microbials16,17 and cardiomyocyte protection18. Using the protocol described here, it is found that Rubicon is upregulated in female primary trophoblasts in response to treatment with increasing concentrations of TNF&#945; up to 250 pg/mL. The regulation of Rubicon may play a role in how female fetuses fare better than males in pregnancies with maternal obesity. Recapitulating inflammation associated with maternal obesity ex vivo by exposing human trophoblasts to exogenous TNF&#945; provides a platform to study the impact of the obese intrauterine environment on the regulation of critical pathways in trophoblasts and by extension, placental function.

<table><tbody><tr><td>10X HBSS</td><td>Gibco</td><td>14185-052</td><td></td></tr><tr><td>CaCl2 (anhyd.)</td><td>Sigma-Aldrich</td><td>C1016-100G</td><td></td></tr><tr><td>MgSO4 (anhyd.)</td><td>Sigma-Aldrich</td><td>M7506-500G</td><td></td></tr><tr><td>Hepes</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>DNAse</td><td>Worthington Biochemical Corp.</td><td>LS002139</td><td></td></tr><tr><td>Protease/Phosphatase inhibitors</td><td>Thermofisher Scientific</td><td>88668</td><td></td></tr><tr><td>Tris HCl</td><td>Invitrogen</td><td>15506-017</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15576-028</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653-1KG</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP166-600</td><td></td></tr><tr><td>Sodium deoxycholate.</td><td>Fisher Scientific</td><td>AAJ6228822</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>Iscove&amp;rsquo;s Modified Dulbecco&amp;rsquo;s Medium (IMDM)</td><td>Gibco</td><td>12440-046</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>Neonatal Calf Serum (NCS)</td><td>Gibco</td><td>26010-074</td><td></td></tr><tr><td>Penicillin/Streptomycin (Pen/Strep)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>10% Formalin</td><td>Fisher Scientific</td><td>23-427-098</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650-100ML</td><td></td></tr><tr><td>TNF&amp;alpha;</td><td>Sigma-Aldrich</td><td>SRP3177-50UG</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>70013-032</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;EDTA vacutainer blood collection tubes</td><td>BD</td><td>366450</td><td></td></tr><tr><td>Percoll (Density Gradient Media, DGM)</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>6 well plates</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Cell strainers</td><td>Fisher Scientific</td><td>22363549</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes 2.0 mL, natural</td><td>Fisher Scientific</td><td>22363352</td><td></td></tr><tr><td>Trypan Blue</td><td>Corning</td><td>25-900-Cl</td><td></td></tr><tr><td>Bio-Rad Mini-PROTEAN Tetra System</td><td>Bio-Rad</td><td>1658001FC</td><td></td></tr><tr><td>Bio-Rad Mini Trans-Blot Cell</td><td>Bio-Rad</td><td>1658033</td><td></td></tr><tr><td>TGX FastCast Acrylamide Kit, 12%</td><td>Bio-Rad</td><td>1610175</td><td></td></tr><tr><td>Mini-Protean 3 Multi-Casting Chamber</td><td>Bio-Rad</td><td>1654112</td><td></td></tr><tr><td>4X Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148-100ML</td><td></td></tr><tr><td>Glycine</td><td>Bio-Rad</td><td>1610718</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P7949-500ML</td><td></td></tr><tr><td>Instant Nonfat Dry Milk</td><td>Carnation</td><td></td><td></td></tr><tr><td>Rubicon (D9F7) Rabbit mAb</td><td>Cell Signalling Technology</td><td>8465S</td><td></td></tr><tr><td>Monoclonal Anti-&amp;beta;-Actin antibody produced in mouse</td><td>Sigma-Aldrich</td><td>A2228-100UL</td><td></td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signalling Technology</td><td>7074S</td><td></td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody</td><td>Cell Signalling Technology</td><td>7076S</td><td></td></tr><tr><td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td><td>Thermo Scientific</td><td>34578</td><td></td></tr></tbody></table>

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.

Term human placentas from lean (pre-pregnancy body mass index (BMI) &#60;25) mothers with uncomplicated pregnancies carrying female offspring were collected and sampled within 15 minutes of delivery by cesarean section (no labor). The placentas were examined for the absence of calcifications and typical development: weighing between 300-600 g with the umbilical cord and membranes removed, round in shape, between 15 - 25 cm in diameter, and umbilical cord inserted into the middle of the placenta. Villous tissue was dissected away from the basal and chorionic plates in 2-3 samples from across the placenta (Figure 1), yielding approximately 100 g of villous tissue as starting material for primary trophoblast isolation. Within 20 minutes of sampling villous tissue, the procedure to isolate primary trophoblasts was started as described here, yielding between 0.8 - 1 x 108 viable cells. The cells were seeded in 6-well culture plates at a density of 3 x 106 (3.3 x 105 cells per cm2). After 24 h&#160;of culture, the cells were examined under a microscope for attachment and proper trophoblast morphology was confirmed (individual round cells). The culture media was replaced with complete media containing a series of concentrations of TNF&#945; between 125-104 pg/mL so that at least two wells were included per concentration and vehicle control (complete media only).
Twenty-four hours&#160;following TNF&#945;-treatment (48 h&#160; of culture), all TNF&#945; medias were replaced with complete media. No appreciable cell death was observed due to treatment with TNF&#945; at concentrations at or below 103 pg/mL. Treatment with 104 pg/mL TNF&#945; was moderately cytotoxic and the cytotoxic effects of this TNF&#945; concentration did not persist after the media was changed as evidenced by Lactate Dehydrogenase (LDH) assays (data not shown). At 72 h of culture, cells were examined for syncytialization under a microscope. Immunocytochemistry for syncytialization and fibroblast contamination revealed relatively pure isolation of trophoblasts (Figure 3). Cellular lysates were harvested according to the protocol described here, yielding between 3-8 &#181;g/&#181;L of total protein per preparation as determined by a BCA assay (data not shown). Western blot analysis in cell lysates from female trophoblasts treated with TNF&#945; showed an upregulation of Rubicon expression in response to concentrations of TNF&#945; up to 250 pg/mL and subsequent downregulation of Rubicon expression at TNF&#945; concentrations greater than 250 pg/mL (Figure 4A and B, 104 pg/mL excluded from analysis based on cytotoxic effects). Likewise, Rubicon is significantly upregulated in flash-frozen villous tissue biopsies from placentas from obese pregnancies with female fetuses compared to lean controls as evidenced by Western blot analysis (Figure 4C and D, n = 6 placentas per BMI class, ANOVA, P&#60;0.05).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Concentration (%)</td>
			<td>90% DGM (ml)</td>
			<td>1x HBSS (ml)</td>
			<td>Layer Thickness (ml)</td>
		</tr>
		<tr>
			<td>4x gradient</td>
			<td>4x gradient</td>
			<td>Total 34.5 ml</td>
		</tr>
		<tr>
			<td>70</td>
			<td>14</td>
			<td>4</td>
			<td>4.5</td>
		</tr>
		<tr>
			<td>60</td>
			<td>8</td>
			<td>4</td>
			<td>3</td>
		</tr>
		<tr>
			<td>55</td>
			<td>7.33</td>
			<td>4.67</td>
			<td>3</td>
		</tr>
		<tr>
			<td>50</td>
			<td>3.34</td>
			<td>2.67</td>
			<td>3</td>
		</tr>
		<tr>
			<td>45</td>
			<td>6</td>
			<td>6</td>
			<td>3 (13.5 ml mark)</td>
		</tr>
		<tr>
			<td>40</td>
			<td>5.33</td>
			<td>6.67</td>
			<td>3</td>
		</tr>
		<tr>
			<td>35</td>
			<td>4.67</td>
			<td>7.33</td>
			<td>3 (19.5 ml mark)</td>
		</tr>
		<tr>
			<td>30</td>
			<td>8</td>
			<td>16</td>
			<td>6</td>
		</tr>
		<tr>
			<td>20</td>
			<td>2.67</td>
			<td>9.33</td>
			<td>3</td>
		</tr>
		<tr>
			<td>10</td>
			<td>1.33</td>
			<td>10.67</td>
			<td>3</td>
		</tr>
	</tbody>
</table>
Table 1. Specifications for Making Density Gradients for Density Centrifugation of Primary Trophoblasts. 
From left to right, column one specifies density gradient media (DGM, see table of the essential supplies, reagents, and equipment, supplementary material) concentration expressed as percentages of DGM in HBSS. Column two specifies the volume of DGM while column three specifies the volume of HBSS required for making the appropriate percentage of DGM solution. Column four specifies the volume to be added to the 50 mL conical tube to build the gradient, beginning with the most dense layer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Trypsin</td>
			<td>HBSS</td>
			<td>DNase</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion</td>
			<td>(total activity; BAEE units)</td>
			<td>volume (ml)</td>
			<td>(total activity; Kunits)</td>
			<td>Total Volume 
			/digestion</td>
		</tr>
		<tr>
			<td>1</td>
			<td>619037 (23.01 ml)</td>
			<td>141.76 ml</td>
			<td>62594 (0.230 ml)</td>
			<td>165 ml</td>
		</tr>
		<tr>
			<td>2</td>
			<td>412691 (15.34 ml)</td>
			<td>94.51 ml</td>
			<td>41729 (0.154 ml)</td>
			<td>110 ml</td>
		</tr>
		<tr>
			<td>3</td>
			<td>313270 (11.65 ml)</td>
			<td>71.74 ml</td>
			<td>31676 (0.116 ml)</td>
			<td>83.5 ml</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>1345000 (50 ml)</td>
			<td>308 ml</td>
			<td>136000 (0.5 ml)</td>
			<td>358.5 ml</td>
		</tr>
	</tbody>
</table>
Table 2. Specifications for the Preparation of Digestion Solution for Primary Trophoblast Isolation Based on the Specific Activity of DNase and Trypsin. 
From left to right, the first column specifies the number of the digestions, the second column specifies the trypsin activity required per digestion, the third column specifies the total volume of supplemented HBSS to be added for the appropriate digestion, the fourth column specifies the DNase activity required per digestion, and the final column specifies the volume of digestion solution to be added to the placental tissue for the appropriate digestion.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HBSS (supplemented with Ca+2 and Mg+2)</td>
			<td>Sample Buffer</td>
		</tr>
		<tr>
			<td>10% 10x HBSS</td>
			<td>90% 4X Laemmli dye</td>
		</tr>
		<tr>
			<td>1.26 mM CaCl2 (anhyd.)</td>
			<td>10% 2-Mercaptoethanol</td>
		</tr>
		<tr>
			<td>0.80 mM MgSO4 (anhyd.)</td>
			<td></td>
		</tr>
		<tr>
			<td>20.77 mM HEPES</td>
			<td></td>
		</tr>
		<tr>
			<td>pH to 7.4 with 10N NaOH 
			Make volume up to 1 L with sterile ddH2O 
			Sterile filter into a sterile bottle</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Media</td>
			<td>Running Buffer</td>
		</tr>
		<tr>
			<td>Remove 11% v/v IMDM</td>
			<td>25 mM Tris Base</td>
		</tr>
		<tr>
			<td>Add 10% v/v FBS</td>
			<td>190 mM Glycine</td>
		</tr>
		<tr>
			<td>Add 1% 10,000 U/mL Penicillin/Streptomycin (100 U/mL final)</td>
			<td>0.1% SDS</td>
		</tr>
		<tr>
			<td></td>
			<td>pH to 8.3</td>
		</tr>
		<tr>
			<td>Freezing Media</td>
			<td></td>
		</tr>
		<tr>
			<td>90% v/v FBS</td>
			<td>Transfer Buffer</td>
		</tr>
		<tr>
			<td>10% v/v DMSO</td>
			<td>25 mM Tris</td>
		</tr>
		<tr>
			<td></td>
			<td>190 mM Glycine</td>
		</tr>
		<tr>
			<td>Digestion Buffer</td>
			<td>20% Methanol</td>
		</tr>
		<tr>
			<td>50 mL Trypsin (26,900 BAEE units/mL)</td>
			<td>pH to 8.3</td>
		</tr>
		<tr>
			<td>0.5 mL DNAse (272,000 K units/mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Bring to 358.5 ml in supplemented HBSS</td>
			<td>TBS</td>
		</tr>
		<tr>
			<td></td>
			<td>20 mM Tris</td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>150 mM NaCl</td>
		</tr>
		<tr>
			<td>25 mM Tris-HCl</td>
			<td>pH to 7.6</td>
		</tr>
		<tr>
			<td>5 mM EDTA</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mM NaCl</td>
			<td>TBST</td>
		</tr>
		<tr>
			<td>0.1% SDS</td>
			<td>TBS with 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>0.5% Sodium deoxycholate</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Triton X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>1 tablet of protease/phosphatase inhibitor per 10 ml RIPA Buffer</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3. Solutions Required for Isolation and Culture of Primary Trophoblasts followed by Western Blotting. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>% DGM</td>
			<td>ml mark</td>
			<td>Cell type</td>
		</tr>
		<tr>
			<td>10</td>
			<td>31.5-34.5</td>
			<td>Debris</td>
		</tr>
		<tr>
			<td>20</td>
			<td>28.5-31.5</td>
			<td></td>
		</tr>
		<tr>
			<td>30</td>
			<td>22.5-28.5</td>
			<td></td>
		</tr>
		<tr>
			<td>35</td>
			<td>19.5-22.5</td>
			<td>Trophoblasts</td>
		</tr>
		<tr>
			<td>40</td>
			<td>16.5-19.5</td>
			<td></td>
		</tr>
		<tr>
			<td>45</td>
			<td>13.5-16.5</td>
			<td></td>
		</tr>
		<tr>
			<td>50</td>
			<td>10.5-13.5</td>
			<td>Lymphocytes</td>
		</tr>
		<tr>
			<td>55</td>
			<td>7.5-10.5</td>
			<td></td>
		</tr>
		<tr>
			<td>60</td>
			<td>4.5-7.5</td>
			<td>Red blood cells</td>
		</tr>
		<tr>
			<td>70</td>
			<td>Below 4.5</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 4. Sedimentation of Trophoblasts by Density Centrifugation. 
From left to right, the first column specifies the percentage of DGM (Table 1), the second column specifies the mL mark where the corresponding percentage of DGM is found on a 50 mL conical tube, and the third column specifies what cell type sediments at the corresponding percentage of DGM and mL mark on a 50 mL conical tube.Trophoblasts sediment between 50- &#160;35% DGM, forming distinct opaque bands. Collecting DGM above or below this range will result in contamination of cellular debris and other cell types such as lymphocytes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56484/56484fig1.jpg" /> 
Figure 1. Villous Tissue is Isolated from the Term Human Placenta by Removing the Chorionic and Basal Plates. 
A) With the chorionic plate (fetal side) facing upwards, a full thickness sample is excised from the placenta. B) A sample of villous tissue is obtained by removing the chorionic and basal plates. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56484/56484fig1large.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/56484/56484fig2.jpg" /> 
Figure 2. Centrifugation of Cells in Digestion Solution over Newborn Calf Serum results in a Multilayered Cell Pellet. 
A) Newborn calf serum (NCS) is layered underneath the cell suspension in digest solution in a 50 mL conical tube. B) Centrifugation of (A) results in a multilayered cell pellet. The bottom-most layer is deep in red color and consists of red blood cells. The layer above includes trophoblasts and is white or beige in color. Above the trophoblast layer is NCS followed by digestion solution (supernatant) to the top of the tube. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56484/56484fig2large.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/56484/56484fig3.jpg" /> 
Figure 3. Immunocytological Analysis of Syncytialization and Fibroblast Content in Primary Human Trophoblast Cultures. 
A) Representative image of Cytokeratin-7 (red) in cytotrophoblasts after 24 h of culture. B) Representative image of Cytokeratin-7 (red) in syncytiotrophoblasts after 72 h of culture shows multinucleated masses of cells that have fused. C) Representative image of Vimentin (red) in syncytiotrophoblasts after 72 h of culture. Images were acquired on a fluorescent microscope with DAPI (blue) nuclear counterstain. Visualized at 10X magnification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56484/56484fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56484/56484fig4.jpg" /> 
Figure 4. Regulation of Rubicon Expression in Response to TNF&#945;-Treatment in Female Trophoblasts and Endogenous Rubicon Expression in Villous Tissue from Lean Versus Obese Pregnancies with Female Fetuses. 
Primary trophoblasts from term placentas from lean mothers with healthy pregnancies carrying a female fetus were isolated and treated with 125, 250, 500, 103, and 104 pg/mL TNF&#945; (or vehicle control). A) Representative Western blot for Rubicon in female trophoblast lysates treated with TNF&#945;. &#946;-actin was used as a loading control. B) Rubicon expression in response to TNF&#945;-treatment in female trophoblasts was quantified from Western blots and normalized to &#946;-actin. Values are mean Rubicon expression per TNF&#945; concentration &#177; S.E. in n=3 placentas. C) Western blots for Rubicon in whole tissue lysates from flash frozen biopsies of villous tissue from lean versus obese pregnancies with female fetuses (F1 -F12). Lactate dehydrogenase A (LDHA) was used as a loading control. D) Rubicon expression in Western blots from (C) was quantified and normalized to LDHA. Values are mean Rubicon expression per BMI classification &#177; S.E. in n=6 placentas per BMI class (ANOVA, *P&#60;0.05). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56484/56484fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta at JoVE.com

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

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 ...

a-primary-human-trophoblast-model-to-study-effect-inflammation

Nanyang Technological University

Research

JoVE Journal

Medicine

11.3K Views.  Oregon Health and Science University. 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 TNFα recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

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

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 ...

Developmental Biology

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 ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Genetics

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.

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 ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Immunology and Infection

Improving Ex Vivo Placental Explant Culture Using a Flow-Based Approach

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models inadequately reflect the dynamic in utero setting, where the placenta encounters constant slight shear stress due to plasma or blood flow. To address this limitation, a flow culture system has been devised to bring ex vivo placental explant cultivation closer to the in utero flow conditions experienced within the maternal body. Within this approach, placental explants are cultivated in a sequence of five interconnected flow chambers. This setting&#160;maintains physiological oxygen concentrations and a consistent flow rate. The collected data reveals that under flow conditions, the preservation of tissue morphology exhibits notable enhancement compared to conventional static methods. This innovative technique introduces a straightforward means of ex vivo placental explant culture, offering a more faithful representation of the dynamic in vivo environment. Moreover, this study introduces new&#160;possibilities for investigating the functional dynamics of the feto-maternal interface. By embracing feasible dynamic methodologies, a deeper comprehension of placental biology is facilitated, underscoring its relevance for maternal-fetal health.

Author Spotlight: Advancing Research Through Single Cell Sequencing and Spatial Histology in Placental Tissues 

Here is a protocol for culturing placental explants under constant flow conditions. This approach enhances traditional static villous culture systems by enabling the replication of dynamic physiological environments.

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

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

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

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

Summary

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Chapters in this video

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

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

Mouse In Vivo Placental Targeted CRISPR Manipulation

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

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

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

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development